Environmental Biotechnology: : Basic Concepts and Applications [1 ed.] 9781783323227, 9781783322602

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Environmental Biotechnology Basic Concepts and Applications

Environmental Biotechnology Basic Concepts and Applications

Viswanath Buddolla

α Alpha Science International Ltd. Oxford, U.K.

Environmental Biotechnology: Basic Concepts and Applications 544 pgs.  |  50 figs.

Viswanath Buddolla Department of Bionanotechnology Gachon University Republic of Korea Copyright © 2017 ALPHA SCIENCE INTERNATIONAL LTD. 7200 The Quorum, Oxford Business Park North Garsington Road, Oxford OX4 2JZ, U.K. www.alphasci.com ISBN 978-1-78332-260-2 E-ISBN 978-1-78332-322-7 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the publisher.

Preface

Biotechnology is the integration of natural sciences and engineering in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. It is versatile and has been assessed a key area which has greatly impacted various technologies based on the application of biological processes in manufacturing, agriculture, food processing, medicine, environmental protection, resource conservation. This new wave of technological changes has determined dramatic improvements in various sectors since it can provide entirely novel opportunities for sustainable production of existing and new products and services. In addition, environmental concerns help drive the use of biotechnology not only for pollution control, but prevent pollution and minimize waste in the first place, as well as for environmentally friendly production of chemical and biomonitoring. This book describes the state-of-the-art and possibilities of environmental biotechnology and explain its various areas together with their related issues and implications. Considering the number of problems that define and concretize the field of environmental biotechnology, the role of some bioprocesses and biosystems for environmental protection, control and health, based on the utilization of living organisms, are analyzed. Environmental remediation, pollution prevention, detection and monitoring are evaluated considering the achievements, as well as the perspectives in the development of biotechnology. Various relevant topics have been chosen to illustrate each of the main areas of environmental biotechnology: wastewater treatment, soil treatment, solid waste treatment, and waste gas treatment, dealing with both the microbiological and process engineering aspects. The distinct role of environmental biotechnology in the future is emphasized considering the opportunities to contribute with new solutions and directions in remediation of contaminated environments, minimizing

vi

Preface

future waste release and creating pollution prevention alternatives. To take advantage of these opportunities, innovative new strategies, which advance the use of molecular biological methods and genetic engineering technology, are examined. These methods would improve the understanding of existing biological processes in order to increase their efficiency, productivity, and flexibility. Examples of the development and implementation of such strategies, are included. In addition, the contribution of environmental biotechnology to the progress of a more sustainable society is revealed in this book. Viswanath Buddolla

Contents

Preface........................................................................................................................... v 1. Introduction to Environmental Biotechnology............................1.1—1.37 1.1 Biotechnology for Environment...................................................... 1.3 1.2 Biotechnology, Agriculture and Environmental Pollution.......... 1.5 1.3 Challenges Imposed on the Environment by Human Activities............................................................................................. 1.6 1.4 Contributions of Biotechnology to Improve Agricultural Productivity........................................................................................1.7 1.4.1 Biotechnology in reducing the use of chemical pesticides, herbicides and fertilizers.................................1.9 1.4.2 Biofertilizers.......................................................................... 1.9 1.5 Biotechnology and Livestock Production in the Improvement of Environmental Conditions...............................1.11 1.6 Biotechnology and the Removal of Toxic Chemicals from the Environment..................................................................... 1.11 1.7 Biotechnology and the Removal of Heavy Metal Pollution from the Environment....................................................1.12 1.8 Biotechnology and Desulphurization of Fossil Fuels................. 1.13 1.9 Biotechnology and Ecosystem Modeling..................................... 1.13 1.10 Microbial Biotechnology in the Monitoring of Environmental Pollution.................................................................1.13 1.11 Microbial Biotechnology in the Bioassay of Environ mental Toxicity.................................................................................1.14 1.12 Biotechnology and Control of Oil Spillage..................................1.14

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1.13 Bioremediation and their Importance in Environment Protection..........................................................................................1.15 1.13.1 Factors affecting biodegradation.....................................1.15 1.13.2 In situ bioremediation....................................................... 1.16 1.13.2.1 Intrinsic bioremediation.................................... 1.16 1.13.2.2 Engineered in situ bioremediation................... 1.16 1.13.3 Ex-situ bioremediation...................................................... 1.17 1.13.3.1 Solid phase treatment.........................................1.17 1.13.3.2 Slurry phase treatment ......................................1.17 1.13.4 Use of genetic engineering and genetic mani pulations for more efficient bioremediation.................. 1.18 1.13.5 Biotechnology to reduce atmospheric Carbon dioxide (CO2) ..................................................................... 1.19 1.13.6 Treatment of sewage using microorganisms.................1.20 1.13.7 Treatment of industrial effluents using biotechnology.....................................................................1.21 1.13.8 Use of biotechnology for toxic site reclamation............ 1.22 1.13.9 Use of biotechnology in the removal of oil and grease deposits................................................................... 1.23 1.13.10 Tannery effluents and their treatment............................ 1.24 1.13.11 Bioremediation of Radioactive Contaminants...............1.25 1.14 Use of Biosensors to Detect Environmental Pollutants..............1.26 1.14.1 Principle of a biosensor..................................................... 1.27 1.14.2 Use of selected and engineered microbes for removal and recovery of strategic and precious metals from contaminated degraded lands................... 1.28 1.15 Biotechnology and the Saving of Resources and Energy...........1.29 1.16 Fears and Concerns About Biotechnology Approach in Achieving a Safe Environment and Agriculture......................... 1.30 1.17 Fears and Concerns about Environmental Impact of Bioremediation................................................................................. 1.31 1.18 Role of Biotechnology in Restoration of Degraded Lands.........1.31 1.18.1 Use of micropropagation and Mycorrhiza for reforestation........................................................................1.31 1.18.2 Improvement of soil infertility through the use of nitrogen fixing bacteria, Rhizobium in association with leguminous trees and Frankia in association with non-leguminous species.................1.32

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1.18.3 Development of plants tolerant to abiotic stress which can be grown on degraded lands........................1.32 1.19 Biomimicry and Biotechnology.....................................................1.33 1.20 Biotechnology in the Conservation of Biodiversity.................... 1.34 1.21 Basic Tools and Methodologies Associated with Environmental Biotechnology....................................................... 1.35 1.22 Developing Environmentally Sound Biotechnologies in India ..............................................................................................1.36 2. Environmental Microbiology—Soil.............................................. 2.1—2.71 2.1 Soil and Soil Microorganisms.......................................................... 2.2 2.1.1 The characteristics of soil microorganisms......................2.2 2.1.1.1 Viruses....................................................................2.3 2.1.1.2 Bacteria................................................................... 2.3 2.1.1.3 Actinomycetes....................................................... 2.5 2.1.1.4 Fungi.......................................................................2.5 2.1.1.5 Soil phytoedaphon...............................................2.6 2.1.1.6 Fauna of soil.......................................................... 2.7 2.1.2 The Number of Soil Microorganisms................................ 2.9 2.1.2.1 Viruses....................................................................2.9 2.1.2.2 Bacteria................................................................... 2.9 2.1.2.3 Fungi.......................................................................2.9 2.1.2.4 Algae.....................................................................2.10 2.1.2.5 Soil fauna............................................................. 2.10 2.1.3 Activity of Microorganisms in soil..................................2.10 2.1.3.1 Cellulose decomposition...................................2.11 2.1.3.2 Lignin decomposition........................................2.11 2.1.3.3 Synthesis and humus decomposition.............. 2.12 2.1.3.3.1 The synthesis of humus (Humification)..................................... 2.12 2.1.3.3.2 Decomposition of humus..................2.13 2.1.3.4 Atmospheric nitrogen fixation..........................2.14 2.1.3.5 Free-living N2 assimilators (non symbiotic nitrogen fixation).............................. 2.15 2.1.3.6 Ammonification..................................................2.15 2.1.3.7 Nitrification.........................................................2.15 2.1.3.8 Denitrification ....................................................2.16 2.1.4 Interactions among Soil Microorganisms....................... 2.16

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2.1.4.1 Beneficial Associations/Interactions................. 2.17 2.1.4.1.1 Mutualism (Symbiosis)......................2.17 2.1.4.1.2 Commensalisms................................. 2.17 2.1.4.1.3 Proto-cooperation............................... 2.17 2.1.4.2 Detrimental (Harmful) Associations/ Interactions............................................................. 18 2.1.4.2.1 Antagonism......................................... 2.18 2.1.4.2.2 Ammensalism..................................... 2.18 2.1.4.2.3 Ammensalism Competition..............2.18 2.1.4.2.4 Parasitism............................................ 2.19 2.1.4.2.5 Predation............................................. 2.19 2.1.5 Mutual interaction of plants and microorganisms.......2.19 2.1.5.1 The symbiosis of microbes and plants – bacterrhiza...........................................................2.19 2.1.5.1.1 Rhizosphere......................................... 2.20 2.1.5.2 The symbiosis of fungi and plants................... 2.21 2.1.5.2.1 Ectotrophic mycorrhiza..................... 2.21 2.1.5.2.2 Endotrophic mycorrhiza...................2.21 2.1.6 Soil Bioremediation...........................................................2.22 2.1.6.1 Microorganisms used in remediation technologies......................................................... 2.22 2.1.6.2 Stimulation of bioremediation.......................... 2.24 2.1.6.3 Classification of bioremediation methods......2.24 2.1.6.3.1 In situ methods................................... 2.25 2.1.6.3.2 Ex situ methods.................................. 2.28 2.1.7 Role of Soil Microorganisms in Biodegradation of Pesticides and Herbicides............................................2.30 2.1.7.1 Effects of pesticides............................................2.30 2.1.7.2 Persistence of pesticides in soil.........................2.31 2.1.7.3 Biodegradation of Pesticides in Soil.................2.31 2.1.7.4 Criteria for Bioremediation/Biodegradation..2.32 2.1.7.5 Strategies for Bioremediation...........................2.33 2.1.8 Microbial Ecology of Petroleum Contaminant Plumes ................................................................................ 2.33 2.1.9 Molecular Microbial Ecology...........................................2.34 2.1.10 Soil Microorganisms as Biofertilizers.............................2.36 2.1.10.1 Role of Biofertilizers in soil fertility and Agriculture..........................................................2.36

Contents

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2.1.10.2 Quality Control Measures (as per ISI Specifications)...................................................... 2.38 2.1.11 Factors Affecting Distribution, Activity and Population of Soil Microorganisms.................................2.38 2.1.12 Rhizosphere Concept and It’s Historical Background......................................................................... 2.42 2.1.12.1 Microorganisms in the Rhizosphere and Rhizosphere Effect.............................................. 2.43 2.1.12.2 Factors affecting microbial flora of the Rhizosphere/Rhizosphere Effect...................... 2.45 2.1.12.3 Alterations in Rhizosphere Microflora............2.47 2.1.12.4 Associative and Antagonistic activities in the Rhizosphere..................................................2.48 2.1.12.5 Rhizosphere in relation to Plant Pathogens.... 2.49 2.1.13 Soil Microorganisms in Cycling of Elements or Plant Nutrient..................................................................... 2.51 2.1.13.1 Nitrogen Cycle....................................................2.51 2.1.13.2 Sulphur Cycle/Sulphur Transformations........2.55 2.1.13.3 Phosphorus Cycle or Transformation..............2.57 2.1.13.4 Iron Cycle or Transformation............................ 2.58 2.1.14 Organic Matter................................................................... 2.59 2.1.14.1 Microbiology of decomposition of various constituents in organic matter..........................2.62 2.1.15 Notable contributions made by several scientists in the field of soil microbiology ...................................... 2.64 2.1.16 Notable contributions made by Indian scientists in the field of soil microbiology....................................... 2.69 3. Environmental Microbiology—Water and Air............................3.1—3.42 3.1 Water and Water Microorganisms .................................................. 3.1 3.1.1 Types of microorganisms in water.................................... 3.2 3.1.1.1 Groups of water organisms................................. 3.2 3.1.2 Factors limiting growth of microorganisms in water..... 3.4 3.1.3 Characterization of water microorganisms...................... 3.5 3.1.4 Sources and types of pollutants....................................... 3.11 3.1.5 Self-purification of surfacewaters.................................... 3.11 3.1.6 Water-transmitted pathogenic microorganisms............3.14 3.1.7 Sanitary quality of water................................................... 3.17 3.1.8 Wastewater treatment....................................................... 3.21

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3.1.9 Biological methods of wastewater treatment ................3.25 3.1.10 Artificial methods of wastewater treatment..................3.28 3.1.11 Methods of chemical wastewater treatment.................. 3.31 3.1.12 Nucleic Acid - Based Techniques for Analyzing the Diversity, Structure, and Dynamics of Microbial Communities in Wastewater Treatment............................................................................3.33 3.2 Air As An Environment of Microorganisms................................3.39 3.2.1 Adaptation of microorganisms to the air environment.......................................................................3.39 4. Environmental Microbiology—Methods and Applications.....4.1—4.56 4.1 Microorganisms-Metal Transformations........................................ 4.1 4.2 Microorganisms in Environmental Monitoring............................4.6 4.3 Applications of the Polymerase Chain Reaction in Environmental Microbiology......................................................... 4.15 4.4 Pesticides and Other Pollutants Degradation by Microorganisms and Genetically Engineered Microbes............4.28 4.5 Degradation of Oil by Microorganisms for the Production of Useful Products.......................................................4.34 4.6 Degradation of Plastics by Microorganisms for Production of Useful Products.......................................................4.37 4.7 Recovery of Minerals By Microbes................................................4.38 4.8 Bioindicators of Hazardous Pollutants.........................................4.46 5. Beneficial and Effective Microorganisms for a Sustainable Agriculture and Environment......................................................... 5.1—5.18 5.1 The Concept of Effective Microorganisms: Their Role and Application..................................................................................5.3 5.2 Utilization of Beneficial Microorganisms in Agriculture............. 5.3 5.3 Beneficial and Effective Microorganisms for a Sustainable Agriculture..................................................................... 5.5 5.4 Beneficial Microorganisms for Soil Quality and a more Sustainable Agriculture..................................................................... 5.6 5.5 Controlling The Soil Microflora: Principles and Strategies.........5.7 5.6 Controlling The Soil Microflora for Optimum Crop Production and Protection................................................................ 5.9 5.7 Application of Beneficial And Effective Microorganisms..........5.11 5.8 Classification of Soils Based on Their Microbiological Properties..........................................................................................5.12

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5.8.1 Functions of Microorganisms: Putrefaction, Fermentation, and Synthesis............................................5.13 5.8.2 Relationships Between Putrefaction, Fermentation, and Synthesis...................................................................... 5.14 5.9 Classification of Soils Based on the Functions of Microorganisms...............................................................................5.15 6. Phytoremediation.............................................................................. 6.1—6.13 6.1 Principal Mechanism of Phytoremediation...................................6.2 6.2 Phytoremediation Processes ...........................................................6.2 6.2.1  Rhizofiltration.......................................................................6.3 6.2.2  Phytostabilization................................................................ 6.3 6.2.3  Phytoextraction.................................................................... 6.3 6.2.4  Phytovolatilisation...............................................................6.4 6.2.5  Phytotransformation or Phytostimulation or Rhizodegradation ............................................................... 6.4 6.3 Phytoremediaiton of Organic Pollutants........................................ 6.5 6.4 Plants’ Response to Heavy Metals...................................................6.7 6.4.1  Metal excluders.................................................................... 6.8 6.4.2  Metal indicators...................................................................6.8 6.4.3  Metal accumulator plant species....................................... 6.8 6.5 Hydraulic Control of Pollutants .....................................................6.9 6.5.1  Riparian corridors................................................................ 6.9 6.5.2  Vegetative cover...................................................................6.9 6.6 Advantages and Disadvantages of Phytoremediation............... 6.10 6.7 Phytoremediation & Biotechnology.............................................. 6.10 6.7.1 Risk Assessment................................................................. 6.11 6.7.2 Future of Phytoremediation.............................................6.12 7. Solid Waste Disposal and Management.......................................7.1—7.31 7.1 Types of Solid Waste..........................................................................7.2 7.1.1 Municipal solid waste......................................................... 7.2 7.1.2 Industrial solid waste ......................................................... 7.3 7.1.3 Biomedical Waste .................................................................7.4 7.2 Solid Waste Disposal and Management: .......................................7.5 7.4 Biological Reprocessing of Solid Waste Disposal ....................... 7.10 7.4.1 Composting........................................................................ 7.11 7.5 Biotechnological Methods of Solid Waste (Agriculture, Domestic and Industrial) Degradation.........................................7.13

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7.6 7.7 7.8

Importance, Health Impacts and Awareness of Waste Management.........................................................................7.14 Health Impacts of Solid Waste....................................................... 7.15 Key Concepts in Municipal Waste Reduction............................. 7.17

7.9 Wastewater........................................................................................ 7.18

7.9.1

Wastewater treatment and Management....................... 7.19

7.9.2

Water Purification by Waterweeds and Membrane Filters...............................................................7.25



7.9.3

Indicator Organisms.......................................................... 7.28



7.9.4

Reclaim of Treated Wastewater........................................7.29



7.9.5

Uses and benefits of reclaimed water.............................7.30

8.

Biological Methods of Pest Management.....................................8.1—8.27



8.1

Advantages of Microbial Insecticides.............................................8.9



8.2

Disadvantages of Microbial Insecticides...................................... 8.10



8.3

Insect Growth Regulators...............................................................8.10



8.4

Use of Pheromones for Pest Management...................................8.14



8.5

Pheromones: Insect Pest Management......................................... 8.15



8.6  Biological Control of Weeds...........................................................8.16



8.6.1 The process...........................................................................8.17



8.7

Chromosomal Manipulation .........................................................8.19



8.8

Sterile Male Technology.................................................................. 8.20

8.9

Environmental Epidemiological Surveys as Indices of Health Hazards for Environmental Pollution.............................8.21



8.10 Toxicity...............................................................................................8.24



8.10.1 Types of toxicity................................................................. 8.25



8.10.2 Factors influencing toxicity..............................................8.25



8.11 Measuring Toxicity...........................................................................8.25



8.11.1 Toxic..................................................................................... 8.27



8.11.2 Highly toxic........................................................................8.27

9. Algae-Biotechnology........................................................................9.1—9.32

9.1

Algae as A Source of Food and Feed...............................................9.4



9.1.1

Microalgae nutritional composition.................................. 9.5

9.2

Mass Cultivation of Commercially Valuble Marine Microalgae for Agar Agar, Alginates ............................................. 9.9



9.2.1

Other applications.............................................................9.18

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9.3

Commercial Application of Microalgae and their Products......9.20



9.3.1

9.4 9.5

9.3.2 Future Development of Microalgal Applications.........9.26 Mass Cultivation of Microalgae as a Source of Protein and Feed............................................................................................ 9.28 Microalgae as a Source of Feed......................................................9.29

Current commercial uses of algae...................................9.21

10. Concepts and Scope of Plant Biotechnology...........................10.1—10.23 10.1 Applications of Genetic Engineering Technology for Crop Improvement.......................................................................... 10.2 10.2 Production of Transgenic Plants with Improved Yields and Nutritional Quality.................................................................. 10.5 10.3 Transgenic Plants for The Production of Viral Antigens.......... 10.10 10.3.1 Edible Vaccines.................................................................10.13 10.4 Biotechnology in Agriculture—Merits and Demerits .............10.15 10.4.1 Transgenic Crops in the U.S. Market............................ 10.17 10.4.2 Possible Risks Associated with using Transgenic Crops in Agriculture .......................................................10.19 10.4.3 Health-related Issues ...................................................... 10.19 10.4.4 Environmental and Ecological Issues .......................... 10.20 10.4.5 Social Issues .....................................................................10.21 11. Animal Biotechnology..................................................................11.1—11.23 11.1 History of Animal Cell Culture .....................................................11.1 11.2 Animal Cell Culture ........................................................................ 11.2 11.3 Requirements for Animal Cell Culture ........................................11.4 11.3.1 Synthetic Media.................................................................. 11.5 11.4 Cell-Based Therapy.......................................................................... 11.6 11.5 Applications of Animal Cell Culture.............................................11.6 11.5.1 Somatic Cell Fusion........................................................... 11.6 11.5.2 Blood Factor VIII ...............................................................11.7 11.5.3 Erythropoietin (EPO) ........................................................11.8 11.5.4 The production of Monoclonal Antibodies using Hybridoma Technology....................................................11.8 11.6 Scale-Up of Animal Cell Culture .................................................. 11.9 11.6.1 Roller Bottles ....................................................................11.10 11.6.2 Micro Carrier Beads.........................................................11.11 11.6.3 Spinner cultures...............................................................11.11

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11.7 Types of Cell Cultures....................................................................11.11 11.8 Characterization of Cell Lines...................................................... 11.13 11.9 Stem Cell Technology....................................................................11.14 11.9.1 Genetic Engineering of Animal Cells and their Applications ...........................................................11.16 11.9.2 Manipulation of Gene Expression in Eukaryotes ...... 11.16 11.9.3 Collection and purification process of Recombinant proteins ....................................................11.17 11.9.4 Organ culture and Histotypic cultures ........................11.17 11.9.5 Organ culture ..................................................................11.18 11.9.6 Techniques and Procedure for organ culture ............. 11.18 11.9.7 The advantages of organ culture ..................................11.19 11.9.8 Limitations of organ culture .......................................... 11.19 11.9.9 Histotypic cultures .........................................................11.19 11.9.10 Organotypic cultures ......................................................11.20 11.10 Cell and Tissue Engineering........................................................11.20 11.10.1 Design and Engineering of Tissues ..............................11.20 12. Biotechnology of Aquaculture....................................................12.1—12.27 12.1 Production of Transgenic Fish....................................................... 12.6 12.2 Pearl Oyster Culture........................................................................ 12.9 12.2.1 Early Development and Larval Rearing....................... 12.11 12.3 Shrimp Farming.............................................................................12.13 12.3.1 Shrimp Nutritional Value Details.................................. 12.17 12.4 Growth and Reproduction of Edible Crustaceans.................... 12.18 12.5 Neuroendocrine Principles Involved in the Regulation of Growth, Reproduction and Metabolism of Prawns and Crabs (Edible Crustaceans).......................................................... 12.22 12.5.1 Inhibitory Principles........................................................12.23 12.5.2 Stimulatory principles.....................................................12.25 12.5.3 Inhibitory principles........................................................12.25 13. Nutrient Film Culture Techniques............................................. 13.1—13.18 13.1 Plant Diseases................................................................................... 13.2 13.2 Phytodiagnostics Based on Immunological and Molecular Techniques.....................................................................13.4 13.3 Molecular or Nucleic acid-based techniques............................... 13.6 13.3.1 Polymerase Chain Reaction (PCR)................................... 13.7

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13.3.2 Real-time PCR..................................................................... 13.8 13.3.3 Rolling Circle Amplification (RCA)............................... 13.11 13.4 Antagonistic Fungi........................................................................13.14 13.5 Antagonistic Bacteria..................................................................... 13.16 13.6 Antifeedants................................................................................... 13.16 13.7 Insecticidal Activities of The Compounds of Botanics (Botanical Insecticdes)...................................................................13.17 13.8 Predators......................................................................................... 13.18 14.

Biotechnology: Industrial Sustainability.................................14.1—14.15 14.1 Industrial Sustainability..................................................................14.1 14.2 Technology, Cleaner Production and Sustainability...................14.3 14.3 Learning from Nature: Biomimicry and Biotechnology............14.5 14.4 Bio-Safety..........................................................................................14.6

15. Ethical Issues in Environmental Biotechnology.....................15.1—15.12 15.1 Release of Genetically Modified Organisms (GMOS).................15.4 15.1.1 Effects on the Environment..............................................15.5 15.1.2 Effects on Human Health................................................. 15.5 15.1.3 Environmental Biotechnology—Biosafety Management.......................................................................15.9 15.1.4 Environmental Biotechnology & Intellectual Property Rights (IPRs).....................................................15.11 Appendix Useful Terms and their Meanings of Environmental Biotechnology........................................................................... A.1—A.68 Index .................................................................................................................. I.1—I.3

CHAPTER

1

Introduction to Environmental Biotechnology

The word “environment” is derived from the French word “environ”. The meaning of the French word is somewhat related to “encompass”, “encircle”, etc. It is believed to have been introduced into the subject by biologist Jacob Van Erkulin in the early 1900s and the term “environment” is defined as our surroundings, which includes the abiotic component (the non-living) and the biotic component (the living) around us. The abiotic environment includes water, air and soil while the biotic environment consists of all living organisms—plants, animals and micro-organisms. The environment is a very important component necessary for the existence of both human and other biotic organisms. Contrary to its name, biotechnology is not a single technology. Rather, it is a group of technologies that share two (common) characteristics working with living cells and their molecules and having a wide range of practical uses that can improve our lives. Biotechnology can be broadly defined as “using organisms or their products for commercial purposes.” Production may be carried out by using intact organisms of bacteria, fungi and other microbes, or by using natural substances created by the organisms, such as enzymes. Environmental biotechnology is “the integration of natural sciences and engineering in order to achieve the application of organisms, cells, parts thereof and molecular analogues for the protection and restoration of the quality of our environment”. The International Society for Environmental Biotechnology defines Environmental Biotechnology as the development, use and regulation of biological systems for remediation of contaminated environments (land, air and water) and for environment-friendly processes (green manufacturing technologies and sustainable development). It can also be described as ‘‘the optimal use of nature, in the form of plants, animals, bacteria, fungi and algae, to produce renewable energy, food and nutrients in a synergistic integrated cycle of profit-making processes where the waste of each process becomes the feedstock for another process’’. The prime target of this science is the abatement of pollution through bioremedation/biotreatment or supporting as resources for human use

1.2

Environmental Biotechnology

in non-polluting ways. It can also help in cleaner production of existing products. On the whole, it encompasses aspects of natural resources management, the treatment of waste and control of pollution. Thus, the major areas of understanding are environmental pollution abatement through biodegradation, biotransformation, bioaccumulation of toxicity like organics, metals, oil, and hydrocarbons, dyes, detergents, etc.; Energy management through production of non-conventional non-polluting energy like biodiesel methanol, biogas, biohydrogen etc.; Agricultural application of biofertilizer, biopesticides, or bioorganics of multiple users. Recovery of resources from toxic or non-toxic wastes through biotechnological approach; Biosensor approach of pollution monitoring and several other allied issues. Environmental Biotechnologies are competing with great success against traditional technologies recent days and are providing solutions to acute problems through the so called ‘end of pipe’ treatment technologies and bioremediation. Environmental biotechnology can also provide a natural way of addressing mainly environmental problems ranging from the identification of biohazards to bioremediation techniques for industrial, agricultural and domestic ends like municipal effluents and residues. Thus, environmental use of biotechnology includes the development, biosecurity use, and regulation of biological systems for remediation of contaminated environments like land, water, air as well as for use in environmentally sound processes leading to clean technologies and sustainable development. Virtually, all types of human activities generate wastes and this places a heavy burden on the environment. So far, we have relied more upon the physical and chemical methods of pollution control. However, micro-organisms are likely to prove as more suitable tools for pollution control due to their versatility and adaptability. In the field of environmental management, biotechnology have helped in environmental monitoring, degradation of wastes, substitution of nonrenewable resource base with a renewable one and production of eco-friendly products and processes. The application of biotechnologies is frought with risks that are difficult to define, much less to assess. The introduction of genetically engineered microbes can create new ecological niches and bring about large-scale transformations in the structure and function of the ecosystem as a whole. There are many unknown risks and hazards associated with the accidental or deliberate release of self-propagating, genetically engineered novel forms of life into the biosphere. In the event of disastrous consequences, it is likely that people will be harmed who had little gain from the use of biotechnology. In recent years, considerable development in terms of R&D and its application on “Environmental Biotechnology” is made round the world. The major areas of application are microbial biodegradation of pollutants, development of appropriate technology for biofertilizers and biopesticides production and its use in the field of agriculture and horticulture; production of single cell protein, etc. Considerable discussion occurred in recognition and support

Introduction to Environmental Biotechnology

1.3

of the tremendous potential that environmental biotechnology offered particularly in the development of the next generation of pollution prevention, pollution abatement, and sustainable development of green technologies. The emergence and acceptance of the concept of sustainable development warrants that the scope of environmental biotechnology be enlarged to address issues like environmental monitoring, restoration of environmental quality, resource/ residue/waste-recovery/utilization/treatment, and substitution of the nonrenewable resource base with renewable resources.

1.1

BIOTECHNOLOGY FOR ENVIRONMENT

Industrial growth, economic development, consumerisation indicate a country’s progress and life standard of its individuals. Industrial growth in the 20th century has brought along with it new problems, too. Water pollution, air pollution, land pollution, noise pollution, radioactive pollution, solid wastes, depletion of resources, scarcity of good quality water, proliferating health hazards, are all results or consequences of stupendous industrial activities with less attention to their negative impacts on the humans and the environment. Nature’s built-in mechanisms and self-regulation ability has been thrown out of gear by the quantity and complexity of wastes generated by the modern society. In economic terms, we only consider material costs and energy costs involved and fail to pay attention to the costs involved in the form of loss of environmental quality. Urbanization, wrong agricultural practices, etc.; are responsible for pollution. As technological progress has followed the industrial revolution, solving of environmental problem must follow technological progress. Industrial processes and products thereof, both must become environmentally friendly and least damaging. Pollution may be defined as an undesirable change in physical, chemical or biological characteristics of air, water and land that can harm human life, the lives of desirable species, our industrial processes, living conditions and cultural assets; or waste our raw material resources. Environment protection means limiting the impairment of environment and it includes conservations of resources. ‘Environment protection’ has three main objectives: • To prevent damage and discomfort; • To improve productivity and pleasure; and • To maintain balances of the ecosystem. Environment protection efforts will pay us back in terms of money, economy, productivity, social justice, cleaner surrounding and sound health. Environmental problems differ only a little with respect to a country; so the problems faced elsewhere and controls applied are equally applicable. Awareness, participation and action in a concerted manner by all who are connected will be needed to solve the pollution problems. ‘Environmental Biotechnology’ involves specific applications of biotechnology to the management of environment and related socio-economic and developmental

1.4

Environmental Biotechnology

issues, keeping in view the concept of sustainable development. ‘Environmental biotechnology encompasses issues like: • Environmental monitoring; • Restoration of environmental quality; • Resource/residue/waste-recovery /utilization/treatment by application of r-DNA technology; • Substitution of non-renewable resources by renewable ones; • Strain improvement for degradation of highly-toxic pollutants with the production of chemicals; • Global changes; • Biological diversity; and • Risk management. Industrial pollution management is thus one amongst the many issues that environmental biotechnology addresses to. For a long time, the point of discussion in environmental pollution as an issue has been symptoms rather than causes of pollution. This naturally influenced our thinking, and emphasis was given on measurement and removal (treatment technologies). Then slowly attention was directed towards the Environment Impact Assessment (EIA) and this could add to the better planning and possibility of better control. And today—we have started to attack the root cause of pollution. We have started discussing of what is described as ‘clean technologies’ which believe in process development and modifications to minimize pollution. The points useful in effective pollution management are: (a) In-process treatment. (b) End-of-pipe treatment. (c) Remediation of polluted sites. (d) Modification of existing processes. (e) Introduction of new processes and products. Though many technologies have been available for clean-up purpose, only a few of them have been proved to be of routine application value. In 1989, the US Environmental Protection Agency published a broad assessment of international technologies (particularly those from Europe, Canada and Japan) for remediation of superfund sites. The criteria used by them for assessment apply in general also while evaluating different technologies for pollution abatement purpose. The criteria are: (i) Function—object and applicability of technology; (ii) Description—details of operating principles and design features; (iii) Performance—demonstration of effectiveness; (iv) Limitations; (v) Economics; and (vi) Status of research, development and availability.

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In addition to the above mentioned points, the choice of technology is also influenced by the social, political, and geographical considerations. The criteria apply to the assessment of technologies in other environmental cleanup work, too.

1.2 BIOTECHNOLOGY, AGRICULTURE AND ENVIRONMENTAL POLLUTION Agriculture is the use of natural resources base for the improvement and increase in production of crops, livestock, fish and trees. In Agricultural biotechnology, improvement is accelerated and production is increased using updated knowledge of living organisms including the genetic code. These include well-established conventional techniques as in biological pest control, fermentation, and production of vaccines and biofertilizers as well as modern techniques like tissue culture, genetic engineering (also called genetic modification), recombinant DNA technology (rDNA) and crop and animal transformation as a result of transgenesis. The importance of these new technologies like biotechnology in food security, environmental sustainability and economic development was captured at the United Nations General Assembly in 2005. Global industrial explosion which is intended to cater to the needs of the world’s increasing population is always associated with environmental pollution. Pollution occurs as a result of improper management of industrial by-products, their accumulation in the environment beyond acceptable limits and therefore, causes hazard and/or nuisance to man. The industrial byproducts that are pollutants may be either organic or inorganic compounds. Man’s environment is composed of abiotic and biotic components. Developing countries are faced with the challenge of rapidly increasing agricultural productivity to help feed their growing populations without depleting the natural resource base. In many countries, agriculture is still subsistent and primitive and this raises concerns on food security, deforestation, rapid population growth, environmental protection, poor soils, stressed environments, unfavorable climatic conditions and improved crops and livestock. For instance, an environment in which pollution of a particular type is maximum. The effluents of a starch industry mixing up with a local water body like a lake or pond. These cause huge deposits of starch which are not easily degraded by micro-organisms except for a few exceptions. Through genetic engineering, a few micro-organisms were isolated from the polluted site and scanned for any significant changes in their genome like evolutions or mutations. The modified genes were then identified because the isolate would have adapted itself to utilize/degrade the starch better than other microbes of the same genus. As a result, the resultant genes are cloned onto industrially significant micro-organisms, which are used for economically significant processes like fermentation, and it can also be applied in pharmaceutical industries.

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Another case study is the incidence of oil spills in the oceans, which require clean-up; microbes isolated from oil rich environments like oil transfer pipelines; oil wells, etc. have been discovered to have the potential to degrade or use it as an energy source and thus serve as a remedy to oil spills. Still another case study is the case of microbes isolated from pesticide-rich soils. These microbes have the potential to utilize the pesticides as a source of energy and so when mixed along with biofertilizers, they would serve as a good insurance against increased pesticide-toxicity levels in agricultural processes. However, there are counter arguments that the newly introduced micro-organisms used for clean-up of oil spillage could create an imbalance in the natural environment concerned. There are also concerns that the mutual harmony in which the organisms existing in that particular environment may be altered and extreme caution should be taken so as not to disturb the mutual relationships that are already established in that environment to which these newly discovered and cloned micro-organisms are introduced. This leads to a suggestion that the positive and negative environmental consequences of environmental and agricultural biotechnology needs to be promptly addressed.

1.3

CHALLENGES IMPOSED ON THE ENVIRONMENT BY HUMAN ACTIVITIES

Human activities constitute one of the major means of introduction of heavy metals into the environment. One of the major developmental challenges facing this decade is how to achieve a cost-effective and environmentallysound strategies to deal with the global waste crisis facing both the developed and developing countries. The crisis has threatened the assimilative and carrying capacity of the earth, our life supporting system. Although the nutrient content of wastes makes them attractive as fertilizers, land application of many industrial wastes and sewage is constrained by the presence of heavy metals, hazardous organic chemicals, salts, and extreme pH. Heavy metal pollution of the environment, even at low levels, and their resulting long-term cumulative health effects are among the leading health concerns all over the world. For example, the bioaccumulation of lead (Pb) in human body interferes with the functioning of mitochondria, thereby impairing respiration, and also causes constipation, swelling of the brain, paralysis and eventual death. This problem is even more pronounced in the developing countries where research efforts towards monitoring the environment have not been given the desired attention by the stakeholders. Heavy metals concentration in the environment cannot be attributed to geological factors alone, as human activities do modify considerably the mineral composition of soils, crops and water. The recent population and industrial growth has led to increasing production of domestic, municipal and industrial wastes, which are indiscriminately dumped in landfill and water bodies without treatment.

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Municipal waste contains such heavy metals as Arsenic (As), Cadmium (Cd), Copper (Cu), Iron (Fe), Gold (Av), Manganese (Mn), Lead (Pb), Nickel (Ni) and Zinc (Zn) which end up in the soil as the sink when they are leached out from the dumpsites. Soil is a vital resource for sustaining two human needs of quality food supply and quality environment. Plants grown on a land polluted with municipal, domestic or industrial wastes can absorb heavy metals in the form of mobile ions present in the soil solution through their roots or through foliar absorption. These absorbed metals get bioaccumulated in the roots, stems, fruits, grains and leaves of plants. Plants are known to take up and accumulate heavy metals from contaminated soils. The consumption of such plants could particularly be hazardous because the accumulated metals in edible plants may end up in human food chain with the attendant adverse effects on human and animal health. A promising cost-effective plant-based technology for the clean-up of heavy metal pollution is phytoremediation. Phytoremediation has attracted attention in recent years because of the low cost of implementation and is particularly attractive in the tropics, where normal climatic conditions favour plant growth and microbial activity. Plants that sprout and grow in metal-laden soils are tolerant to metal pollution in soil and are ‘candidates’ for remediation strategies and management for heavy metals contaminated soils.

1.4 CONTRIBUTIONS OF BIOTECHNOLOGY TO IMPROVE AGRICULTURAL PRODUCTIVITY Biotechnology is regarded as a means to the rapid increase in agricultural production through addressing the production constraints of small-scale or resource-poor farmers who contribute more than 70% of the food produced in developing countries. Biotechnology is applicable to all areas and fields of human endeavours. Agricultural biotechnology has the potential to address some of the problems of developing countries like food insecurity; unfavourable environmental and climatic conditions, etc. and improve agricultural productivity. Agricultural biotechnology has provided animal agriculture with safer, more efficacious vaccines against pseudo rabies, enteric collibacilosis and foot and mouth disease (FMD). Disease detection in crops and animals are more efficiently and rapidly done using DNA probes. Biotechnology acts as a key tool to breakthrough in medical and veterinary research. Crops are now routinely genetically modified for insect and pest resistance, delayed ripening, herbicide tolerance and maximal production under stressed environments. Molecular mapping of crops and farm animals has markedly cut down breeding time and enhanced man’s understanding and manipulation of genes. Nutrition is one of the most serious limitations to livestock production in developing countries. Plants generally contain anti-nutrients acquired from fertilizers, pesticides and several naturally occurring chemicals. Some of these chemicals are known as

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secondary metabolites and they have been shown to be highly biologically active. Examples of these secondary plant metabolites are saponins, tannins, flavonoids, alkaloids, oxalates, phytates, trypsin (protease) inhibitors, cyanogenic glycosides, etc. Some of these chemicals have been shown to be deleterious to health or evidently advantageous to human and animal health if consumed at appropriate amounts. These anti-nutritional factors affect the overall nutritional value of human foods and animal feeds. Some of these plant components have the potential to precipitate adverse effects on the productivity of farm livestock. Conventional plant breeding methods has been used to reduce and in some cases, eliminate such anti-nutritive factors (ANF). An example is the introduction of cultivars of oilseed rape, which are low in or free from erucic acid and glucosinolates. A combination of genetic engineering and conventional plant breeding methods could lead to substantial reduction or removal of the major anti-nutritive factors in plant species of importance in animal feeds. Transgenic rumen microbes could also play a role in the detoxification of plant poisons or inactivation of anti-nutritional factors. Successful introduction of a caprine rumen inoculum obtained in Hawaii into the bovine rumen in Australia detoxified 3 hydroxy 4(IH) pyridine (3,4 DHP), a breakdown product of the non-protein amino acid mimosine found in Leucaena forage. In animal production, biotechnology techniques applied include gene cloning, embryo transfer, artificial insemination, milk modification, etc. In animal health, biotechnology techniques are used for the fast and accurate diagnosis and treatment of diseases. Gene therapy, vaccine production, production of recombinant pharmaceuticals, etc. are examples. Biotechnology can help promote sustainable and safe agriculture and environment, respectively, globally in two ways:

1. By increasing food production within existing land area under plough, making it unnecessary to use marginal land or environmentallysensitive methods and areas. This leads to conservation of bioresources thereby avoiding soil erosion.



2. Using environment-friendly crops such as insect-resistant, herbicide tolerant species, as well as crops that can fix nitrogen leading to purification of the environment.

Consequently, less chemicals like pesticides, herbicides and synthetic nitrogen fertilizers are used. Agricultural biotechnology has long been a source of innovation in the production and processing of agricultural products and has profound impact on the livestock sector. Globally, if hunger and malnutrition is to be reduced drastically, agriculture must be tailored to meet the future demands of increased population. The increase in human population increases the demand for land, space and available resources and primitive agricultural practices cause desertification, environmental pollution and produces resultant effects on climate, ecosystems, biogeochemical cycles and

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human health. Sustainable agricultural practices targeted towards improved agricultural productivity, under clean, safe and environment-friendly conditions must be introduced into the global agricultural system in order to reduce the adverse effects of environmental pollution on human health and the climate (global warming). The new techniques of biotechnology provide innovations that complement the weaknesses of conventional agricultural practices and should be adopted for increased food production. Many scientists would agree that biotechnology is an important contributor to a sustainable agriculture system because it can produce more food with a lesser environmental impact as compared to conventional agriculture.

1.4.1 Biotechnology in reducing the use of chemical pesticides, herbicides and fertilizers A lot of debate is going on the overuse of chemical herbicides, pesticides and fertilizers. They become an environmental hazard because they undergo degradation by microorganisms and ultraviolet light which releases toxic chemicals in the environment. Using biotechnology, bacterial pesticides and viral pesticides are being developed which will help in reducing the use of chemical pesticides. Several companies in USA like Monsanto, Mycogen, Ecogen, Repligen, Zoecon, etc. are actively involved in the development of biological pesticides. The trials are going on to use the genetically engineered live soil bacteria for coating seeds before planting. Another method being tried is to kill the recombinant bacteria and apply them to the leaves of crop plants. Both these approaches protect the toxin from degradation by microbes or ultraviolet rays once applied to the crop plants. The company Ecogen Inc. was involved in developing biological pesticides against the two major crop pests, budworm and ballworm, by transferring a gene from Bacillus thuringiensis (Bt), into either a naturally occurring soil bacterium or in to a strain of Pseudomonas. Bt insecticides are already being marketed for past few years and in future these will be modified using genetic engineering and will be used against a variety of insects. Genetically engineered insect resistant plants have been successfully produced which will further help in reducing the use of insecticides in the future. The experiments are going on to develop environmentally safe herbicides. In order to use these herbicides for crop protection programme, genetically engineered herbicide resistant plants have been produced in a variety of crop plants. This will ensure the use of environmentally safer herbicides.

1.4.2 Biofertilizers Biofertilizers are also being used in place of chemical fertilizers to further reduce the environmental hazards caused due to chemical fertilizers. The term biofertilizers is used to refer to the nutrient inputs of biological origin to

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support plant growth which is generally achieved by the addition of microbial inoculants as a source of biofertlizers. Biofertilizers broadly includes the following categories:

• Symbiotic nitrogen fixers: The diazotrophic microorganisms are the symbiotic nitrogen fixers that serve as biofertilizers e.g. Rhizobium sp., Bradyrhizopium sp.



• Asymbiotic nitrogen fixers: The asymbiotic nitrogen fixing bacteria can directly convert the gaseous nitrogen to nitrogen rich compounds. On the death of these nitrogen fixers, the soil becomes enriched with nitrogenous compounds thereby serving as biofertilizers e.g. Azobacter sp., Azospirillum sp.



• The blue green algae, multiply in the water logging conditions and fixes the nitrogen. They accumulate the biomass which helps in improving the physical properties of the soil. This is useful for reclamation of alkaline soils besides providing partial tolerance to pesticides. The most common blue green algae are Azobacter sp. and Azospirillum sp. Azolla, which is an aquatic fern contains an endophytic cynobacterium Anabaena azollae in the leaf cavities providing symbiotic relationship. Azolla with Anaebaena is useful as biofertilizer.



• Phosphate solubilizing bacteria: Some bacteria like Thiobacillus, Bacillus are capable of converting non-available inorganic phosphorus present in the soil to organic or inorganic form of phosphate. These bacteria can also produce siderophores, which chelates with iron, and makes it unavailable to pathogenic bacteria. Siderophores are iron-binding low molecular weight (400–1,000 Daltons) peptides synthesized by some soil bacteria.



• Organic fertilizers: Certain types of organic wastes are used as fertilizers e.g. animal dung (cow dung, elephant dung, etc.), urine, urban garbage, sewage, crop residues and oil cakes. All these wastes can be converted into organic manures.

Advantages of using biofertilizers

• Biofertilizers improve the tolerance of plants against toxic heavy metals.



• It is possible to reclaim salinity or alkalinity of soil by using biofertilizers.



• Use of biofertilizers helps in controlling environmental pollution.



• Fertility of soil is increased year after year.



• Low cost and easy to produce.



• Biofertilizers increase the physico-chemical properties of the soil, soil texture and water holding capacity.

Some of the limitations encountered while using the biofertilizers are that they alone cannot meet the total needs of the plants for nutrient supply

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and also they do not produce the spectacular results as observed in synthetic fertilizers. It is important to evolve an approach which can maximize the use of biofertilizers and reduce the dependency on the chemical fertilizers in the near future without affecting the crop productivity. This will help us to solve the environment related problems caused due to overuse of chemical fertilizers.

1.5 BIOTECHNOLOGY AND LIVESTOCK PRODUCTION IN THE IMPROVEMENT OF ENVIRONMENTAL CONDITIONS Livestock recycle nutrients on the farm, produce valuable output from land that is not suitable for sustained crop production and provide energy and capital for successful farm operations. Livestock can also help maintain soil fertility in soils lacking adequate organic content or nutrients. Adding animal manure to the soil increases the nutrient retention capacity (or cationexchange capacity), improves the soil’s physical condition by increasing its water-holding capacity and improves soil structure. Animal manure provides favourable conditions for microflora and fauna in soils. Grazing animals improve soil cover by dispersing seeds, controlling shrub growth, breaking up soil crusts and removing biomass that otherwise might be fuel for bush fires. These activities stimulate grass tilling and improve seed germination and thus improve land quality and vegetation growth. Livestock production also enables farmers to allocate plant nutrients across time and space by way of grazing to produce manure, and in the land land that cannot sustain crop production. This makes other land more productive. Grazing livestock can also accelerate transformation of nutrients in crop by-products to fertilizer, thus speeding up the process of land recovery between crops. As disease constraints are also removed, large breeds of livestock can be integrated into crop operations for providing farm power and manure. Biotechnology has enhanced increased animal production through Artificial insemination (AI) and also improved animal health and disease control through the production of DNA recombinant vaccines. Crops improved through biotechnology are producing higher yields worldwide to help feed a hungry and growing world.

1.6

BIOTECHNOLOGY AND THE REMOVAL OF TOXIC CHEMICALS FROM THE ENVIRONMENT

Microorganisms have broadened the environments they live in by evolving enzymes that allow them to metabolize numerous man-made chemicals (that is, xenobiotics). Bioremediation is the use of microorganisms or microbial processes to detoxify and degrade environmental contaminants. Microorganisms have been used for the routine treatment and transformation of waste products for several decades. The fixed-film and activated sludge treatment systems depend on the metabolic activities of microorganisms which degrade the wastes entering the treatment facility. Specialized waste treatment

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plant containing selected and acclimated microbial populations are often used to treat industrial effluents. The innovation in bioremediation has been applied to the remediation of soils, groundwater and similar environmental media. Bioremediation techniques depend on having the right microorganisms in the right place with the right environmental condition for degradation to occur. The right microorganisms are those bacteria and fungi which possess the physiological and metabolic capability to degrade the contaminants. Already, bacteria with natural abilities to digest certain chemicals are being used to clean up industrial sites. By means of genetic engineering, biotechnology has brought about the rapid production of bacteria.

1.7 BIOTECHNOLOGY AND THE REMOVAL OF HEAVY METAL POLLUTION FROM THE ENVIRONMENT Basically, the heavy metals of environmental interest include mercury, vanadium, nickel, cobalt, lead, cadmium, chromium, tin, etc. Some harmful compounds that cause serious environmental pollutions and disaster like Dichlorodiphenyltrichloroethane (DDT) and lead (Pb) could be safely removed by means of genetic engineering of bacteria manufactured for that purpose. The ability of microorganisms to accumulate metals and their potential use in the decontamination of environments impacted by toxic metals has been reported by many scientists. Microorganisms remove toxic metals by various mechanisms such as adsorption to cell surfaces, complexation of exopolysaccharides, intracellular accumulation, biosynthesis of metallothionins and other proteins that trap metals and transform them to volatile compounds. Micrococcus luteus and Azotobacter sp. have been shown to immobilize large quantities of lead from sites containing high concentrations of lead salts, without a detectable effect on viability. Uranium, copper and cobalt could be removed by polyacrylamide immobilized cells of Streptomyces albus. Microbial processes can also mediate the precipitation of metals from aqueous solutions. Certain bacteria extracellular products may interact with free or absorbed metal cations forming insoluble metal precipitates. The major mechanism involved in such precipitation is through the formation of hydrogen sulphide and the immobilization of metal cations as metal sulphides. Certain fungi that produce oxalic acid (oxalates) facilitate the immobilization of metals such as metal oxalate crystals. Microbes can also catalyze a range of metal transformations, which are useful for waste treatment. These transformations include oxidation, reduction and alkylation reactions. Bacteria, fungi, algae or protozoa, in the oxidation reactions, can deposit ferrous and manganese ions. Geobacter metallireducens remove uranium, a radioactive waste, from drainage waters in mining operations and from contaminated ground waters.

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1.8 BIOTECHNOLOGY AND DESULPHURIZATION OF FOSSIL FUELS The removal of inorganic sulphur from coal is mediated by microbial oxidation of sulphur. The direct oxidation of inorganic sulphur by Thiobacillus sp. is a membranebound reaction and requires direct contact of the substrate with the bacterium. As a result of this, the attachment of the culture to coal particle is the absolute requirement. Mixed and pure cultures of a variety of microorganisms (heterotrophic bacteria) can be used to remove organic sulphur from coal and oil. However, sulphur removal has also been reported under anaerobic microbial action.

1.9

BIOTECHNOLOGY AND ECOSYSTEM MODELING

An ecosystem consists of producers, consumers, decomposers and detritivores and their physical environment, all interacting through energy flow and materials recycling. A food web is a network of crossing, interlinked food chains involving primary producers, consumers and decomposers. Disturbances to one part of an ecosystem can have unexpected effects on other, seemingly unrelated parts. Ecosystem modeling is an approach to predict unforeseen effects. By this method, researchers identify crucial bits of information about different ecosystem components. They use computer programs and models to combine the information and then use the resulting data to predict the outcome of the next disturbance. Biotechnology techniques like bioinformatics are useful in ecosystem modeling. Bioinformatics deals in gene database management, gene mapping, coding, sequence alignment, etc.

1.10 MICROBIAL BIOTECHNOLOGY IN THE MONITORING OF ENVIRONMENTAL POLLUTION A number of microbial parameters are used for the detection and monitoring of pollutants, especially in water bodies. Some micro-organisms serve as indicators of organic pollution while others serve as indicators of inorganic pollution. Some of the parameters used for the monitoring of organic pollution are heterotrophic bacteria, total and faecal coliforms and faecal streptococci. Parameters used for monitoring inorganic pollution include nitrifying bacteria, sulphur-oxidizing bacteria, sulphate-reducing bacteria (SRB), iron bacteria, etc. The presence of faecal coliforms in numbers above the World Health Organization (WHO) standard for portable water is indicative of faecal contamination of human origin. Heterotrophic microorganisms are organisms that derive their energy from the oxidation of organic molecules. Their presence in large numbers in aquatic systems is indicative of organic pollution. The presence of biodegradable carbon sources supports the proliferation of heterotrophs in

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aquatic systems. In the case of xenobiotics, the few species that can degrade them may produce by-products during metabolism that may support other microbial species. Thus, a high heterotrophic microbial count is suggestive of high level of organics in aquatic system while low count is suggestive of either a low level of organic pollution or the presence of persistent organic matter within the aquatic system.

1.11 MICROBIAL BIOTECHNOLOGY IN THE BIOASSAY OF ENVIRONMENTAL TOXICITY Toxic industrial wastes are a threat to both the biological waste treatment systems and the environment of their ultimate disposal. As a result, bioassays are very necessary to generate data that could be used for the prediction of environmental effects of waste and regulation of discharges. Although fishes have been the most popular test organisms, standard organisms for aquatic bioassays also include phytoplanktons, zooplanktons, molluscs, insects and crustaceans. The use of microbes (especially bacteria) as bioassay organism is gaining wide acceptance and offers a number of advantages over the standard organisms. Bacteria are easily handled and require relatively small space for culturing and/or testing, compared with other bioassays. Moreover, the short life cycle means fast experimental results, thus enabling the laboratory to process more samples. The simple and rapid bacteria bioassay techniques include Nitrobacter assay, Microtox tests, the Toxi-chromotest and the Ames/Salmonella test. The Nitrobacter bioassay relies on the quantification of Nitrobacter activity determined by measuring the toxicant effect on the rate of nitrite utilization. Photobacterium phosphoreum is the basis of the Microtox assay. Toxichromotest is based on the inhibition of beta galactosidase biosynthesis in E. coli or biosynthesis of enzymes, such as tryptophanse and alpha-glucosidase, under the control of operons (other than the Lac operon) by environmental pollutants.

1.12 BIOTECHNOLOGY AND CONTROL OF OIL SPILLAGE Microorganisms can now be genetically engineered for use in oil recovery, pollution control, mineral leaching and recovery. In the petroleum industry, micro organisms can also be genetically engineered to produce chemicals useful for enhanced oil recovery. Cleaning up oil spills could, in the future, be left to genetically-engineered bacteria. In the mining industries, microorganisms with the property of enhanced leaching ability could be designed. Microorganisms can bind metals to their surfaces and concentrate them internally. As a result of this, genetically improved strains can be used to recover valuable metals or remove polluting metals from dilute solution as in industrial waste. Research is already being carried out to improve the naturally-occurring bacteria that can ‘eat oil’, for use following an oil spill. By

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applying bacteria to oil covered beaches, the complex oil molecules would be broken down into harmless sugars. Many microorganisms can degrade various kinds of environmental pollutants into relatively harmless materials before the death of the microorganisms. This property could also be used in overcoming the environmental hazards of DDT, lead and other environmental pollutants like toxic wastes globally. Strains of bacteria which can degrade fuel hydrocarbons have been designed and the use of genetically engineered micro-organisms to clean up oil spillages or treat sewages has been proposed and is undergoing production/ manufacturing.

1.13 BIOREMEDIATION AND THEIR IMPORTANCE IN ENVIRONMENT PROTECTION Bioremediation is defined as ‘the process of using microorganisms to remove the environmental pollutants where microbes serve as scavengers’. • The removal of organic wastes by microbes leads to environmental cleanup. The other names/terms used for bioremediation are biotreatment, bioreclamation, and biorestoration. • The term “Xenobiotics” (xenos means foreign) refers to the unnatural, foreign and synthetic chemicals, such as pesticides, herbicides, refrigerants, solvents and other organic compounds. • The microbial degradation of xenobiotics also helps in reducing the environmental pollution. Pseudomonas which is a soil microorganism effectively degrades xenobiotics. • Different strains of Pseudomonas that are capable of detoxifying more than 100 organic compounds (e.g. phenols, biphenyls, organophosphates, naphthalene, etc.) have been identified. • Some other microbial strains are also known to have the capacity to degrade xenobiotics such as Mycobacterium, Alcaligenes, Norcardia, etc.

1.13.1 Factors affecting biodegradation The factors that affect the biodegradation are: • the chemical nature of xenobiotics, • the concentration and supply of nutrients, • O2, temperature, pH, redox potential and • the capability of the individual microorganism. The chemical nature of xenobiotics is very important because it was found out that the presence of halogens, e.g. in aromatic compounds, inhibits biodegradation. The water-soluble compounds are more easily degradable whereas the presence of cyclic ring structure and the length chains or branches decrease the efficiency of biodegradation. The aliphatic compounds are more easily degraded than the aromatic ones.

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Bio-stimulation: It is a process by which the microbial activity can be enhanced by increased supply of nutrients or by addition of certain stimulating agents like electron acceptors, surfactants, etc. Bio-augmentation: It is possible to increase biodegradation through manipulation of genes i.e. using genetically engineered microorganisms and by using a range of microorganisms in biodegradation reaction. Depending on the method followed to clean up the environment, bioremediation is carried out in two ways:

1.13.2 In situ bioremediation In situ bioremediation involves a direct approach for the microbial degradation of xenobiotics at the site of pollution which could be soil, water etc. The adequate amount of essential nutrients is supplied at the site which promotes the microbial growth at the site itself. The in situ bioremediation is generally used for clean up of oil spillages, beaches, etc. There are two types of in situ bioremediation 1. Intrinsic bioremediation 2. Engineered in situ bioremediation

1.13.2.1  Intrinsic bioremediation The microorganisms which are used for biodegradation are tested for the natural capability to bring about biodegradation. So, the inherent metabolic ability of the microorganisms to degrade certain pollutants is the intrinsic bioremediation. The ability of surface bacteria to degrade a given mixture of pollutants in ground water is dependent on the type and concentration of compounds, electron acceptor and the duration of bacteria exposed to contamination. Therefore, the ability of indigenous bacteria degrading contaminants can be determined in laboratory by using the techniques of plate count and microcosm studies. The conditions of site that favour intrinsic bioremediation are groundwater flow throughout the year, carbonate minerals to buffer acidity produced during biodegradation, supply of electron acceptors and nutrients for microbial growth, and absence of toxic compounds. 1.13.2.2  Engineered in situ bioremediation When the bioremediation process is engineered to increase the metabolic degradation efficiency (of pollutants), it is called engineered in situ bioremediation. This is done by supplying sufficient amount of nutrients and oxygen supply, adding electron acceptors, and maintaining optimal temperature and pH. This is done to overcome the slow and limited bioremediation capability of microorganisms.  Advantages of in situ bioremediation • The method ensures minimal exposure to public or site personnels. • There is limited or minimal disruption to the site of bioremediation.

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• Due to these factors, it is cost effective.



• The simultaneous treatment of contaminated soil and water is possible.

Disadvantages of in situ bioremediation

• The sites are directly exposed to environmental factors like temperature, oxygen supply, etc.



• The seasonal variation of microbial activity exists.



• Problematic application of treatment additives like nutrients, surfactants, oxygen, etc.



• It is a very tedious and time-consuming process.

1.13.3  Ex-situ bioremediation In this, the waste and the toxic material is collected from the polluted sites and the selected range of microorganisms carry out the bioremediation at designated place. This process is an improved method over the in situ bioremediation method. On the basis of phases of contaminated materials under treatment, ex-situ bioremediation is classified into two : • Solid phase system and • Slurry phase system.

1.13.3.1  Solid phase treatment • This system includes land treatment and soil piles comprising of organic wastes like leaves, animal manures, agricultural wastes, domestic and industrial wastes, sewage sludge, and municipal solid wastes. • The traditional clean-up practice involves the informal processing of the organic materials and production of composts which may be used as soil amendment. • Composting is a self-heating, substrate-dense, managed microbial system which is used to treat large amount of contaminated solid material. • Composting can be done in open system i.e. land treatment and/or in closed treatment system. • The hazardous compounds reported to disappear through composting include aliphatic and aromatic hydrocarbons and certain halogenated compounds. • The possible routes leading to the disappearance of hazardous compounds include volatilization, assimilation, adsorption, polymerization and leaching. 1.13.3.2  Slurry phase treatment • This is a triphasic treatment system involving three major components— water, suspended particulate matter and air.

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• Here, water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved.



• Suspended particulate matter includes a biologically inert substratum consisting of contaminants and biomass attached to soil matrix or free in suspending medium.



• The contaminated solid materials, microorganisms and water formulated into slurry are brought within a bioreactor i.e. fermenter.



• Biologically, there are three types of slurry-phase bioreactors: aerated lagoons, low-shear airlift reactor, and fluidized-bed soil reactor.



• The first two types are in use of full-scale bioremediation, while the third one is in developmental stage.

Advantages of ex-situ bioremediation

• As the time required is short, it is a more efficient process.



• It can be controlled in a much better way.



• The process can be improved by enrichment with desired and more efficient microorganisms.

Disadvantages of ex-situ bioremediation

• The sites of pollution remain highly disturbed.



• Once the process is complete, the degraded waste disposal becomes a major problem.



• It is a costly process.

Several types of reactions occur during the bioremediation/microbial degradation

(a) Aerobic bioremediation- When the biodegradation requires oxygen (O2) for the oxidation of organic compounds, it is called aerobic bioremediation. Enzymes like monooxygenases and dioxygenases are involved and act on aliphatic and aromatic compounds.

(b) Anaerobic bioremediation: This does not require oxygen. The degradation process is slow but more cost-effective since continuous supply of oxygen is not required.

(c) Sequential bioremediation: Some of the xenobiotic degradations require both aerobic as well as anaerobic processes which very effectively reduce the toxicity e.g. tetrachloromethane and tetrachloroethane undergo sequential degradation.

1.13.4 Use of genetic engineering and genetic manipulations for more efficient bioremediation In recent years, efforts have been made to create genetically engineered microorganisms (GEMs) to enhance bioremediation. This is done to overcome

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some of the limitations and problems in bioremediation. These problems are:

• Sometimes the growth of microorganisms gets inhibited or reduced by the xenobiotics.



• No single naturally occurring microorganism has the capability of degrading all the xenobiotics present in the environmental pollution.



• The microbial degradation is a very slow process.



• Sometimes, certain xenobiotics get adsorbed on to the particulate matter of soil and thus become unavailable for microbial degradation.

As the majority of genes responsible for the synthesis of enzymes with biodegradation capability are located on the plasmids, the genetic manipulations of plasmids can lead to the creation of new strains of bacteria with different degradative pathways. In 1970s, Chakrabarty and his team of coworkers reported the development of a new strain of bacterium Pseudomonas by manipulations of plasmid transfer which they named as “superbug”. This superbug had the capability of degrading a number of hydrocarbons of petroleum simultaneously such as camphor, octane, xylene, naphthalene, etc. In 1980, United States granted the patent to this superbug making it the first genetically engineered microorganism to be patented. In certain cases, the process of plasmid transfer was used. E.g., the bacterium containing CAM (camphor degrading) plasmid was conjugated with another bacterium with OCT (octane degrading) plasmid. Due to noncompatibility, these plasmids cannot co-exist in the same bacterium. However, due to the presence of homologous regions of DNA, recombination occurs between these two plasmids which results in a single CAM-OCT plasmid giving the bacterium the capacity to degrade both camphor as well as octane.

1.13.5 Biotechnology to reduce atmospheric Carbon dioxide (CO2)

Carbon dioxide is the gas that is the main cause of greenhouse effect and rise in the atmospheric temperature. During the past 100-150 years, the level of CO2 has increased about 25% with an increase in the atmospheric temperature by about 0.5% which is a clear indication that CO2 is closely linked with global warming. There is a steady increase in the CO2 content due to continuous addition of CO2 from various sources particularly from industrial processes. It is very clear that the reduction in atmospheric CO2 concentration assumes significance. Biotechnological methods have been used to reduce the atmospheric CO2 content at two levels:

(a) Photosynthesis: Plants utilize CO2 during photosynthesis which reduces the CO2 content in the atmosphere. The equation for photosynthesis is: 6CO2 + 6H2O → C6H12O6 + 6O2

The fast growing plants utilize the CO2 more efficiently for photosynthesis. The techniques of micro-propagation and synthetic

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seeds should be used to increase the propagation of such fast growing plants. Further, the CO2 utilization can be increased by enhancing the rate of photosynthesis. The enzyme ribulose biphosphate carboxylase (RUBP-case) is closely linked with CO2 fixation. The attempts are being made to genetically manipulate this enzyme so that the photosynthetic efficiency is increased. Some microalgae like Chlorella pyrenodiosa, Spirulina maxima are known to be more efficient than higher plants in utilizing atmospheric CO2 for photosynthesis and generate more O2 than the amount of CO2 consumed. The growing of these microalgae near the industries and power plants (where the CO2 emission into atmosphere is very high) will help in the reduction of polluting effects of CO2. Using genetic engineering, attempts are being made to develop new strains of these microalgae that can tolerate high concentrations of CO2. A limited success has already been reported in the mutants of Anacystis nidulans and Oocystis sp.

(b) Biological Calcification: Certain deep-sea organisms like corals, green and red algae store CO2 through a process of biological calcification. As the CaCO3 gets precipitated, more and more atmospheric CO2 can be utilized for its formation. The process of calcification is as follows:

H2O + CO2 → H2CO3

H 2CO3 + Ca2+ → CaCO3 + CO2 + H2O

1.13.6 Treatment of sewage using microorganisms The sewage is defined as the wastewater resulting from the various human activities, agriculture, and industries and mainly contains organic and inorganic compounds, toxic substances, heavy metals, and pathogenic organisms. The sewage is treated to get rid of these undesirable substances by subjecting the organic matter to biodegradation by microorganisms. The biodegradation involves the degradation of organic matter to smaller molecules (CO2, NH3, PO4, etc.) and requires constant supply of oxygen. The process of supplying oxygen is expensive, tedious, and requires a lot of expertise and manpower. These problems are overcome by growing microalgae in the ponds and tanks where sewage treatment is carried out. The algae release the O2 while carrying out photosynthesis which ensures a continuous supply of oxygen for biodegradation. The algae are also capable of adsorbing certain heavy toxic metals due to the negative charges on the algal cell surface which can take up the positively charged metals. The algal treatment of sewage also supports fish growth as algae is a good source of food for fishes. The algae used for sewage treatment are Chlorella, Euglene, Chlamydomnas, Scenedesmus, Ulothrix, Thribonima, etc.

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1.13.7 Treatment of industrial effluents using biotechnology

• The industrial effluents should be properly treated in order to control the environmental pollution. • The industrial effluent often contains toxic materials like suspended solids and soluble organic compounds, heavy metals, cyanides, nonbiodegradable chemical and volatile materials like H2 S and SO2, etc. • The soluble organic compounds undergo slow decomposition resulting in oxygen depletion and production of noxious gases. The heavy metals and other toxic organic materials such as chlorinated compounds in effluents from paper industry have adverse effects on the aquatic flora and fauna. • The high levels of nitrogen and phosphorus causes eutrophication which leads to undesirable algal growth and death of animals due to lack of oxygen. Biotechnological techniques are being used to address some of these problems. The biological treatment of effluents has been in use for quite some time in many countries around the world. However, some specific problems which are related to conventional treatment are being solved using biotechnology. Among the substances released in effluents are calcitrants which cannot be degraded using conventional treatment methods. Biotechnology helps in overcoming this problem. • In USA, the company BioTechnica is using lignin degradation for the treatment of substances like polychlorinated biphenyls (PCBs) and dioxin. • In Europe, ICI and Ciba-Geigy are working on enzymatic detoxification (breaking down) of substances such as cyanides and also the byproducts from the synthesis of S-triazine herbicides. Microbial transformation of certain substances such as dibenzofurans, biarylketones halogenated bibenzodioxins, etc. are used to minimize the problem of pollution due to toxic effluents. • E.g., certain strains of Pseudomonas have been isolated which selectively deoxygenate the 1,2 positions of substituted biarylethers and biarylketones. — Microbial degradation of chloro-, dichloro-, and carbontetrachloride, etc. is being also tried to deal with the problem. — The paper and pulp industries release effluents which contain chromophoric compounds and chlorogenated organic materials like chlorolignins, chlorosyringols, chloroaliphatics, catechols etc. which can affect the aquatic system by their inhibitory and mutagenic activities.

— Certain soil-inhabiting fungi, streptomycetes, bacteria and white rot fungi (Ganoderma lacidum, Coriolus (Trametes) versicolor, P.

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chrysosporium, Coprinus macrorhizus, Hericiumerinaceus, etc. have been studied for their ability to decolourise chromophoric substrates. White rot fungi produces a variety of lignin-degrading enzymes (e.g. peroxidases and lacasses) that degrade phenolic substances.

1.13.8 Use of biotechnology for toxic site reclamation Generally, incineration (drying and then burning to ashes in furnace) or chemical treatment are being used to get rid of toxins and waste from the waste disposal sites. Of late, biotechnological techniques involving biodegradation as an alternative approach is being used. Companies like BioTechnica are working on treating polluted site in situ. However, there is a lot of debate over the issue regarding the release of genetically engineered microbes for treatment of toxic sites and the risk involved in the whole procedure. As we know that, the released engineered organisms that have capacity to reproduce, spread to sites other than the initial release sites and may undergo mutations. All this can lead to the risk of developing what are described as “super bugs”. Some of the companies in US are experimenting and conducting their work in the closed reactors in order to further evaluate the risk assessment and cost effectiveness of this approach. In order to solve the problem of soil pollution caused due to extensive use of herbicides, pesticides and insecticides, bioremediation of soil using microorganisms is being carried out. The most common pollutants are: hydrocarbons, chlorinated solvents, polychlorobiphenyls and metals. The bioremediation of soil involves:

• Biostimulation: Biostimulation involves stimulation of microorganisms already present in the soil. This can be done by adding nutrients e.g. nitrogen, phosphorus etc., by supplying co-substrates e.g. methane which can degrade trichloroethylene, or by adding surfactants to disperse the hydrophobic compounds in water.



• Bioaugmentation: Addition of specific microorganisms to the polluted soil constitutes bioaugmentation. Some of the pollutants like polychlorobiphenyls (PCBs), trinitrotoluene (TNT), polyaromatic hydrocarbons (PAHs), etc. are not degraded by only native soil microorganisms so a combination of microorganisms; referred to as “consortium” or “cocktail” of microorganisms; is added to achieve bioaugmentation.



• Bioventing: Bioventing involves aerobic biodegradation of pollutants by circulating air through sub-surfaces of soil and is one of the very cost-effective and efficient technique used for the bioremediation of petroleum-contaminated soils. It is very effectively used for degradation of soluble paraffins and polyaromatic hydrocarbons. • Phytoremediation: Bioremediation by using plants is called phytoremediation. Certain plant species which have the capability to



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stimulate biodegradation of pollutants (especially near the soil adjacent to roots- rhizosphere) are cultivated near the sites of polluted soil. This is a cheap and environmentally friendly process but takes a long time to finish the clean-up process. • Land farming: Land farming is a technique for the bioremediation of hydrocarbon contaminated soils. In this, the soil is excavated, mixed with microorganisms and nutrients and spread out on a liner just below the polluted soil. • Use of slurry-phase bioreactors: In this process, the excavated polluted soil is subjected to bioremediation under optimal controlled conditions in specifically designed bioreactors. Engineered bacteria used for the degradation of xenobiotics and toxic wastes Bacterium

Substrate that can be degraded

Pseudomonas capacia

2,4,5-trichloro-phenoxyacetic acid

P. putida and other spp. (also E. coli)

2,2,5-dichloropropionate; mono and dichloroaromatics

Alcaligenes sp.

Dichlorophenoxyacetic acid, mixed chlorophenols; 1,4-dichlorobenzene

Acinetobacter sp.

4-chlorobenzene

Pseudomonas capacia

2,4,5-trichoro-phenoxyacetic acid

1.13.9 Use of biotechnology in the removal of oil and grease deposits





• The oil spills from oil tankers on land surface as well as in seas and oceans are a major environmental hazard. This not only kills the aquatic flora and fauna by destroying the habitat but also creates health problems for the local inhabitants. • Traditionally, chemical dispersants are being used as remediation efforts. However, these chemical dispersants are also toxic in nature and they persist in the environment for a long time. • The present techniques of washing the oil off the gravel and cleaning the area of oil spills, is very expensive and time-consuming. • In order to overcome some of these problems, the oleophilic fertilizers are being developed which allow rapid growth and multiplication of microbes which further leads to the increase in the biodegradation process for removal of oil. • In recent years, using genetic engineering, oil utilizing microorganisms have been produced which can grow rapidly on oil. • The genetically engineered microbes for cleaning oil spills are mixed with straw. At the site of oil spill, the straw mixed with microbes are scattered over the oil spilled area.

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• The straw soaks the oily water and the microbes break the oil into nontoxic and non-polluting materials thereby cleaning up the site.



• Some of the oil-utilizing microbes can also produce surface-active compounds that can emulsify oil in water and thereby removing the oil.



• A strain of Pseudomonas aeruginosa produces a glycolipid emulsifier that reduces the surface tension of oil-water interface, which helps in the removal of oil from water. This microbial emulsifier is nontoxic and biodegradable and has shown promising results in the laboratory experiments.



• Some of the microorganisms which are capable of degrading petroleum include Pseudomonas, various Corynebacteria, Mycobacteria and some yeasts. The two methods for bioremediation of oil spills are:



(a) using a consortium of bacteria, and



(b) using genetically engineered bacteria/microbial strains.

Both bacterial and fungal cultures from the petroleum sludge have been isolated. The fungal culture could degrade 0.4% sludge in 3 weeks. Degradation of petroleum sludge occurred within two weeks when the bacterial culture (Bacillus circulans CI) was used. A significant degradation of petroleum sludge was observed in 10 days when the fungus + B. circulans and a prepared surfactant were exogenuously added to petroleum sludge.

1.13.10  Tannery effluents and their treatment The environment, we know, is under increasing pressure from solid and liquid effluents from the leather industry, an offshoot of the natural resources (animals). They are categorized as:

• Solids: Suspended solids; Settleable solids; Gross solids.



• Sulphides (S2–): They result from the use of sodium sulphide and sodium hydrosulphide and the breakdown of hair and in the unhairing process. In the alkaline condition, they remain largely in solution and when the pH drops below 9.5, hydrogen sulphide, the obnoxious gas evolves.



• Neutral salts: Sulphates (SO24–) and Chlorides (Cl–).



• Oils and grease.



• Chromium compounds: Chrome (3+) and Chrome (6+).



• Other metals: Al, Zr, etc.



• Solvents.

The tannery effluents damage the normal functioning of a river, destroy its ecosystem and are the main causes of imminent systems collapse of the

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Ganges. They are responsible for polluting the aquifers to such a significant extent that it is becoming a serious environmental hazard (water becomes non-potable). Pollution remediation of the tannery effluents is very complex. A multi-prong treatment is thus called for a combination of nanotechnology and microbial technology with prior processing by cycloning, flotation, microflotation or electroflotation. The suspended solids can be separated by an initial filtration process followed by flotation, microflotation or electroflotation or a combination depending on their surface properties and their electrostatic behaviour whereas the semi-colloidal solids, i.e. the protein residues can be separated by cycloning. During cycloning, the protein material will be separated out at the outlet (at the top of the cyclone). Injection of black iron (Fe0) nanoparticles (ZVI) or Ni/Fe bimetallic nanites15, produced in the laboratory, into the contaminant in an effluent treatment basin (where oxygen is available) with non-porous boundary zones will immobilize the metals, particularly the hazardous chromium, and detoxify and dechlorinate the other pollutants available. Biodegradation by microorganisms (discussed earlier under biosorption) will follow the nanoremediation process if it still contains partial degradation products that are considered hazardous. The flow of the nanotreated material to a second basin is, therefore, imperative. When the engineering design is carried out, the material after treatment can be flowed out into the open system. Microorganisms like Bacillus, Escherichia, Enterobacter, Micrococcus, Arthrobacter, Pseudomonas, etc. and fungi like Neurospora can be utilized for biodegradation of the metals available in these effluents, after they have been exposed to ZVI remediation technology. The heap soil washing technology using leaching microbes is receiving much attention these days for the remediation of large volumes of heavy metals and radioactive elements in contaminated soil. From another perspective, such bioremediation is gaining recognition as a metallurgical process for recovery of metals.

1.13.11  Bioremediation of Radioactive Contaminants Among the most toxic chemicals of global concern are the radionuclides found in contaminated waste streams and groundwaters. For example, former nuclearweapons facilities are severely contaminated with plutonium, uranium, and neptunium because of past practices that released mixtures of radionuclides and other organic and inorganic wastes into adjacent soil and groundwater. The greatest potential for clean-up of such sites is in situ bioremediation, which exploits the reactions of microorganisms to directly or indirectly alter the chemical form of the radionuclides, rendering them immobile and less toxic. The microorganisms interact directly with the radionuclides by catalyzing chemical redox transformations. The microorganisms act indirectly by producing acids, bases, and complexing ligands that react with the radionuclides.

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Radionuclide bioremediation is a complicated situation, since the fate of radionuclide depends on so many different reactions, which proceed through multiple steps and at vastly different rates. Such a complex scenario can be understood and controlled only by using mathematical modeling. Such a unique biogeochemical mathematical model, CCBATCH, was developed by Dr. Rittmann and colleagues to connect all the different types of reactions that control the fate of radionuclides and a large range of metals. Current research is focused on applying CCBATCH towards bioremediation of plutonium, one of the most toxic materials. To create a clean environment for all the people around the world, it is necessary to mitigate waste problems left behind as legacies of dangerous past activities, such as making nuclear weapons. When understood well, such as with CCBATCH, in situ bioremediation has great promise to deal with one of the most difficult challenges — groundwater and soil contamination by radionuclides, such as plutonium.

1.14 USE OF BIOSENSORS TO DETECT ENVIRONMENTAL POLLUTANTS Biosensors are biophysical devices which can detect the presence of specific substances e.g. sugars, proteins, hormones, pollutants and a variety of toxins in the environment. They are also capable of measuring the quantities of these specific substances in the environment. Technically, a “Biosensor” is defined as “an analytical device containing an immobilized biological material (which could be an enzyme, or antibody, or nucleic acid, or hormone, or an organelle/whole cell), which can specifically interact with an analyte and produce physical, chemical or electrical signals that can be measured”. An analyte is the compound (e.g. glucose, urea, drug, pesticide) whose concentration has to be measured. Biosensors basically involve the quantitative analysis of various substances by converting their biological actions into measurable signals. Generally the performance of the biosensors is mostly dependent on the specificity and sensitivity of the biological reaction, besides the stability of the enzyme. A biosensor or an enzyme or an antibody is associated with microchip devices which is used for quantitative estimation of the substance. A biosensor equipment has the following components

• biological component—enzyme, cell, etc.



• a physical component—a device for measuring the quantity of this product, thus indirectly giving an estimate of the substrate e.g. transducer, amplifier etc.

The biosensors are being used in the area of medicine, industry, etc. however, their use in environmental monitoring is of great benefit. Special kits

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have been designed to identify the specific pollutants in the environment. E.g., special cost-effective enzymatic tests are available which can detect pesticide contamination in water.

1.14.1 Principle of a biosensor The biological material in use (e.g. an enzyme) is immobilized by conventional methods like physical or membrane entrapment, non-covalent or covalent binding. A contact is made between the immobilized biological material and the transducer. The analyte binds to the biological material to form a bound analyte which in turn produces the electronic response that can be measured. Sometimes, the analyte is converted to a product which could be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer then converts the product linked changes into electrical signals which can be amplified and measured. A good example of a biosensor in frequent use is the glucose oxidase enzyme. The enzyme is immobilized on an electrode surface which acts as an electrocatalyst for oxidation of glucose. The biosensor gives reproducible electrical signal for glucose concentrations as low as 0.15 mM. Another area where biosensors are being used is “Biomonitoring” or “biological monitoring”. Biomonitoring is defined as the measurement and assessment of work place agents or their metabolites either in tissues, secreta, excreta, or any combination of these systems in occupationally exposed human subjects. The “Biological effect monitoring” refers to the biological effects of these toxic agents in the workers exposed to these agents. A continuous evaluation of biological monitoring methods is done in order to assess the risk effectiveness of these tests against the various kinds of exposures to toxins. The use of genetic engineering to create organisms specifically designed for bio remediation also has great potential. The bacterium Deinococcus radiodurans, which is the most radioresistant organism known, has been modified to consume and digest toluene and ionic mercury from highly radioactive nuclear waste. Some of the important biosensors used in environmental pollution monitoring are: (a) Gas biosensors: In order to detect gases such as sulphur dioxide (SO2), methane, carbon dioxide, etc., microbial biosensors have been developed. Thiobacillus-based biosensors can detect the pollutant SO2, whereas methane (CH4) can be detected by immobilized Methalomonas. A particular strain of Pseudomonas is used to monitor carbon dioxide levels. (b) Immunoassay biosensors: Immunoelectrodes as biosensors are used to detect low concentrations of pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines, malathion and carbamates, by using immunoassay methods.

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(c) BOD biosensor: Biological oxygen demand (BOD) is widely used as a test to detect the levels of organic pollution. This requires five days of incubation but a BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes only 15 minutes to detect organic pollution. (d) Miscellaneous biosensors: A graphite electrode with Cynobacterium and Synechococcus has been developed to measure the degree of electron transport inhibition during photosynthesis due to certain pollutants e.g. herbicides. To detect phenol, phenol oxidase enzyme obtained from potatoes and mushrooms is used as a biosensor. Biosensors for the detection of polychlorinated biphenyls (PCBs) and chlorinated hydrocarbons and certain other organic compounds have also been developed. Biosensors employing acetylcholine esterase which can be obtained from bovine RBC can be used for the detection of organophosphorus compounds in water.

1.14.2 Use of selected and engineered microbes for removal and recovery of strategic and precious metals from contaminated degraded lands The domestic and industrial effluents often contain harmful heavy metals. These heavy metals cause soil contamination when these effluents are used for irrigation purposes. The biotechnological methods and procedures are being developed to prevent the contamination by these heavy metals and also restore the contaminated soils. This involves the selective use of engineered microbes. Plasmids have been constructed which can enhance the recovery of gold from arsenopyrite ores, by Thiobacillus ferroxidans. Ganoderma lucidum which is a wood rotting macrofungus, is a highly potential biosorbent material for heavy metals and thus can be used to control contamination by heavy metals. The metal pollution occurs through several processes. As the living organisms including man are constantly exposed to metals, they accumulate by a process referred to as ‘bioaccumulation.’ The continuous exposure and accumulation of a given metal leads to increase in it’s concentration which is referred to as ‘biomagnification’. Biomagnification occurs through food chain and the human gets the maximum impact due to it’s being on top of the food chain. The ‘biomethylation’ is carried out by microorganisms in the soil and water and involves the process of transfer of methyl groups from organic compounds to metals. Some phytoplanktons (plants that float freely on water surface) and some benthics (plants attached to some substratum at the bottom of aquatic bodies) microorganisms can take up the metals from the wastewater ponds. These natural bioscavengers not only control the water pollution by absorbing metals but also contributes in the recovery of industrially important metals from the effluents. The microorganisms like algae can absorb metals form the fresh water e.g. Chlorella vulgaris takes up copper, mercury, uranium. Certain fungal

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species like Rhizopus, Aspergillus, Pencillium, Neurospora are good absorbers of heavy metals like lead, mercury, etc. Some of the bacterial species are capable of accumulating metals on cell walls such as E. coli can take up mercury while Bacillus circulans can accumulate copper. The mechanism of metal scavenging by these microorganisms is very complex and involves multiple steps. Some of the microorganisms bioaccumulate these metals on their cell walls whereas some others have the capacity to transport these metals to intracellular and intercellular free space and cellular organelles. In certain cases, some of the metals occur as immobilized metal containing crystals e.g heavy metal complexes of calcium oxalate crystals. Some of the fungal and algal species synthesize metal binding proteins or peptides. ‘Phytochelatin’ is an ubiquitious metal chelating protein present in all plants and acts as a common buffering molecule for the homeostasis of metals. It is rich in cysteine and can form salt metal complexes through sulfhydral (SH) groups. Due to this property, phytochelatin can be used as a biomarker for metal pollution detection. The mechanisms involved in the removal of metals by microorganisms are: adsorption, complexation, precipitation and volatilization.

• The process of adsorption involves the binding of metal ions to the negatively charged cell surfaces of microorganisms.



• The process of complexation leads to production of organic acids e.g. citric acid, oxalic acid, gluconic acid, lactic acid, malic acid, etc. which chelate the metal ions.



• In precipitation, the metals are precipitated as hydroxides or sulfates by some bacteria such as which produce ammonia, organic bases or H2S. e.g. Desulfovibrio and Desulfotomaculum transform SO4 to H2S which promotes extracellular precipitation of insoluble metal sulfides. Klebsiella aerogenes detoxifies cadmium sulphate which precipitates on cell surface.



• Volatilization involves bacteria that cause methylation of Hg2+ and convert it to dimethyl mercury which is a volatile compound.

Whole cell of Bacillus subtilis have been shown to reduce gold from Au3+ to Au° through extracellular enzymatic biotransformation. Under anoxic environment, sulphate-reducing bacteria (Desulfovibrio) oxidize organic matter using sulphate as an electron acceptor. In yeast, Saccharomyces cerevisiae, removal of metals is done by their precipitation as sulphides e.g. Cu2+ is precipitated as CuS.

1.15 BIOTECHNOLOGY AND THE SAVING OF RESOURCES AND ENERGY Breeding of insect and pest–resistant crop strains help promote a safe environment, save money and conserve resources. Industrial processes are

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very complex and chemists make use of inorganic catalysts which speed up the rate of reaction when making new chemicals. These catalysts often need high temperatures, and acid or alkaline conditions, in order to work efficiently. In future, genetically engineered organisms may be able to work effectively at lower temperatures, and require less extreme conditions. This will save money and resources, and will also produce fewer hazardous byproducts. For example, in paper-making, the wood pulp has to be treated with chemicals which break up the fibres and remove the lignin (the substance that makes up the wood). The pulp is bleached so that the finished paper is white. This process produces a large volume of chemical waste that has to be treated before it is ready for disposal. Enzymes have been discovered in fungi which may be suitable for use as biological alternatives to some of these chemicals. In the near future, using the modern plant breeding techniques, it may be possible to breed trees which have less lignin, and so require fewer chemicals and less energy to produce the pulp. Plastic is made from oil and its manufacture uses a lot of energy and produces a lot of polluting byproducts. There is now hope that some forms of plastic will be made by living organisms. One biodegradable plastic, called Biopol (trade name) is made by bacteria. One way to make larger quantities of this plastic at lower cost might be to insert the gene into potatoes. This would save energy, and reduce both cost and pollution. As the supply of fossil fuels (oil, gas and coal) dwindles as a result of the global financial crisis, genetically engineered organisms may be manufactured to produce far more materials like plastics at less energy, reduced cost and minimal environmental pollution.

1.16 FEARS AND CONCERNS ABOUT BIOTECHNOLOGY APPROACH IN ACHIEVING A SAFE ENVIRONMENT AND AGRICULTURE There are some fears and concerns about biotechnology and safe environment and agriculture. For instance, plant breeding, an agricultural biotechnology approach has some concerns. Genetically engineered organisms are living things and so are much less predictable than artificial materials and chemicals. They can reproduce, move and even mutate. Developments in genetic engineering take place in carefully controlled laboratory conditions. However, once a new or modified organism has been developed, it is likely to be grown outside and once released into the environment, it cannot be recalled. The organism could change or interbreed with others, creating new species. Geneticists should therefore be cautious and assess the possible risks involved so that genetically engineered plants cause no more harm than the chemicals they are replacing. It has been found that genetic alteration of plants to resist viruses can stimulate the virus to mutate into a more virulent form, one that might even attack other plant species. If the genes for insect- and weed killer resistance, introduced into crop plants, find their way into weeds, this could

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result in development of super-weeds, which would be impossible to kill using traditional weed killers. Some scientists think that genetically engineered plants and animals will threaten the survival of other species and reduce diversity (the number of different plant and animal species). The genetically engineered plants, which may be more resistant to disease and pests, when grown around the world, may cross to the wild species. The wild population would be contaminated which could affect local habitats and the species that grow there. For example, oilseed, rape, cross-breeds easily with wild relatives. This may mean that genetically engineered oilseed rape would breed with related plants, and the new gene for resistance to weed killer could spread into the wild population. Some scientists predict that, within just one year, a large percentage of weeds growing near the crop would have acquired this gene. There is no way of stopping genetically engineered crops from breeding with wild plants.

1.17 FEARS AND CONCERNS ABOUT ENVIRONMENTAL IMPACT OF BIOREMEDIATION Bioremediation is the use of microorganisms or microbial processes to detoxify and degrade environmental contaminants. Microorganisms have been used for the routine treatment and transformation of waste products for several decades. Although bioremediation represents a promising and largely untapped environmental biotechnology, it has some disadvantages. Additives added to enhance the functioning of one particular group of micro-organisms during in situ bioremediation, may be disruptive to other organisms inhabiting that same environment. Also, stimulated microbial population or geneticallymodified micro-organisms introduced into the environment after a certain point of time may become difficult to remove. Bioremediation is generally very costly and labour-intensive, and can take several months for the remediation to reach acceptable levels.

1.18 ROLE OF BIOTECHNOLOGY IN RESTORATION OF DEGRADED LANDS The urbanization and increased human activity has led to degradation of habitats. The restoration of the degraded lands can be carried out by using biotechnology which involves the manipulations of biological systems. This restoration could be carried out by the following biotechnological methods:

1.18.1 Use of micropropagation and Mycorrhiza for reforestation One of the approaches to tackle this problem is to develop strong and superior species which have the capability to grow well on degraded lands. This can be done by using mass multiplication which involves starting aseptic culture, multiplication of shoot using shoot apical meristems or buds, rooting of in vitro

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formed shoots, transfer, acclimatization and adaptation of micro propagated plantlets in the field. Using this methodology, an estimated 500 million plants of diverse nature have been produced. Mycorrhizae, which are symbiotic non-pathogenic associations between plant roots and fungi, improve the seedling survival and growth by enhancing uptake of nutrients and water. They also lengthen the root life and provide protection against the pathogens. A list of fungi which can efficiently form mycorrhizae has been prepared. These fungi can be used as inocula which are applied to roots of seedlings, to allow formation of mycorrhizae. The experimental infection of micro propagated plants during rooting increases their survival chances in the field, which is very important in case of plantations on degraded lands.

1.18.2 Improvement of soil infertility through the use of nitrogen fixing bacteria, Rhizobium in association with leguminous trees and Frankia in association with non-leguminous species Biotechnological methods are being developed to help the non-leguminous plants to survive under adverse conditions such as low nutrient supply. There are about 160 species of angiosperms, which are known to form nitrogen fixing root nodules with the Actinomycetes bacteria belonging to the genus Frankia which is being used for this purpose. Frankia helps in nitrogen fixation in non-leguminous plant species; therefore, it can be used for land reclamation through reforestation due to high biomass production without the need of expensive nitrogen fertilizers.

1.18.3 Development of plants tolerant to abiotic stress which can be grown on degraded lands The techniques like tissue culture and genetic engineering are being used to develop plants resistant to abiotic stresses e.g. salinity, acidity, aluminium toxicity, etc. The cell lines which exhibit resistance to salt stress are selected and then used for plantation on degraded lands. E.g., Brassica spp., Citrus aurantium, Nicotiana tabacum etc. Research is going on to understand the molecular basis of salt tolerance and to isolate genes responsible for this attribute so that salt tolerant plants can be developed using genetic engineering. In vitro selection for tolerance to abiotic stress like aluminium toxicity has been successful in certain plant species e.g. tomato, rice, barley, rice and wheat. “Triticale”, which is a man made synthetic crop, has been found suitable for growing on acid soils, dry and sandy soils, on alkaline and calcareous soils and on mineral deficient and high-boron soils especially in countries like Kenya, Ethiopia, Ecuador, Mexico, Brazil, etc. In China, a number of new stress resistant varieties of rice, wheat and tobacco have been developed using anther culture.

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1.19 BIOMIMICRY AND BIOTECHNOLOGY It is difficult to achieve a four-fold improvement in environmental performance through incremental improvements in conventional production technologies. Improvements of this magnitude usually call for a paradigm shift. For a growing number of companies, the inspiration for such a paradigm shift is coming from the products and processes found in natural ecosystems and the organisms that live in them. Biomimicry is the name coined for this approach in which industrial production systems imitate nature. Industrial biotechnology is that set of technologies that come from adapting and modifying the biological organisms, processes, products, and systems found in nature for the purpose of producing goods and services. The organisms, processes, products and systems found in natural ecosystems have evolved over millions of years to become highly efficient. For example, all energy in natural ecosystems is renewable and is initially captured from sunlight through photosynthesis. Also, all bio-organic chemicals and materials are renewable, biodegradable and recycled. There is no such thing as “waste”—the by-products of one organism are the nutrients for another. Most, if not all, metabolic processes are catalyzed by enzymes and are highly specific and efficient. Biotechnology has evolved over the last 25-30 years into a set of powerful tools for developing and optimizing the efficiency of bioprocesses and the specific characteristics of bio products. This increase in efficiency and specificity has great potential for moving industry along the path to sustainability. Increased efficiency allows for greater use of renewable resources without leading to their depletion, degradation of the environment and a negative impact on quality of life. Biotechnology can become an important tool for decoupling economic growth from degradation of the environment and the quality of life. Biotechnology can also enable the design of processes and products whose performance cannot be achieved using conventional chemistry or petroleum as feedstock. Here are some examples of some of the industrial efficiency tools now coming from the application of biotechnology:

• Enzymes extracted from naturally occurring micro-organisms, plants and animals can be used biologically to catalyze chemical reactions with high efficiency and specificity. Compared to conventional chemical processes, bio-catalytic processes usually consume less energy, produce less waste and use less organic solvents (that then require treatment and disposal).



• By imitating natural selection and evolution, the performance of naturally occurring enzymes can be improved. Enzymes can rapidly be ‘evolved’ (this technique is called “molecular evolution”) through mutation or genetic engineering and selected using high-throughput

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screening to catalyze specific chemical reactions and to optimize their performance under certain conditions such as elevated temperature.

• The metabolic pathways of micro-organisms can also be modified by genetic engineering. The aim is to turn each cell into a highly efficient “mini reactor” that produces in one step and at high yield what would take an organic chemist a number of steps with much lower yield (this technique is called “metabolic engineering”).



• A further improvement on metabolic engineering involves engineering the enzymes in the optimal configuration onto the cell membrane, and when the cell is ruptured, the cell membrane becomes a bio-catalytic surface that provides the high efficiency of metabolic engineering without the energy penalty of keeping the organism alive.



• Plant biomass can be processed and converted by fermentation and other processes into chemicals, fuels and materials that are renewable, and result in no net emissions of greenhouse gases. Also, these biologically derived products (“bio products”) are generally less toxic and less persistent than their petrochemical counterparts.



• Groups of companies can mimic the co-operative action of organisms in natural ecosystems by clustering around the processing of a feedstock such as biomass, so the by-product of one is the starting material for another. Also, energy, such as waste heat, can be used efficiently. This approach is called “industrial ecology”.



• The ability to “evolve” bioprocesses and bio production systems allows for major improvements in both economic and environmental performance. This permits a manufacturing facility to increase its profitability and capacity while maintaining or even reducing its environmental footprint.

1.20 BIOTECHNOLOGY IN THE CONSERVATION OF BIODIVERSITY The extinction of wild species due to the destruction of habitats and ecosystems has raised serious concerns about the biodiversity in general. Biodiversity provides genes from wild species for biotechnology exercises and experiments hence, biotechnology and biodiversity are interrelated. Besides taking steps to minimize and regulate the factors responsible for causing loss of biodiversity, efforts are on to develop the techniques of conservation of biodiversity. One of the methods involves the establishment of “gene banks” leading to “in situ conservation” and “ex situ conservation”. The in situ conservation involves the conservation of plants and animals in their natural habitat and ecosystems. The ex situ conservation includes conservation of species away from their habitats. The ex situ conservation uses sample populations and establishes the “gene banks” which include resource centers, zoos, botanical gardens, national parks, culture collection centers, etc.

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Biotechnology offers special methods to conserve both animal and plant genetic resources especially in the conservation of endangered plant species. The tissue culture method is being used to multiply an endangered plant species. The method of embryo transfer and artificial insemination is used for the multiplication of endangered animal species. The way these fears and concerns about application of biotechnology to achieving a safe environment and agriculture are addressed will have a remarkable impact on the future of biotechnology. A detailed analysis of both the advantages and the disadvantages would assist in directing the future of environmental and agricultural biotechnology, since the overall goal is to achieve a safe environment and improved agricultural productivity.

1.21 BASIC TOOLS AND METHODOLOGIES ASSOCIATED WITH ENVIRONMENTAL BIOTECHNOLOGY It is understood that the scope of biotechnology is widening with every passing day. This is largely because more and more intensive research in this field has given birth to new ideas, innovations, and need of approaching new philosophies and new allied fields to make the maximum use of biotechnological methodologies and tools for the benefit of mankind. As far as the use of biotechnology in the fields of genetics, breeding, agriculture, animal husbandry, tissue culture and cloning is concerned, this is quite known and common now. However, another exciting and comparatively newer field of biotechnology, where it has shown an immense progress and productivity over a very short period, is environmental biotechnology. Environmental biotechnology, in fact, environmental biology, deals with the application of biotechnological methodologies to make use of living organisms and living systems for the remediation of environmental pollution ensuring a clean and human-friendly environment. This means that on one hand, this area of science cleans and repairs the damaged environment, and on the other, it reduces the hazardous implications of technological processes and renders them environmental friendly. Environmental biotechnological techniques make use of microorganisms and plants, and thereby preserve energy.  A conventional example of the use of environmental biotechnology is the release of guppy fish (Poecilia reticulata) in ponds and lakes. Guppy fish eats mosquitoes and its larvae. This reduces mosquito population in the respective area and helps contain malaria, dengue and other diseases which are dependent on mosquitoes for their spread. Another technique makes use of microorganisms to extract heavy metals from low grade ores. Chemoautotrophic bacteria derive energy from the breakdown of inorganic chemicals, which in this case, are ores of heavy metals. The action of such bacteria on these ores releases the metal itself. It is also an example of symbioses, where the bacteria derive their energy from the breakdown of ore and release useful heavy metals. This reiterates the fact that environmental biotechnology

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Environmental Biotechnology

serves to preserve energy and resources. Copper, uranium, cobalt, lead, nickel and gold can be extracted in this way. Thiobacillus ferrioxidans is used to extract copper and uranium. Some species of bacteria also accumulate metals. This property enables them to be used in both, the extraction of metals, as well as detoxification of waste. For example, some species of Pseudomonas accumulate mercury and uranium, while those of Thiobacillus accumulate silver.  Another useful implication of environmental biotechnology, and a major one at that, is bioremediation. It is defined as the use of biological agents, such as bacteria or plants, to remove or neutralize contaminants in polluted soil or water. This technique usually breaks down the pollutants into smaller harmless compounds such as water and carbon dioxide. However, depending on the various types and nature of processes used, it can also be used to produce methane and hydrogen, which are very valuable and highly worthy fuels. This takes place under the especially maintained anaerobic conditions, and therefore, bacteria that thrive best under such conditions, are selectively used for the purpose. Bioremediation is used to clean oil spills and beaches; treat sewage water and decontaminate soil, air and water.  Apart from this, environmental biotechnology has helped to make paper and plastic industries environmental friendly. It has introduced biological organisms and microbes in place of compounds that are costly, consume a lot of energy and cause pollution if they escape into the environment. For example, when a lignin degrading and modifying enzyme isolated from a fungus was used in pulp processes, it reduced energy costs, increased the life of the system and reduced the risks associated with bleach. Likewise, in plastic industry, glucose is replacing ethylene and propylene as raw material, and microbes are being used to convert it into alkene oxides. However, with its vast area of applications and so many advantages, environmental biotechnology also poses some complications. Microorganisms used in the aforementioned techniques are more often than not genetically engineered to make the processes efficient. Such organisms being genetically different form their natural contemporaries pose a threat to the balance of the ecosystem. Thus, it can be concluded in a very fair and confident way that environmental biotechnology has some very promising and extremely production future concerns associated with the environmental remediation of our planet. 

1.22 DEVELOPING ENVIRONMENTALLY SOUND BIOTECHNOLOGIES IN INDIA India’s National Environmental Engineering Research Institute has developed a number of environmentally sound biotechnologies – demonstrating that not all advances take place in developed countries. They include the following:

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1.37

• A chemo-biochemical technology for desulphurizing gaseous fuels and emissions containing hydrogen sulphide, which also recovers elemental sulphur. The process removes 99 per cent of the hydrogen sulphide. The recovered sulphur, with a purity of up to 99.7 per cent, can be used commercially. • A technology for producing biosurfactants—active compounds derived from biological sources which, like synthetic surfactants, exhibit characteristic physical and chemical properties. Biosurfactants can be used in situ to enhance oil recovery, in remediation of oil spills, and as detergents. • The bioremediation of mine spoil dumps, which involves excavating pits on the eroded, stony dumps, filling them with bedding material (organic waste and spoil), and planting selected saplings pretreated with microbial cultures. The process reclaims spoil dumps, mined land and wastelands within three to four years without using chemicals. The degraded ecosystems recover fast, providing carbon dioxide sinks. • A process using a microbial treatment which removes 85 per cent of high pyritic sulphur and 89 per cent of ash from coal before it is burned, leaving coal which is usable in thermal power plants, coal gasification plants and for generating cleaner liquid fuels. The institute’s costbenefit analysis of these and other biotechnologies shows that the initial investments, annual operating and maintenance costs, benefits and investment returns are attractive to small-scale enterprises in developing countries.

CHAPTER

2

Environmental Microbiology—Soil

Microbes are everywhere in the biosphere, and their presence invariably affects the environment that they are growing in. The many and varied metabolic activities of microorganisms assure that they participate in chemical reactions in almost every environment on earth. Microorganisms require an energy producing system (including an electron acceptor) to sustain life and nutrients, including liquid water, in order to grow and reproduce. Since microorganisms have been present on earth longer than other organisms, they have evolved the ability to thrive in almost any environment that meets these minimal criteria. Energy comes from one of two sources, light (photosynthesis), or the oxidation of reduced molecules. Oxidizable molecules may be organic (e.g. sugar, protein or any of the other foods we humans relish) or a variety of inorganic molecules such as sulfur, iron, hydrogen, carbon monoxide, or ammonia or even a combination of organic/inorganic molecules. Microorganisms exist that prosper inside eukaryotic cells, at temperatures >100oC, in the presence of toxic metals like copper or mercury, at pHs ~2.0 and ~11.0, down to 3.5 km below the earth’s surface and in saturated salt solutions at 0°C. Microorganisms have broadened the environments they can live in by evolving enzymes that allow them to utilize sunlight for energy as well as a diversity of electron donor/acceptor pairs; so they can perform energy-yielding oxidative reactions on available energy sources. That this evolution is ongoing is shown by the isolation of microorganism that can metabolize numerous man-made chemicals (ones not found in nature). The range of electron acceptors includes gaseous oxygen, sulfate, nitrate, nitrite, carbon dioxide, carbon monoxide, iron and magnesium. Indeed, evolutionary principles predict that microbes should have evolved to utilize any niche meeting the minimal physical and chemical requirements. Recently, bacteria that live ~3.5 km below the earth’s surface in rocks at high temperatures have been discovered. Since these conditions cover the entire earth—even that portion under the oceans—these bacterial forms may make up the largest single mass of life on (or in) earth.

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Below is an abbreviated list of the roles microbes play in our lives: • They maintain soil fertility and soil tilth. • They clean up all the dead organic material; without them, we would be up to our ears in dead things, like our ancestors. • They fix gaseous nitrogen into forms that can be used by plants to maintain the fertility of soils. • They can be used to extract minerals from ores. • They are the prime food for all the marine and freshwater life; even whales depend on them directly or indirectly for their nutrition.

2.1

SOIL AND SOIL MICROORGANISMS

Soil is the outer, loose material of earth’s surface which is distinctly different from the underlying bedrock and the region which supports plant life. Agriculturally, soil is the region which supports the plant life by providing mechanical support and nutrients required for growth. From the microbiologist viewpoint, soil is one of the most dynamic sites of biological interactions in the nature. It is the region where most of the physical, biological and biochemical reactions related to decomposition of organic weathering of parent rock take place. Living organismsboth plants and animals, constitute an important component of soil. The pioneering investigations of a number of early microbiologists showed for the first time that the soil was not an inert static material but a medium pulsating with life. The soil is now believed to be a dynamic or rather a living system, containing a dynamic population of organisms/microorganisms. Cultivated soil has relatively more population of microorganisms than the fallow land, and the soils rich in organic matter contain much more population than sandy and eroded soils. Microbes in the soil are important to us in maintaining soil fertility / productivity, cycling of nutrient elements in the biosphere and sources of industrial products, such as enzymes, antibiotics, vitamins, hormones, organic acids etc. At the same time, certain soil microbes are the causal agents of human and plant diseases.

2.1.1 The characteristics of soil microorganisms Soil inhabit diverse group of living organisms, both microflora and microfauna • Microflora: — Bacteria — Fungi, Molds, Yeast, Mushroom — Actinomycetes, Streptomyces — Algae, e.g. BGA, Yellow Green Algae, Golden Brown Algae Bacteria is again classified into: (I) Heterotrophic e.g. symbiotic & non - symbiotic N2 fixers, Ammonifiers, Cellulose Decomposers, Denitrifiers (II) Autrotrophic e.g. Nitrosomonas, Nitrobacter, Sulphur oxidizers, etc.

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2.3

• Microfauna: Protozoa, Nematodes

The density of living organisms in soil is very high i.e. as much as billions/gm of soil usually density of organisms is less in cultivated soil than uncultivated/virgin land and population decreases with soil acidity. Top soil, the surface layer contains greater number of microorganisms because it is well supplied with oxygen and nutrients. Lower layer/subsoil is depleted with oxygen and nutrients, hence, it contains fewer organisms. Soil ecosystem comprises of organisms which are both, autotrophs (algae, BOA) and heterotrophs (fungi, bacteria). Autotrophs use inorganic carbon from CO2 and are “primary producers” of organic matter, whereas heterotrophs use organic carbon and are decomposers/consumers.

2.1.1.1 Viruses

• Viruses lead a strictly parasite existence - they reproduce within bacteria, plants, animals and human cells.



• The most important kind of viruses in the soil environment are the viruses living in bacterial cells, called bacteriophages (phages).



• The role of phages in the soil environment depends on their ability to eliminate some populations of bacteria and on selecting the microorganisms both in a negative and positive way. The example of their negative influence are the phages that attack the root nodule bacteria (Rhizobium) which are the cause of the decline of papilionaceous plants crops.

2.1.1.2 Bacteria

• Bacteria constitute the basic mass of all soil microorganisms. They are characterized by high metabolic activity.



• Most soil bacteria are characterized by the ability to adhere to surfaces of the mineral molecules and to the soil colloids.



• Especially high numbers of bacteria gather around the residue of plants’ and animals’ tissues as well as in animal droppings that finds its way into the soil. The environment that is especially suitable for the development of the bacteria are the plants’ roots and their other underground parts.



• Soil bacteria can be subdivided into two groups: those that always occur in each one of the soils’ type (autochthonous) and the ones that grow only after high amount of the organic matter discharge into the soil (zymogenous).



• The largest group of soil bacteria is represented by the Actinomycetes and rod-coccus bacteria that belong to the Arthrobacter genus.

Rod-coccus bacteria: Club-shaped bacteria that belong to the Arthrobacter genus are dominant in numbers representative of the autochthonous soil

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microflora. They make up 2-60% of the whole population of soil microflora and are characterized by the tendency to form branching and coccus forms. The bacteria are polymorphic. In new bacterial cultures, the bacteria grow in a form of long irregular rods whereas in old cultures, they create coccus forms. They are characterized by a high resistance to the environmental factors during the vegetative stage. Also, they are capable of surviving in dry soil for a few months, whereas most of the other bacteria, that do not produce resting spores, die out. The bacteria have the ability to utilize a wide spectrum of organic compounds as a food substrate. They conduct biodegradation of not easily accessible compounds and may utilize many metabolites of other microorganisms including various polymers, growth factors and the amino acids produced by microorganisms. The bacteria which utilize the cellulose (Cellulomonas) also belong to the club-shaped forms.

• Bacteria play an important role in the environment. Many dead materials are decomposed by bacteria. If there were no bacteria, the environment would have been polluted and full of harmful microorganisms. They degrade the dead organic matter and convert it into energy and nutrients. For example, they decompose trees and get their food from them in the form of nutrition.



• Organic carbon present in the environment in the form dead organism is able to eat up all the carbon dioxide from the atmosphere if there were no decomposers in the on earth. It can be imagined that if carbon dioxide gets reduced from the atmosphere, there would have been no photosynthesis in plants and as a result no food would have been produced by plants. Decomposers or bacteria help in cycling of minerals like carbon and sulfur. They are also helpful in making, drugs, antibodies and hormones.



• Nitrogen is the most important component of plants. Plants totally rely on nitrogen for their health and growth and they cannot inhale it directly from the environment. They are dependent on soil for the supply of nitrogen. Through the process of nitrogen fixation, nitrogen from the atmosphere becomes available to the plants. This process takes place through bacteria, for example, Rhizobium and Cyanobacteria. These species of bacteria convert the atmospheric nitrogen into nitrates and nitrites which is the part of their metabolism and make it available to the plants. Some plants have modified themselves so well that they are now able to store the bacteria into their tissues.



• Using soil rich with beneficent bacteria can increase the productivity, growth and health of the plants. Just as carbon dioxide is the useful component of plants, similarly oxygen is also necessary for them because plants need oxygen in the process of respiration. They take oxygen by digesting the sugars present in them and use it for their growth. They absorb oxygen from the roots and if the roots will be able

Environmental Microbiology—Soil

2.5

to absorb nutrients and energy from the soil only then they will be able to provide large amount of oxygen to the plants. Those soils are not fertile which do not have proper structure and organic matter. If these components will be present in the soil, then plants will get plenty of oxygen for respiration.

2.1.1.3 Actinomycetes The Actinomycetes are (chemo) organotrophic bacteria. They form elongated, branched out mycelium-like threads that contain a large number of prokaryotic cells. The width of the threads is 1-5 ìm. They mainly live in soil or upon decomposing plants. Most of them lead a saprophytic type of life, and some are pathogenic to plants and animals (for example: Streptomyces somaliensis, Actinomyces israelii and Nocardia asteroides cause the subcutaneous infections of feet called mycetoma). Their growth abilities in temperatures of 40-50°C give them a wide range of decomposition potential of various substances.

• The Actinomycetes degrade steroids, lignin, chitin, hydrocarbons, fatty and humic acids, which are not easily decomposed by other bacteria.



• During the decomposition of the above, they produce aromatic compounds.



• The characteristic smell of freshly ploughed soil, especially in spring, comes from the actinomycetes bacteria. The smell is caused by the substance called geosmin (1,10- dimetylo-9-dekalol), which is produced by Streptomyces griseus. They are the aerobic bacteria, whereas a small group has the ability to conduct the metabolic processes in anaerobic conditions (Eg. Actinomyces, Micromonospora).



• Many types of actinomycetes produce antibiotics such as erythromycin, neomycin, streptomycin, tetracycline, plus others, as the by-product of metabolism. About 90% of all actinomycetes isolated from soil are Streptomyces.

2.1.1.4 Fungi

• Fungi belong to a group of eucaryotic organisms which are the absolute heterotrophs. Most of them belong to the group of aerobes or fermenting fungi. They take the carbon and energy to build their own cells from the decomposition of the organic compounds. Fungi do not have any chlorophyl. In contrast to bacteria, the fungal cell wall contains chitin, glucans and other polysaccharides.



• They occur mostly in the upper layers of soil, however, they can be found as deep as 1 m.



• They get into symbiotic relationships with algae, insects and higher plants. Many species of fungi are pathogenic to humans, plants and animals.

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Environmental Biotechnology

• Their vegetative forms create thread-like shreds that are more or less branched out and usually multi-cellular. Their thick weaves form mycelium or thallus. The individual cells are the size of about 10ìm. The most common soil fungi are the genera of Penicillium, Aspergillus, Trichoderma, Verticillium, Fusarium, Rhizopus, Mucor, Zygorhynchus, Chaetomium. Fungi grow strongly in acidic soils and have crucial influence on changing of pH reaction.

Role of Fungi in environment:

• Fungi plays significant role in soils and plant nutrition.



• They plays important role in the degradation/decomposition of cellulose, hemicellulose, starch, pectin, lignin in the organic matter added to the soil.



• Lignin which is resistant to decomposition by bacteria is mainly decomposed by fungi.



• They also serve as food for bacteria.



• Certain fungi belonging to sub-division Zygomycotina and Deuteromycotina are predaceous in nature and attack on protozoa & nematodes in soil and thus, maintain biological equilibrium in soil.



• They also plays important role in soil aggregation and in the formation of humus.



• Some soil fungi are parasitic and cause number of plant diseases such as wilts, root rots, damping-off and seedling blights eg. Pythium, Phyiophlhora, Fusarium, Verticillium, etc.



• Number of soil fungi form mycorrhizal association with the roots of higher plants (symbiotic association of a fungus with the roots of a higher plant) and help in mobilization of soil phosphorus and nitrogen eg. Glomus, Gigaspora, Aculospora (Endomycorrhiza) and Amanita, Boletus, Entoloma, Lactarius (Ectomycorrhiza).

2.1.1.5 Soil phytoedaphon Phytoedaphon consists mainly of algae, and to a lesser extent, the higher plants. Algae are the main component of phytoedaphon. They are most numerous upon the surface of soil reaching deeper through ploughing, percolating water, animal activities and the ability to migrate. Two groups are distinguishable: algae colonies that live upon the surface-epiphytoedaphon and the ones that live in deeper layers-endophytoedaphon.

• Soil algae are obligatory photoautotrophs, however, the ones living in deeper layers probably feed heterotrophically.



• They play a major role in soil’s ecosystem and influence its qualities and stability. Through extracellular secretion, they fertilize the soil and take part in nutrient discharge into the environment.

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Environmental Microbiology—Soil



• Some blue-green algae (Cyanobacteria) are capable of fixing the atmospheric nitrogen (Nostoc, Anabaena, Scytonema, Tylypothrix). Soil inhabited by these microorganisms contains 26-400 times more nitrogen. Due to the ability of nitrogen (N2) and carbon dioxide (CO2) assimilation, they may be the first ones to colonize the nitrogen and organic carbon-free ground.



• About two thousand species of algae occur in soil. They are mainly:



— Blue-green algae: Nostoc, Microcoleus, Schizothrix



— Green algae: Ankistrodesmus, Chlorella, Chlorococcum, Chlamydomonas, Characium, Klebsormidium



— Diatoms: Achnanthes, Cymbella, Eunotia, Fragilaria, Hantzschia, Navicula, Nitzschia, Pinnularia



— Yellow-green Pleurochloris



— Euglenoids: Euglena, Peranema



— Red algae: Porphyridium



algae:

Anabaena,

Botrydiopsis,

Scytonema,

Heterothrix,

Tolypothrix,

Heterococcus,

• Among the macrobiotic plant organisms that inhabit the soil environment, higher plants dominate making up the basic element of biocenoses of all the land ecosystems.

Algae plays important role in the maintenance of soil fertility especially in tropical soils. They add organic matter to soil when die and thus increase the amount of organic carbon in soil. Most of soil algae (especially BGA) act as cementing agent in binding soil particles and thereby reduce/prevent soil erosion. Mucilage secreted by the BGA is hygroscopic in nature and thus helps in increasing water retention capacity of soil for longer time/period. Soil algae, through the process of photosynthesis, liberate large quantity of oxygen in the soil environment and thus facilitate the aeration in submerged soils or oxygenate the soil environment. They help in checking the loss of nitrates through leaching and drainage especially in uncropped soils. They help in weathering of rocks and building up of soil structure.

2.1.1.6 Fauna of soil The soil microfauna is represented by the protozoans, which mainly feed on bacteria. Their role is to conduct selection and rejuvenate the population of soil bacteria. Amoebae and flagellates dominate among them. Mesofauna is represented by the nematodes (eelworms), snails, insects, myriapods, mites

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Environmental Biotechnology

and others. They feed upon dead organic matter contributing to the formation of humus. Protozoa: These are unicellular, eukaryotic, colourless, and animal like organisms (Animal kingdom). They are larger than bacteria and size varies from few microns to a few centimeters. Their population in arable soil ranges from l0,000 to 1,00,000 per gram of soil and are abundant in surface soil. They can withstand adverse soil conditions as they are characterized by “cyst stage” in their life cycle. Except few genera which reproduce sexually by fusion of cells, rest of them reproduce asexually by fission/binary fission. Most of the soil protozoa are motile by flagella or cilia or pseudopodia as locomotors organs. Depending upon the type of appendages provided for locomotion, protozoa are: • Rhizopoda (Sarcondia) • Mastigophora • Ciliophora (Ciliata) • Sporophora (not common Inhabitants of soil) • Class - Rhizopoda: Consists protozoa without appendages; usually have naked protoplasm without cell; wall, pseudopodia as temporary locomotory organs are present some times. Important genera are Amoeba, Biomyxa, Euglypha, etc. • Class - Mastigophora: Comprises flagellated protozoa, which are predominant in soil. Important genera are: Allention, Bodo, Cercobodo, Cercomonas, Entosiphon Spiromonas, Spongomions and Testramitus. Many members are saprophytic and some possess chlorophyll and are autotrophic in nature. In this respect, they resemble unicellular algae, and hence, are known as “Phytoflagellates”. • The soil protozoa belonging to the class ciliate/ciliophora are characterized by the presence of cilia (short hair-like appendages) around their body, which helps in locomotion. The important soil inhabitants of this class are Colpidium, Colpoda, Balantiophorus, Gastrostyla, Halteria, Uroleptus, Vortiicella, Pleurotricha etc. Protozoa are abundant in the upper layer (15 cm) of soil. Soil moisture, aeration, temperature and pH are the important factors affecting soil protozoa. • Most of protozoans derive their nutrition by feeding upon or ingesting soil bacteria belonging to the genera Enterobacter, Agrobacterium, Bacillus, Escherichia, Micrococcus, and Pseudomonas and thus, they play important role in maintaining microbial/bacterial equilibrium in the soil. • Some protozoa have been recently used as biological control agents against phytopathogens. • Species of the bacterial genera viz. Enterobacter and Aerobacter are commonly used as the food base for isolation and enumeration of soil protozoans.

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• Several soil protozoa cause diseases in human beings which are carried through water and other vectors, eg. Amoebic dysentery is caused by Entomobea histolytica.

Macrofauna is represented by the earthworms, moles, rodents. The organisms break up soil material and carry it down to a significant depth. The earthworms play the most important role among the invertebrates, by feeding upon dead organic matter and absorbing it along with the mineral part of the soil; non-digested residue mixed with mineral soil and the metabolites are excreted in the form of lumps (coprolites) that contributes to the formation of crumb texture of soil and to its loosening. During the period of one year, earthworms, on the area of one hectare, are able to pass 7 thousand kg of soil through their digestive tracts.

2.1.2 The Number of Soil Microorganisms The number and composition of soil microorganisms depends on the type of soil, its structure, humidity and on the content of the organic matter.

2.1.2.1 Viruses The exact number of viruses in soil is not known. Their mass is estimated at less than 0.01 tons/ha.

2.1.2.2 Bacteria

• The number of bacteria varies from a couple of million to a couple of billion cells per each 1g of soil. The highest number of bacteria occurs in a layer of cultivable soil at the depth of up to about 30 cm. In deeper layers their numbers quickly lower. In the cultivable layer of soil of about 30 cm thick, there may be anywhere from several hundred kg up to a few tons of bacterial mass per each 1 hectare.



• In the vicinity of roots and upon their surface, the bacteria find increased amounts of organic compounds, such as organic acids, amino acids and vitamins that are excreted by plants. Therefore, in the layer around roots, called rhizosphere, the number of bacteria is several times higher than in soil far from the roots.



• In soils rich in organic compounds, there live more bacteria; usually in 1 g of cultivable soil there may be between 0.5-5.0 billion bacteria (1.515 tons/ha).



• Acidic soils contain relatively low number of bacteria and a large number of fungi.

2.1.2.3 Fungi

• Fungi, excluding yeast, occur in the forms of mycelia or spores. The number of such units per 1 gram of soil may reach several tens of

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Environmental Biotechnology

thousand; among that spores constitute anywhere from several to a few dozen per cent depending on the soil’s humidity and the organic substances available.

• Fungi are most widely represented in acidic peat and forest soils. There, they may be more numerous than the bacteria.



• The main mass of fungi is found in the upper 20-30 cm layer. The combined mass of fungi in the upper layers is almost identical to that of bacteria, and in forest soils, it may even be greater. On an average, it is between 0.001-1.0 billion fungi (about 1.5 tons/ha).

2.1.2.4 Algae Algae live mainly in the upper layers of soil anywhere between 0-10 cm where the sunlight penetrates (rarely below 50 cm). Their number may vary between 100 thousand to 3 million per 1 g of soil (0.2 tons/ha). In favourable conditions, for instance in highly irrigated tropical soils, the numbers may be increased.

2.1.2.5 Soil fauna Protozoa can widely develop in adequately humid soils. Their numbers reach anywhere from a few hundred to several million per 1 g of dry soil. The mass of protozoa in soil is between 0.1-0.5 tons/ha, whereas the mass of nematodes is between 0-0.2 tons/ha, earthworms 0-2.5 tons/ha, and other soil animals 0-0.5 tons/ha.

2.1.3 Activity of Microorganisms in soil Microorganisms reproduce and transform the organic matter creating biomass of their own cells and collect substrates essential for replenishing the supplies of humus. Additionally, they decompose and mineralize the organic compounds, consequently recirculating the indispensable elements in plant production based on the assimilation of CO2 from the atmosphere.

• Carbon makes up 50% of mass of the organic matter that gets into soil in the form of plant and animal residues (falling leaves, various remains in meadows and forests, animal corpses, roots and shoots of dead plants).



• The fresh organic matter is composed of monosaccharides (hexose, pentose), polysaccharides (starch, cellulose, hemicellulose, chitin), organic acids, aromatic compounds (lignin, phenols, tannin), hydrophobic compounds (wax, cutin, fat and others).



• Carbon is recovered during the organic compounds decomposition and mineralization processes.



• Depending on its chemical nature, particular components of plants’ mass are decomposed and mineralized at various speeds.

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2.11



• Soluble substances such as sugars, amino acids, organic acids are easily washed away with water from plant and animal residues and then are quickly metabolized by soil microorganisms; they especially regulate the microbiological activities in the rhizosphere.



• Waxes, fats, rubbers and tannin are decomposed with great difficulty due to their high hydrophobic properties. Lignin is the most resistant substance to decompose among the plant materials.

2.1.3.1 Cellulose decomposition

• Cellulose occurs commonly in the walls of plant cells and is associated with hemicellulose and lignin. In the dry mass of green plants, the content of cellulose is at 15-30%, whereas in lignified parts and straw, it can reach 50%.



• Cellulose is a polysaccharide that consists of a long unbranched chain of glucose units.



• Cellulolitic bacteria belong mainly to the genera: Cytophaga, Cellfalcicula, Cellulomonas and Cellvibro.



• The best known cellulolytic system occurs in fungi. The Trichoderma genus releases the most active cellulase enzymes into the environment, then the enzymatic attack occurs away from the cells. Other fungi like Chaetomium, Fusarium and others also take active part in the above process.



• Decomposition may also occur in oxygen-free conditions; it’s conducted by the genera: Acetovibrio, Bacteroides, Clostridium, Ruminococcus. Consequently, a large number of gaseous substances, such as CO2, H2, CH4 are created.



• Decomposition of cellulose occurs faster in soils of neutral or slightly acidic pH and is slowed down in highly acidic soils. • Microorganisms that decompose cellulose change it into simpler compounds and this way they create a nutrient base for all the soil heterotrophs.

2.1.3.2 Lignin decomposition Lignin belongs to a large group of aromatic compounds and, apart from cellulose, is a main component of wood tissues (up to 30% of plant biomass).

• Lignin is a polymer built of phenylopropane units which contain an aromatic ring and the methoxyl groups - OCH3.

• The most active lignin-degrading organisms are the fungi that cause, so called, white rot of wood. They decompose wood to CO2 and H2O. They belong to basidiomycete and ascomycyte groups and are represented by several hundred species. Among the Basidiomycetes, the best

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known ones are: Trametes versicolor, Phanerochaete chrysosporium, oyster mushroom Pleurotus ostreatus—edible mushroom and Lintunula edodes. Among the Ascomycetes, the ones involved are: Xylaria, Libertella and Hypoxylon. Among the mold fungi, Trichoderma lignorum demonstrates the ability to decompose lignin. • The enzymatic complex composition that decomposes lignin is, among others, represented by oxidoreductases which require oxygen or hydrogen peroxide H2O2 for oxidative tearing up of bonds that connect phenylpropane subunits of lignin to each other. In direct decomposition of lignin, the following takes part: laccase (may oxidize the monophenoles), lignin peroxidase and other enzymes that haven’t been elucidated. • The activity of microorganisms that decompose lignin in soil stimulates the production of humus.

2.1.3.3 Synthesis and humus decomposition Humus is an amorphous organic substance, usually dark, that makes up the colloidal system of a large surface area capable of adsorbing ions of water and gases. • It contains fractions of organic substances which have a low ratio of C:N (from 10 to 15), whereas the ratio of these elements in dead plants’ residue is at about C:N=40:1. • Fulvic, humic acids and humins make up the composition of humus. These are the conglomerates of more or less carbonized compounds, which are characterized by the presence of carboxyl, phenyl and methoxyl groups that contain C, O, N, P and S, as well as the aromatic skeleton with numerous side chains. • The main humus forming system is the activity of soil microorganisms: bacteria (including actinomycetes) and fungi. 2.1.3.3.1 The synthesis of humus (Humification) • The process of humus formation is called humification. • The main substrates from which humus compounds are formed are lignin, hydrocarbons and nitrogen compounds. However, the soil type and the climate conditions determine the kind of humus. • Microorganisms conduct the following processes connected to the formation of humus: — they decompose fresh organic matter producing metabolites - the precursors of humus compounds. — they create biomass, which after atrophy and autolysis make up the additional initial substrates needed for the formation of humus. — they catalyze the processes of humus synthesis.

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The basic way of humus compounds formation is through its synthesis from the fragments such as polyphenols with the participation of nitrogen components of protein origin. The source of polyphenols may be the processes of lignin decomposition, hydrocarbon transformation and various microbiological synthesis processes. Many polyphenols form as the metabolites of different microorganisms. The next stage of humification is the oxidation of polyphenols that leads to the formation of chinoid compounds. These transformations are catalyzed by the phenol oxidases produced by different microorganisms such as by the fungus Serpula lacrymans. The final phase of the process is the polymerization of oxidized phenols. Transformation of the organic matter in conditions when oxygen is available leads to the formation of humus and in oxygen-free conditions to peat deposit formation.

2.1.3.3.2 Decomposition of humus

• Degradation of humus occurs in conditions when there is a shortage of fresh organic matter and when there is not an adequate supply of nitrogen in soil.



• It is believed that the decomposition is caused by the autochthonous bacteria, which are adapted to the shortages of the available organic substances and they are consequently utilizing components contained in humus complexes. Particularly high degradation activity is

The role of microorganisms in nitrogen processes in soil—the nitrogen cycle

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demonstrated by the actinomycetes and some other bacteria such as Micrococcus, Corynebacterium and some white rot fungi such as Polysticus.

• It may be assumed, that both in humus formation and during its decomposition, the entire system of soil microflora and microfauna play a collective role.



• Due to the microbiological processes nitrogen from the atmosphere is being incorporated into the compounds of the organic cells (so called nitrogen fixation)



• The organic compounds contained in animal and plant residues are mineralized by microorganisms and then are incorporated into the nitrogen cycle. In this way, the free nitrogen level in the atmosphere remains stable (78%).



• The Nitrogen cycle in the environment is composed of several links such as:



— symbiotic and non-symbiotic fixation of atmospheric N2 by microorganisms.



— microbial decomposition of the organic nitrogen compounds, ammonification – the release of NH3 and of NH4+ions the, utilization of NH4+ ions for the resynthesis of proteins by microorganisms the utilization of NH4+ ions as ammonium salts by plants.



— the nitrification of NH4+ ions , nitrates are created through nitrites.

— the utilization of nitrates by higher plants as well as by some of the microorganisms (transformation of nitrogen into protein). — denitrification.

2.1.3.4 Atmospheric nitrogen fixation The assimilators are capable of fixing of nitrogen only in symbiosis with plants.

• Rhizobium bacteria, living in symbiosis with papilionaceous plants, supply soil with the most nitrogen. They get into the root system of the plant where they multiply forming the long bacteria threads which penetrate plant tissue. The overgrowth of the plant tissue stimulated by bacteria causes the growth of nodules that form specific units upon the roots. Inside the nodules, a part of plant-infecting bacteria transform into bacteroids, which do not reproduce but are continuously active.



• It is believed, that the bacteroids take active part in the process of the atmospheric nitrogen fixation. The bacteroids contain a red dye called leghemoglobin. It is called that way because of the similarities to the hemoglobin. The iron Fe3+ contained in the dye is reduced to Fe2+, thus it is believed, that the leghemoglobin mediates the transfer of electrons to free nitrogen, hence causing its reduction.

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• Inside the plant roots; for instance, of black alder root, nodules contain interacting actinomycetes (Streptomyces alnii).



• Nitrogen fixation by the free living bacteria is similar. The reduction of N2 to NH3 is performed by pyruvate dehydrogenase and nitrogenase enzymes.

2.1.3.5 Free-living N2 assimilators (non-symbiotic nitrogen fixation)

The following heterotrophic bacteria posses the ability to fix free nitrogen from the air and to enrich the soil with nitrogen:

• aerobes — Azotobacter, Azotomonas, Derxia, Achromobacter, Beijerinckia.



• microaerophiles — Pseudomonas, Arthrobacter and Aerobacter.



• anaerobes — some species of Clostridium such as: Cl. butyricum, Cl. pectinovorum.

Flavobacterium,

Mycobacterium,

Within the group of autotrophs, the above capabilities are demonstrated by the photosynthesizing bacteria: Chlorobium, Chromatium and cyanobacteria e.g. Anabaena, Nostoc.

2.1.3.6 Ammonification Ammonification is a process of the ammonium ion NH4+ or of the free ammonia formation. During the first stages of this process, a break down of protein and a liberation of the amino acids occurs. Next, deamination of the amino acids takes place. The proteolytic break down of proteins occurs with a participation of the exocellular enzymes. Formed amino acids are transported to microorganisms’ cells where the process of deamination takes place. Ammonia, being a gas, quickly spreads in dry soils, whereas in humid ones, it dissolves in water forming NH4+. Formed ammonium ions are utilized by the bacteria and plants for the synthesis of amino acids or undergo the process of nitrification.

2.1.3.7 Nitrification Nitrification is a biological process of oxidation of ammonia to nitrate accomplished by nitrificating bacteria (chemolithotrophs). The energy released during this process is utilized by bacteria in the synthesis of organic compounds. The nitrification proceeds in two stages:

1. First, the ammonium is oxidized to nitrite. Bacteria oxidizing NH4 to NO2 are described as the “nitroso”: Nitrosomonas, Nitrosospira, Nitrosocyjastis, Nitrosoglea



2. Second, the formed nitrite is oxidized to form nitrate. Nitrites are oxidized into nitrates by the group of bacteria called “nitro”, such as genera Nitrobacter, Nitrospira, Nitrococcus

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The nitrifying bacteria are sensitive to the acidification of the environment; slowing down of their growth occurs at pH 5.0. The nitrification process may be also conducted by the heterotrophic microorganisms. The biggest group that conductss the heterotrophic nitrification are fungi: Aspergillus flavus, Penicillium, Cephalosporium. The nitrification conducted by fungi is less sensitive to acidification and more resistant to drought. Formed nitrates in soil can be assimilated by the plants, flushed out by water, or decomposed in the process of denitrification.

2.1.3.8 Denitrification Denitrification is the process of nitrate reduction to form molecular nitrogen. The above process is conducted mainly in oxygen-free conditions, and it is when the nitrates are utilized for respiration as the terminal electron acceptors. Several kinds of heterotrophic bacteria belonging to Pseudomonas, Achromobacter, Bacillus, Micrococcus genera are involved in the process of denitrification. The reduction of nitrates occurs in a few stages. During the first stage the nitrates are reduced to nitrites (NO2-), then the nitrites are reduced to nitric oxides (NO, N2O) and down to molecular nitrogen. The process of denitrification is also conducted by some chemoautotrophic bacteria such as Thiobacillus denitrificans. The above bacteria obtain the energy from the oxidation of sulfur compounds to simultaneously reduce nitrates. Denitrification is believed to be a disadvantageous process since it leads to the deprivation of vital nitrogen compounds from plants. The loss of nitrogen from soil due to the denitrification increases with excess soil moistening, oxygen-free conditions, accumulation of nitrates and temperature increase.

2.1.4 INTERACTIONS AMONG SOIL MICROORGANISMS Soil is the largest terrestrial ecosystem where a wide variety of relationships exists between different types of soil organisms. The associations existing between different soil microorganisms, whether of a symbiotic or antagonistic nature, influence the activities of microorganisms in the soil. Microflora composition of any habitat is governed by the biological equilibrium created by the associations and interactions of all individuals found in the community. In soil, many microorganisms live in close proximity and interact among themselves in a different ways. Some of the interactions or associations are mutually beneficial, or mutually detrimental, or neutral. The various types of possible interactions/associations occurring among the microorganisms in soil can be:

(a) beneficial—(i) mutualism, (ii) commensalism and (iii) proto-cooperation or



(b) detrimental/harmful—(i) armensalism, (ii) antagonism, (iii) competition (iv) parasitism and (v) predation

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2.1.4.1 Beneficial Associations/Interactions 2.1.4.1.1 Mutualism (Symbiosis) It is a relationship or a type of symbiosis in which both the interacting organisms/partners are benefited from each other. The way/manner in which benefit is derived depends on the type of interactions. When the benefit is in the term of exchange of nutrients, then the relationship is termed as “syntrophism” (Greek meaning: Syn-mutual and trophe = nourishment), for example, Lichen (association of algae or BGA with fungus) in which algae benefits by protection afforded to it by the fungal hyphae from environmental stresses, while the fungus obtain and use CO2 released by the algae during photosynthesis. Where the blue green algae are the partners in the lichen association, the heterotrophs (Fungus), benefit from the fixed nitrogen by the blue green algae. Microorganisms may also form mutualistic relationships with plants, for example, nitrogen-fixing bacteria i.e. Rhizobium growing in the roots of legumes. In this Rhizobium-legume association, Rhizobium bacteria are benefited by protection from the environmental stresses while, in turn, plant is benefited by getting readily available nitrate nitrogen released by the bacterial partner. The Anabaena-Azolla is an association between the water fern Azolla and the cyanobacterium Anabaena. This association is of great importance in paddy fields, where nitrogen is frequently a limiting nutrient. An actinorrhizal symbiosis of actinomycetes, Frankia, with the roots of Alnus and Casurina (non-legumes) is common in temperate forest ecosystem for soil nitrogen economy. Another type of symbiotic association which exists between the roots of higher plants and fungus is Mycorrhiza. In this association fungus gets essential organic nutrients and protection from roots of the plants and allows them to multiply and, in turn, plants uptake phosphorus, nitrogen and other inorganic nutrients made available by the fungus. 2.1.4.1.2 Commensalisms In this association, one organism/partner in association is benefited by other partner without affecting it. For example, many fungi can degrade cellulose to glucose, which is utilized by many bacteria. Lignin is major constituent of woody plants and is usually resistant to degradation by most of the microorganisms but in forest soils, lignin is readily degraded by a group of Basidiomycetous fungi and the degraded products are used by several other fungi and bacteria which can not utilize lignin directly. This type of association is also found in organic matter decomposition process. 2.1.4.1.3 Proto-cooperation It is mutually beneficial association between two species/partners. Unlike symbiosis, proto-cooperation is not obligatory for their existence or performance of a particular activity. In this type of association, one organism

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favor’s its associate by removing toxic substances from the habitat and simultaneously obtain carbon products made by the another associate/ partner. Nutritional proto-cooperation between bacteria and fungi has been reported for various vitamins, amino and purines in terrestrial ecosystem and are very useful in agriculture. Proto-cooperative associations found beneficial in agriculture are : i) synergism between VAM fungus-legume plants and Rhizobium in which nitrogen fixation and phosphorus availability/uptake is much higher resulting in higher crop yields and improved soil fertility, ii) synergism between PSM-legume plants and Rhizobium and iii) synergism between plant roots and PGPR in rhizosphere, where rhizobacteria restrict the growth of phytopathogens on plant roots and secretes growth promoting substances.

2.1.4.2

Detrimental (Harmful) Associations/Interactions

2.1.4.2.1 Antagonism It is the relationship in which one species of an organism is inhibited or adversely affected by another species in the same environment. In such antagonism, one organism may directly or indirectly inhibit the activities of the other. Antagonistic relations are most common in nature and are also important for the production of antibiotics. The phenomenon of antagonism may be categorized into three i.e. antibiosis, competition and exploitation. In the process of antibiosis, the antibiotics or metabolites produced by one organism inhibits another organism. An antibiotic is a microbial inhibitor of biological origin. Innumerable examples of antibiosis are found in soil. For example, Bacillus species from soil produces an antifungal agent which inhibits growth of several soil fungi. Several species of Streptomyces from soil produce antibacterial and antifungal antibiotics. Most of the commercial antibiotics such as streptomycin, chloramphenicol, Terramycin and cyclohexamide have been produced from the mass culture of Streptomyces. Thus, species of Streptomyces are the largest group of antibiotic producer’s in soil. Another example of antibiosis is inhibition of Verticillium by Trichoderma, inhibition of Rhizoctonia by a bacterium Bacillus subtilis, inhibition of soil fungus Aspergillus terreus by a bacterium Staphylococcus aureus.

2.1.4.2.2 Ammensalism In this interaction/association, one partner suppresses the growth of other partner by producing toxins like antibiotics and harmful gases like ethylene, HCN, Nitrite, etc.

2.1.4.2.3 Ammensalism Competition As soil is inhabited by many different species of microorganisms, there exists an active competition among them for available nutrients and space. The limiting substrate may result in favoring one species over another. Thus, competition

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can be defined as “the injurious effect of one organism on another because of the removal of some resource of the environment”. This phenomenon can result in major fluctuations in the composition of the microbial population in the soil. For example, chlamydospores of Fusarium, oospores of Aphanomyces and conidia of Verticillium dahlae require exogenous nutrients to germinate in soil. But other fungi and soil bacteria deplete these critical nutrients required for spore germination and thereby hinder the spore germination resulting into the decrease in population. Competition for free space has been reported to suppress the fungal population by soil bacteria. Therefore, organisms with inherent ability to grow fast are better competitors.

2.1.4.2.4 Parasitism It is an association in which one organism lives in or on the body of another. The parasite is dependent upon the host and lives in intimate physical contact and forms metabolic association with the host. So, this is a host-parasite relationship in which one (parasite) is benefited while the other (host) is adversely affected, although not necessarily killed. Parasitism is widely spread in soil communities, for example, bacteriophages (viruses which attack bacteria) are stricte intracellular parasites Chytrid fungi, which parasitize algae, as well as other fungi and plants. There are many strains of fungi which are parasitic on algae, plants, animals parasitized by different organism earthworms are parasitized by fungi, bacteria, viruses etc. 2.1.4.2.5 Predation Predation is an association/exploitation in which the predator organism directly feeds upon and kills the prey organism. It is one of the most dramatic interrelationship among microorganisms in nature, for example, the nematophagous fungi are the best examples of predatory soil fungi. Species of Arthrobotrytis and Dactylella are known as nematode trapping fungi. Other examples of microbial predators are the protozoa and slime mold fungi which feed on bacteria and reduce their population. The bacteriophages may also be considered as predators of bacteria.

2.1.5 Mutual interaction of plants and microorganisms The most typical example of direct interaction of bacteria with plants are the following symbiosis: • bacterrhiza—the symbiosis of plants and bacteria. • mycorrhiza—the symbiosis of plants and fungi.

2.1.5.1 The symbiosis of microbes and plants – bacterrhiza • Symbiotic agreements are clearly exemplified in the rhizosphere, which is the area incorporating the outer surface of plants’ roots and the adjacent soil.

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• The symbiosis occurs when microorganisms settle in plants’ root systems. Both the plants and microorganisms may greatly benefit from such interaction. • The best example of such interaction is the symbiosis of nodule bacteria Rhizobium with the papilionaceous plants. • The symbiotic Rhizobium bacteria are among the best known nitrogenfixing organisms. Rhizobium belongs to the heterotrophic and aerobic bacteria. While developing inside the plant tissues, they obtain the energy and carbon needed from its host. On the other hand, the nitrogen assimilated from the air by the bacteria is utilized by the plant. • The Rhizobium bacteria may live in soil for years without having any contact with a plant, by utilizing monosaccharides and mannitol as the source of C and energy. In such conditions, they do not exhibit any abilities to reduce and fix nitrogen, but they obtain it from the substrate in the form of ammonium nitrogen. Nevertheless, once in the vicinity of a papilionaceous plant, with which they can interact, they penetrate its root system and form nodules that participate in atmospheric nitrogen fixation. • The symbiotic system is formed only with particular types of papilionaceous plants and the suitable species of Rhizobium genus.

2.1.5.1.1 Rhizosphere The rhizosphere is the layer of soil around the roots where among others, in great concentrations, live bacteria, fungi, protozoa, nematodes, mites, springtails, which usually form groups of species characteristic to a given plant. • The rhizosphere is occupied by a large variety of forms, however the Pseudomonas and Achromobacter as well as the denitrifiers are the most numerous, and less numerous are the Arthrobacter and Bacillus forms. The above organisms utilize nutrients released by the roots. The increased number of microorganisms is accompanied by higher activity of soil’s fauna, especially of those organisms which feed upon roots and microorganisms. • The number of bacteria in the rhizosphere may even be 1000 times higher than outside the rhizosphere. The ratio of bacteria from within the rhizosphere to the number of bacteria from outside is called the rhizosphere effect and it is marked with the R/S symbol (R – rhizosphere, S – soil). • Microorganisms of the rhizosphere also have a big effect on plants. They lead to a continuous breakdown of organic and mineral compounds, which become available to plants. Moreover, they produce organic and non-organic acids, influence the dissolution of mineral salts and protect the plants against the phytopathogens.

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• An unfavorable effect is due to the metabolic activity of microflora that cause the depletion of the oxygen required for development of denitrifiers. As a consequence, some phytotoxic substances may be produced, like alcohols, antibiotics or phenolic compounds.

2.1.5.2 The symbiosis of fungi and plants Mycorrhiza is the interaction of fungi with vascular plants. In this type of interaction, both organisms benefit. Fungi grow into plant roots. They penetrate its cells and stimulate their growth by producing auxin hormone. Due to mycorrhiza, plants obtain larger absorbing surface and better access to nutrients being broken down and absorbed by fungi. Plants supply fungi with organic substances in the form of assimilation products transported from leaves to roots. Mycorrhiza is a widespread phenomenon; it concerns not only roots of trees, bushes, species of flowers but also cultivated plants such as grains and potatoes. There are two types of mycorrhiza: • Ectotrophic, • Endotrophic. 2.1.5.2.1 Ectotrophic mycorrhiza Fungus develops upon the surface of plant roots, creating a kind of muff composed of intertwined threads of mycelium. The outer hyphae of this mycelium penetrate the soil, while the inner ones penetrate the surface layers of root tissues. • As the result of the mycorrhiza, roots lose their root hairs and become shorter since the functions of roots are taken over by the fungi. Because mycelium’s suction force is much stronger than that of the roots the plant is better supplied with water and mineral salts than in the cases when there is no mycorrhiza. • For plants interacting with mycorrhizal fungi, the absorption of nitrogen may increase by 90%, phosphorus by 20% and potassium by 75%. • Fungi also produce substances that stimulate roots’ growth, and some are able to fix nitrogen. That kind of symbiotic fungi mainly belong to the Basidiomycetes. 2.1.5.2.2 Endotrophic mycorrhiza Endotrophic mycorrhiza occurs usually among green plants and some deciduous trees. • In this type of interaction, the plant roots do not differ externally from those without mycorrhiza. • The inside of the root cells is filled with a thick network of intertwined hyphae which are partially digested by the plant. • Endotrophic mycorrhiza is formed by the Fungi imperfecti.

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2.1.6  Soil Bioremediation Bioremediation is a group of treatment methods or processes designed to enhance the natural microbial degradation of organic contaminants. The microorganisms carry out the degradation of harmful substances to a less toxic or non-toxic state. Microorganisms utilize the environment-polluting organic compounds as food substrates. After the degradation of polluting compounds, the population of microorganisms reduces. Dead microorganisms or low numbers of microorganisms once lack food substrates, no longer pose any danger to the environment. The goal of bioremediation is the neutralization of the organic pollution to achieve undetectable concentrations or concentrations permissible by the national regulations of particular countries. Bioremediation is utilized for cleanup of grounds and groundwaters as well as sewage and sludge. During the utilization of bioremediation for the purpose of pollution neutralization, the following conditions must be met: (a) the environment undergoing bioremediation should contain microorganisms characterized by the specific catabolic processes,

(b) microorganisms utilized within the bioremediation process should be capable of efficiently converting chemical compounds and reducing their concentration down to the level allowed by the regulations,



(c) metabolites produced during the biodegradation should not have toxic, mutagenic or carcinogenic properties, and

(d) the conditions in the immediate area, where the process is being conducted, should be favourable to the growth and activity of the microorganisms (adequate nutrients, acceptable pH, oxygen or other electron acceptor, acceptable redox level, favorable moisture). The rate of biodegradation may be limited by: temperature, the toxicity of concentrated contaminants, or mass transfer limitations.

2.1.6.1 Microorganisms used in remediation technologies The bioremediation processes may be conducted by the autochthonous microorganisms, which naturally inhabit the soil/water environment undergoing purification, or by other microorganisms, that derive from different environments. However, in both the cases, they are characterized by a high ‘xenobiotics’ degradation activity. The choice of strains capable of being used for the inoculation of the contaminated grounds creates many problems.

• Apart from the high efficiency in ‘xenobiotics’ decomposition, the chosen strains should also possess many additional features that enable their adaptation and development in a new environment.



• One of the conditions for the adaptation of the inoculants in soil is the lack of antagonistic interaction with the natural water and soil microflora.

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• Moreover, they can not be pathogenic microorganisms nor ones, which, during growth on hydrocarbons, produce substances with cytotoxic, mutagenic or carcinogenic properties. The selection of microorganisms specialized in degradation of particular compounds is based on the processes of their adaptation or genetic operation. Substances making up the environment’s contamination, as a rule, are composed of many compounds. Therefore, a complete soil cleanup requires a special, carefully selected, mixture of microorganisms. Utilization of mutated microorganisms is useful only in ex situ methods where precise conditions of the process control and the prevention of contamination by dangerous mutants exists. In order to avoid problems related to the introduction of foreign organisms into the environment, mixtures of microorganisms should be created to degrade the contamination. Moreover, the techniques used to allow microorganisms adapted to new conditions should constantly be improved. The initial high number of microorganisms in soil may be obtained at the beginning of the purification process by the inoculation of the ground with microorganisms. This allows a faster and more effective bioremediation process. Inoculation of the soil is carried out after growing the microorganisms in a bioreactor. The cultured natural autochthonous microorganisms or specially prepared strains derived from a culture collection and/or the commercial preparations are utilized in the process of inoculation. The strains are stored in the lyophilised form, frozen or placed in a special suspension. The microorganisms may be incorporated into both the soil and the water environment in the form of a suspension or placed on a solid support (immobilized). The microbial cultures usually contain a mixture of bacteria, nutrients, a solid support and possibly enzymes. Depending on the composition of these biopreparations used for bioremediation, the above can be subdivided into microbiological (bacterial), enzymatic and bacterial-enzymatic. The advantage of microbiological preparations in relation to enzymatic preparations is the fact that the microorganisms multiply in a previously clean environment whereas the enzymatic preparations are added, in specific dosages, without the possibility of multiplication. One of the conditions which have to be met in order to obtain effective biodegradation is the bacteria’s accessibility to the contaminated layers of soil. Migration within the soil depends on the number of microorganisms as well as on the type of soil. The effects of the biodegradation process of xenobiotics in soil are dependent on the method used to inoculate the soil with microorganisms. Even distribution of microorganisms within the soil and their contact with nutrients contained in the soil has to be provided. Surface introduction of the inoculants is not highly effective due to slow migration of microorganisms, especially in soils, which contain clay and silt. In order to speed up the process of migration, the following are applied: • injections with the use of high-pressure equipment, • immobilization of microorganisms on solid supports,

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• electromagnetic field to speed up bacterial migration in the soil.

Due to the fact that biopreparations are composed of living organisms, their application requires thorough knowledge of the subject as well as a careful supervision of the cleaning process.

2.1.6.2 Stimulation of bioremediation Bioremediation processes are designed to optimise the conditions for microbial growth and degradation of contaminants. Microbial growth and metabolism of contaminants require macronutrients (phosphorus and nitrogen) and micronutrients. The demand for nutrients is specific for each case. It has been determined that the ratio of carbon to nitrogen and phosphorus within the soil should range from 100:10:1(by weight). Therefore, the basic condition for a proper biostimulation is the control of nitrogen and phosphorus concentrations in the soil by application of mineral fertilizers. The most suitable ones, for the above purpose, are ammonium sulphate and sodium phosphate (sources of nitrogen and phosphorus). Moreover, magnesium sulphate, sodium carbonate, calcium chloride, iron sulphate are also used for the purpose. The choice of appropriate dosages of the biogenic substances ought to be very precise and readjusted to the soil conditions, since it has been shown that, for example, nitrogen compounds in excessive numbers may slow down the biodegradation process. The selection of nutrients with an optimum composition is conducted in laboratories. The type of missing nutrients is established by growing bacteria in the presence of various sources of biogenic elements and by careful supervision of their growth. In addition, pollution reduction, oxygen consumption and dioxide release are measured.

2.1.6.3 Classification of bioremediation methods The division of bioremediation methods may be done in accordance with the level of environment oxygenation as well as the location of the cleaning process. Depending on the level of environmental oxygenation, the types of biological purification can be divided into aerobic, anaerobic/aerobic, and anaerobic. Aerobic microorganisms use oxygen as an electron acceptor. Anaerobic microorganisms use other electron acceptors such as nitrate, sulphate, iron, manganese, or certain organic compounds. Facultative organisms can use oxygen, if it is present or other electron acceptors, if it is not. Most soil bioremediation processes are aerobic. In anaerobic conditions the microbiological decomposition of pollutants is slower and the end products may have a toxic character. The source of oxygen utilized by microorganisms in the respiration processes may be the atmospheric air that gets into the soil passively or by force. The passive route depends on natural ground penetration by the atmospheric air. Active increase of the delivered oxygen can be done by mechanical mixing of the surface layers of soil (harrowing, ploughing etc.), the introduction of special perforated spray lances driven directly into the

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soil or aeration with compressors and fans. In addition, the environmental enrichment in oxygen may take place because of the utilization of hydrogen peroxide, which breaks down in soil to water and oxygen. Depending on the level of contamination as well as the character of the recultivated environment, bioremediation may take place through both, in situ or ex situ methods. In the first one, in situ, the point is that the pollution is eliminated directly at the place where it occurs. However, in the second method, the contaminated ground or waters are excavated before the actual regeneration procedures. In situ techniques do not require excavation of the contaminated soils; so may be less expensive, create less dust, and it is possible to treat a large volume of soil and cause less release of contaminants than by ex situ methods. But in situ techniques are slower than ex situ, may be difficult to manage, and are most effective at sites with permeable soil. Ex situ methods are utilized where there is danger of toxic pollutant migration into the groundwaters and when the process of detoxification must be conducted in a short period of time.

2.1.6.3.1 In situ methods In situ methods are based mainly on biostimulation of the organic pollutant degradation processes by enriching waters or soils in biogenic elements, by acidity correction as well as by their aeration. The following in situ methods can be used: Agricultural methods: Those are the most commonly used methods of soil decontamination. They are effective with almost all components of fuels. Lighter products, such as gasoline, are eliminated by vaporization or, to lesser extent, by biodegradation. When it comes to heavy products, like diesel fuels and kerosene, contamination is eliminated mainly by biodegradation. There are many variations of agricultural methods depending on the technical solutions. The simplest variation depends on spreading the contaminated soil in a thin layer of no more than 0.5m thick followed by a period ploughing or deep harrowing in order to aerate the soil. This activates the microflora by delivering essential nutrients, oxygenating the soil and by deacidification when needed. Nutrients are replenished by application of nitrogenous, phosphate or potassium fertilizers, when necessary. Soil deacidification can be accomplished by liming. Planting grasses or papilionaceous plants on the contaminated grounds may also assist the cleaning processes. Detailed programs for fertilization, mechanical and other treatments are designed for each individual case. Bio-extraction: The situation is significantly more complicated when the need for removal of contaminants from within deeper layers of soils arises. The growth stimulation of microorganisms decomposing, for example, petroleum products, is more difficult; nevertheless, the methods are similar. It is also essential in this case to deliver missing nutrients, supply oxygen and, if necessary, inoculate the soil with microorganisms able to actively decompose the contaminants. The processes of soil venting and rinsing with

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solution containing nutrients or active bacterial cultures are utilized, as well. For the above, the following technical equipment is necessary: suction and positive displacement pumps, sumps, and screens preventing the spreading of contaminant within soils. The condition of an effective cleaning process is also a proper geological configuration of the terrain, which allows a controlled flow of the medium (air, water vapour, solutions). The natural decomposition can be accelerated by utilizing the so-called bio-extraction. Optimization of the process can be accomplished by rinsing the soil and water environments through forced infiltration of groundwater (water stimulated bioremediation in-situ) and aeration (soil bio-ventilation). Microorganisms and nutrients delivered with water stimulate biodegradation of petroleum products while the air enriches soil in oxygen thus assisting in the biodegradation.

Water stimulated bioremediation The goal of the above process is to force a vertical and then horizontal flow of water along with the organic contaminants in the water and soil environment.

Water stimulated soil bioremediation

This method is utilized for the cleaning of the environment from petroleum related non-soluble substances gathered at the surface of groundwaters. During the process, the groundwater is pumped out up to the surface, purified, oxygenated, and returned back to the source after nutrient enrichment. In other cases, the flow of the underground waters may be utilized. The waters are obtained from wells, which are situated in the lowest points. These waters are then sent to bio-reactors, in which the process of biodegradation of petroleumrelated products occurs. The same water is then returned back to its source.

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The above method is a combination of in situ and ex situ methods which are complementary to each other, which in turn, allows for optimisation of the process. Water stimulated bioremediation can be assisted by surfactants. It has been shown that synthetic surfactants and biosurfactants accelerate the processes of hydrophobic contaminants biodegradation, particularly of heavy fractions of petroleum products. Increased solubility and emulsion formation result in better mobility of petroleum products in soil and in larger specific surfaces accessible to microorganisms. Surfactants can also increase the permeability of soils. Bioventilation: The acceleration of the natural processes of biodegradation may be assisted by soil ventilation. Ventilation is a physical method, which may be utilized as an independent decontamination technique used for the maximization of volatilization of low molecular mass hydrocarbons (for example, products based on gasoline or solvents). However, during this process, a very little biodegradation occurs.

Bioventilation

The ventilation process permits the removal of volatile petroleum products from the aeration and saturation zones, while at the same time enriching the ground with air and increasing the level of oxygenation. The aeration process can be both, active or passive. In the case of passive ventilation, the aeration is done by the utilization of perforated pipe networks. Active ventilation however, depends on the creation of negative pressure (extraction of the ground air) or positive pressure (air injection). The effectiveness of the soil and water environment bioventilation depends on the level of oxygen in the air contained in the soil, level of bio-genes, reduction-oxidation conditions, presence of surface-active agents, level of saturation (moisture), pH and temperature.

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The most effective way of supplying oxygen to the contaminated ground is by introducing it with compressed air. Ozone (O3) is also utilized as an alternative source of oxygen. However, there is some danger in using ozone as the source of oxygen since it possesses some toxic properties.

2.1.6.3.2 Ex situ methods Ex situ methods can be implemented when the danger of toxic pollutant migration into the groundwaters occurs and when the process of detoxification has to be completed in a short period of time. Bioreactor method: The decontamination process is most effective when run in specially designed bioreactors. They provide effective control of all parameters, delivery of required ingredients, oxygenation and inoculation of the soil. The cost of this method is however rather high due to the technological requirements as well as the need for transportation of heavy masses. Therefore, the above method is not often used and is usually employed for smaller amounts of soil.

Schematic representation of the bioreactor method

Among the technologies based on ex situ methods, there are bio-reactors and lagoons used for decontamination of oily sewage waters and reactors for treatment of semi-liquid masses of contaminated soil, sludge and deposits. Solid materials undergo mixing with liquids followed by aeration. In both types of bioreactors, the level of dissolved oxygen may be controlled just like the pH and the concentration of inorganic nutrients. This technique is similar to the one that treats municipal wastes by utilizing the activated sludge method. Moreover, the technique may be aided by surfactants or dispersants in order to desorb the hydrocarbons from the solid particles, and to increase the degree of dispersion of water insoluble oil contaminants. Surface-active agents used in these methods may be synthetic or natural. Bio-surfactants have found ever-increasing use due to the fact that they are more environmentally friendly and are biodegradable.

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Bioreactors may also be utilized for decontamination of groundwaters. They are either stationary or portable, which makes the process easier and less expensive. Portable bioreactors are used for decontamination of ground waters after pumping out the material from the water-bearing bed or for decontamination of waters derived from the process of contaminated ground rinsing. There are two different types of bioreactors, fluidized and immobilized beds. The choice of bed depends mainly on the type of contaminants and their concentration in water that is going to be processed. The immobilized biomass is a mixture of selected microorganisms capable of biodegradation of particular pollutants. The microorganisms may be trapped inside the carrier’s structure (natural polymers such as agar, alginate, collagen, or synthetic polymers such as polyacrylamide gels, polyurethanes and others) which is called active immobilization. In the passive immobilization, microorganisms are bound to the surface of a porous material (activated carbon, for instance) or may create a gelatinous film upon the surface of stationary elements (ceramic rings, plastic tiles, polyurethane foam). In such bioreactors, the contaminated water flows countercurrent to the air through the solid bed containing the immobilized microorganisms. Biopile method: The method consists of transferring the contaminated soil or ground into a specially prepared place.

Schematic representation of the biopile method

The excavated soil is arranged in the form of an elongated heap inside a foil-lined ditch that is equipped with drainage and ventilation systems. The process is also aided by aeration as well as water and nutrients addition. The

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air is pumped in through the system of perforated pipes equipped with a blow-fan that creates negative pressure at the bottom of the heap. In simpler technologies, the actual aeration is ensured by mechanical shifting of the soil. The heap is usually covered with a foil tunnel equipped with a system of sprinklers through which water and nutrients are delivered. In many recently used technologies, water circulates in a closed system. Reflux from within the ground is filtered into bioreactors and then pumped into the reservoir from which, after enrichment in nutrients and growth of the microorganisms, it is returned by the sprinkler system onto the heap.

2.1.7 Role of Soil Microorganisms in Biodegradation of Pesticides and Herbicides Pesticides are the chemical substances that kill pests and herbicides are the chemicals that kill weeds. In the context of soil, pests are fungi, bacteria insects, worms, and nematodes, etc. that cause damage to field crops. Thus, in broad sense, pesticides are insecticides, fungicides, bactericides, herbicides and nematicides that are used to control or inhibit plant diseases and insect pests. Although wide-scale application of pesticides and herbicides is an essential part of augmenting crop yields, excessive use of these chemicals leads to the microbial imbalance, environmental pollution and health hazards. An ideal pesticide should have the ability to destroy target pest quickly and should be able to degrade non-toxic substances as quickly as possible. The ultimate “sink” of the pesticides applied in agriculture and public health care is soil. Soil being the storehouse of multitudes of microbes, in quantity and quality, receives the chemicals in various forms and acts as a scavenger of harmful substances. The efficiency and the competence to handle the chemicals vary with the soil and its physical, chemical and biological characteristics.

2.1.7.1 Effects of pesticides Pesticides reaching the soil in significant quantities have direct effect on soil microbiological aspects, which in turn influence plant growth. Some of the most important effects caused by pesticides are : • alterations in ecological balance of the soil microflora, • continued application of large quantities of pesticides may cause ever lasting changes in the soil microflora, • adverse effect on soil fertility and crop productivity, • inhibition of N2 fixing soil microorganisms such as Rhizobium, Azotobacter, Azospirillum, etc. and cellulolytic and phosphatesolubilizing microorganisms, • suppression of nitrifying bacteria, Nitrosomonas and Nitrobacter, by soil fumigants ethylene bromide, telone, and vapam have also been reported,

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• alterations in nitrogen balance of the soil,



• interference with ammonification in soil,



• adverse effect on mycorrhizal symbioses in plants and nodulation in legumes, and



• alterations in the rhizosphere microflora, both quantitatively and qualitatively.

2.1.7.2 Persistence of pesticides in soil How long an insecticide, fungicide, or herbicide persists in soil is of great importance in relation to pest management and environmental pollution. Persistence of pesticides in soil for longer period is undesirable because of the reasons: a) accumulation of the chemicals in soil to highly toxic levels, b) may be assimilated by the plants and get accumulated in edible plant products, c) accumulation in the edible portions of the root crops, d) to be get eroded with soil particles and may enter into the water streams, and finally leading to the soil, water and air pollutions. The effective persistence of pesticides in soil varies from a week to several years depending upon structure and properties of the constituents in the pesticide and availability of moisture in soil. For instance, the highly toxic phosphates do not persist for more than three months while chlorinated hydrocarbon insecticides (eg. DOT, aldrin, chlordane, etc.) are known to persist at least for 4-5 years and, some times, more than 15 years. From the agricultural point of view, longer persistence, of pesticides leading to accumulation of residues in soil may result into increased absorption of such toxic chemicals by plants to the level at which the consumption of plant products may prove deleterious/hazardous to human beings as well as livestock. There is a chronic problem of agricultural chemical having entered in food chain at highly inadmissible levels in India, Pakistan, Bangladesh and several other developing countries in the world. For example, intensive use of DDT to control insect pests and mercurial fungicides to control diseases in agriculture had been known to persist for longer period and thereby got accumulated in the food chain leading to food contamination and health hazards. Therefore, DDT and mercurial fungicides have been banned for use in agriculture as well as in public health department.

2.1.7.3 Biodegradation of Pesticides in Soil Pesticides reaching the soil are acted upon by several physical, chemical, and biological forces. However, physical and chemical forces are acting upon/ degrading the pesticides to some extent microorganisms play major role in the degradation of pesticides. Many soil microorganisms have the ability to act upon pesticides and convert them into simpler non-toxic compounds. This process of degradation of pesticides and conversion into non-toxic compounds by microorganisms is known as “biodegradation”. Not all pesticides reaching

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the soil are biodegradable and such chemicals that show complete resistance to biodegradation are called “recalcitrant”. The chemical reactions leading to biodegradation of pesticides fall into several broad categories: • Detoxification: Conversion of the pesticide molecule to a non-toxic compound. Detoxification is not synonymous with degradation. Since a single change in the side chain of a complex molecule may render the chemical non-toxic. • Degradation: The breaking down/transformation of a complex substrate into simpler products leading finally to mineralization. Degradation is often considered to be synonymous with mineralization, e.g. Thirum (fungicide) is degraded by a strain of Pseudomonas and the degradation products are dimethlamine, proteins, sulpholipids, etc. • Conjugation (complex formation or addition reaction): In which an organism make the substrate more complex or combines the pesticide with cell metabolites. Conjugation or the formation of addition product is accomplished by those organisms catalyzing the reaction of addition of an amino acid, organic acid or methyl crown to the substrate; for e.g., in the microbial metabolism of sodium dimethly dithiocarbamate, the organism combines the fungicide with an amino acid molecule, normally present in the cell, and thereby, inactivate, the pesticides/ chemical. • Activation: It is the conversion of non-toxic substrate into a toxic molecule, for eg. Herbicide, 4-butyric acid (2, 4-D B) and the insecticide Phorate are transformed and activated microbiologically in soil to give metabolites that are toxic to weeds and insects. • Changing the spectrum of toxicity: Some fungicides/pesticides are designed to control one particular group of organisms/pests, but they are metabolized to yield products inhibitory to entirely dissimilar groups of organisms, for e.g. the fungicide PCNB is converted in soil to chlorinated benzoic acids that kill plants. Biodegradation of pesticides/herbicides is greatly influenced by the soil factors like moisture, temperature, pH and organic matter content, in addition to microbial population and pesticide solubility. Optimum temperature, moisture and organic matter in soil provide congenial environment for the break down or retention of any pesticide added in the soil. Most of the organic pesticides degrade within a short period (3-6 months) under tropical conditions. Metabolic activities of bacteria, fungi and actinomycetes have significant role in the degradation of pesticides.

2.1.7.4 Criteria for Bioremediation/Biodegradation For successful biodegradation of pesticide in soil, following aspects must be taken into consideration: (i) Organisms must have necessary catabolic

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activity required for degradation of contaminant at fast rate to bring down the concentration of contaminant, ii) the target contaminant must be bioavailable, iii) soil conditions must be congenial for microbial /plant growth and enzymatic activity and iv) cost of bioremediation must be less than other technologies for removal of contaminants. According to Gales (1952) principal of microbial infallibility, for every naturally occurring organic compound, there is a microbe/enzyme system capable its degradation.

2.1.7.5 Strategies for Bioremediation For the successful biodegradation/bioremediation of a given contaminant following strategies are needed.

• Passive/intrinsic Bioremediation: It is the natural bioremediation of contaminant by tile indigenous microorganisms and the rate of degradation is very slow.



• Biostimulation: Practice of addition of nitrogen and phosphorus to stimulate indigenous microorganisms in soil.



• Bioventing: Process/way of biostimulation by which gases stimulants like oxygen and methane are added or forced into soil to stimulate microbial activity.



• Bioaugmentation: It is the inoculation/introduction of microorganisms in the contaminated site/soil to facilitate biodegradation.



• Composting: Piles of contaminated soils are constructed and treated with aerobic thermophilic microorganisms to degrade contaminants. Periodic physical mixing and moistening of piles is done to promote microbial activity.



• Phytoremediation: Can be achieved directly by planting plants which hyperaccumulate heavy metals or indirectly by plants stimulating microorganisms in the rhizosphere.



• Bioremediation: Process of detoxification of toxic/unwanted chemicals/contaminants in the soil and other environment by using microorganisms.



• Mineralization: Complete conversion of an organic contaminant to its inorganic constituent by a species or group of microorganisms.

2.1.8 Microbial Ecology of Petroleum Contaminant Plumes The aerobic biodegradation of petroleum in groundwater systems is effected by microorganisms which metabolize PHCs for organic carbon and energy. The microorganisms involved are primarily procaryotic soil bacteria such as Nocardia, Pseudomonads, Acinetobacter, Flavobacterium, Microcossus, Anthrobacter, and Corynebacterium. Petroleum-degrading soil bacteria consist of two different groups distinguished by unique respiratory capabilities.

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• Obligate aerobic microbes consist of those soil bacteria which metabolize organic carbon only under oxic conditions, whereas • facultative anaerobic microbes consist of those bacteria which metabolize organic carbon under either, oxic or anoxic conditions. — Intrinsic biodegradation is typically effected by a bacteria group rather than a single bacterium. This is because ultimate bio-oxidation to carbon dioxide and water involves a series of biotransformations in which one bacteria converts one group of petroleum hydrocarbons to intermediate compounds. The intermediate compounds are themselves metabolized by a different bacteria. — In uncontaminated groundwater systems, indigenous microbes obtain organic carbon and energy from dissolved organic carbon (DOC). The DOC leaches from soil organic matter in the unsaturated zone. In petroleum-contaminated groundwater systems, certain bacteria having the genetic capability to metabolize petroleum constituents are stimulated by the supplemental organic carbon supplied by PHCs. — Bacteria metabolize DOC and PHCs by breaking carbon-carbon and carbon-hydrogen covalent bonds. PHCs amenable to intrinsic biodegradation include the aliphatic hydrocarbons with carbon number ranges of C10 to C25 and the aromatic hydrocarbons benzene, toluene, ethyl benzene, and xylenes (BTEX). • During bio-oxidation of DOC/PHCs, microbes use O2 as a terminal electron acceptor to collect electrons released during metabolism, and ambient inorganic nutrients and organic carbon to maintain cell tissue and increase biomass. Although oxgyen is consumed in this process, nutrients are generally conserved as they are recycled during production of waste materials and cellular tissue. Alternate terminal electron acceptors include nitrate/nitrite, sulfate/sulfite, and carbon dioxide. However, O2 is generally the most energetic electron acceptor for stimulating biodegradation.

2.1.9 Molecular Microbial Ecology The scientific core of environmental biotechnology is microbial ecology—a scientific discipline dedicated to understanding complex communities of microorganisms: which microorganisms are present, what is their metabolic potential, what part of the potential are they realizing, and how they interact with each other and their environment. Fundamental scientific research in microbial ecology provides us with a deep understanding of how the complex communities work. Fortunately, we have powerful new tools to help us analyze these intriguing organisms and their community organization. Among the tools we can use, are molecular methods that probe the genetic information of

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the microorganisms in microbial communities. By targeting genomic DNA, we can identify which microorganisms are present and what reactions they can perform; through the use of RNA-based techniques we can identify the members in the community that are actively growing and what reactions they are performing. Using in-situ techniques, we can also investigate the metabolic interactions that take place among microorganisms in a mixed community. Applying these molecular tools to understand microbial communities is called molecular microbial ecology, and it is a critical research strength of the Center for Environmental Biotechnology.

PCR is used to detect the presence of a specific group of microorganisms

In particular, the Center has exceptional capabilities to investigate changes in microbial structure through the use of molecular techniques that target the 16S rRNA gene and the 16S rRNA, such as denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH). DGGE is especially powerful for doing “detective work” to identify important, but uncharacterized strains. Real-time PCR is powerful for quantifying the different types of microorganisms. FISH allows us to do 3-dimensinal visualization of the communities and understand the interactions among different strains. The Center also targets specific metabolic genes and their expression to investigate the role of critical enzymes in detoxification and energy generating reactions occurring in natural or engineered techniques including reverse transcriptase PCR, RNA or cDNA microarrays, and realtime PCR to quantify over- and under-expression of specific genes and for microarray validation. Identification of specific gene-targets will allow us to investigate critical factors affecting the performance of natural or engineered

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microbial systems. The expression of such critical genes can be used to monitor and evaluate the success of engineered processes.

2.1.10 Soil Microorganisms as Biofertilizers Biofertilizers are microbial inoculants or carrier-based preparations containing living or latent cells of efficient strains of nitrogen-fixing, (phosphate is solublizing) and cellulose decomposing microorganisms intended for seed or soil application and designed to improve soil fertility and plant growth by increasing the number and biological activity of beneficial microorganisms in the soil. • The objects behind the application of biofertilizers/microbial inoculants to seed, soil or compost pit is to increase the number and biological/ metabolic activity of useful microorganisms that accelerate certain microbial processes to augment the extent of availability of nutrients in the available forms which can be easily assimilated by plants. The need for the use of Biofertilizers has arisen primarily due to two reasons, i.e., though chemical fertilizers increase soil fertility, crop productivity and production, but increased/intensive use of chemical fertilizers has caused serious concern for soil texture, soil fertility and other environmental problems and that use of Biofertilizers is both economical as well as environment friendly. Therefore, an integrated approach of applying both chemical fertilizers and Biofertilizers is the best way of integrated nutrient supply in agriculture. Organic fertilizers (manure, compost, vermicompost) are also considered as Biofertilizers, which are rendered in available forms due to the interactions of microorganisms or their association with plants. Biofertilizers, thus include (i) Symbiotic nitrogen fixers viz. Rhizobium sp., (ii) Non-symbiotic, freeliving nitrogen fixers viz. Azotobacter, Azospirillum etc., (iii) BGA-inoculants viz., Azolla-Anabaena, (iv) Phosphate, solubilizing microorganisms (PSM) viz. Bacillus, Pseudomonas, Penicillium, Aspergillus, etc. (v) Mycorrhiza, (vi) Cellulolytic microorganisms and (vii) Organic fertilizers. Nobbe and Hiltner (1895, USA) produced the first Rhizobium biofertilizer under the brand name “Nitragin” for 17 different legumes.

2.1.10.1 Role of Biofertilizers in soil fertility and Agriculture Biofertilizers are known to play a number of vital roles in-soil fertility, crop productivity and production in agriculture as they are eco-friendly and can not, at any cost, replace chemical fertilizers that are indispensable for getting

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maximum crop yields. Some of the important functions or roles of Biofertilizers in agriculture are:

• They supplement chemical fertilizers for meeting the integrated nutrient demand of the crops.



• They can add 20-200 kg N/ha year (eg. Rhizobium sp 50-100 kg N/ha yr; Azospirillum , Azotobacter: 20-40 kg N/ha /yr; Azolla : 40-80 kg N/ha; BGA :20-30 kg N/ha) under optimum soil conditions and thereby increase 15-25 per cent of total crop yield.



• They can, at best, minimize the use of chemical fertilizers, not exceeding 40-50 kg N/ha under ideal agronomic and pest-free conditions.



• Application of Biofertilizers results in increased mineral and water uptake, root development, vegetative growth and nitrogen fixation.



• Some Biofertilizers (eg, Rhizobium BGA, Azotobacter sp) stimulate production of growth promoting substance like vitamin-B complex, Indole acetic acid (IAA) and Gibberellic acids, etc.



• Phosphate-mobilizing or phosphorus-solubilizing Biofertilizers/ microorganisms (bacteria, fungi, mycorrhiza, etc.) converts insoluble soil phosphate into soluble forms by secreting several organic acids and under optimum conditions, they can solubilize/mobilize about 30-50 kg P2O5/ha due to which crop yield may increase by 10 to 20%.



• Mycorrhiza or VA-mycorrhiza (VAM fungi), when used as Biofertilizers enhance uptake of P, Zn, S and water, leading to uniform crop growth and increased yield and also enhance resistance to root diseases and improve hardiness of transplant stock.



• They liberate growth-promoting substances and vitamins and help to maintain soil fertility.



• They act as antagonists and suppress the incidence of soil borne plant pathogens and thus, help in the bio-control of diseases.



• Nitrogen-fixing, phosphate-mobilizing and cellulolytic microorganisms in biofertilizers enhance the availability of plant nutrients in the soil and thus, sustain the agricultural production and farming system.



• They are cheaper, pollution-free and renewable energy sources.



• They improve physical properties of soil, soil tilth and soil health, in general.



• They improve soil fertility and soil productivity.



• Blue green algae like Nostoc, Anabaena, and Scytonema are often employed in the reclamation of alkaline soils.



• Bio-inoculants, containing cellulolytic and lignolytic microorganisms, enhance the degradation/decomposition of organic matter in soil, as well as enhance the rate of decomposition in compost pit.

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• BGA plays a vital role in the nitrogen economy of rice fields in tropical regions.



• Azotobacter inoculants, when applied to many non-leguminous crop plants, promote seed germination and initial vigor of plants by producing growth-promoting substances.



•  Azolla-Anabaena grows profusely as a floating plant in the flooded rice fields and can fix 100-150 kg N/ha/year in approximately 40-60 tonnes of biomass produced.



• Plays important role in the recycling of plant nutrients.

2.1.10.2 Quality Control Measures (as per ISI Specifications)

• Since, Biofertilizers contain live cells, care should be taken during their transportation and storage. • They should be kept in a cold place and not exposed to sunlight. • Biofertilizers for legumes are crop-specific; therefore, they must be used for the crop for which they are meant. • Biofertilizers, when used under adverse soil conditions, appropriate remedial measures (liming and use of Gypsum) should be followed. • Biofertilizers must be carrier-based. • Carrier material used should be in form of powder (75-106 micron size. • They should contain minimum of 10^8 viable cells of microorganisms /gram of the carrier material on dry weight basis. • They should have a minimum period of six months expiry from date of its • They should be free from any contaminant /contamination with other microorganisms. • pH should be in the range of 6.0-7.5. • They should induce desired beneficial effects on all those crops, species/ cultivars listed on the packet before the expiry date. • They should be packed in 50-75 micron low density polythene packets

2.1.11 Factors Affecting Distribution, Activity and Population of Soil Microorganisms Soil microorganisms (Flora & Fauna), just like higher plants, depend entirely on soil for their nutrition, growth and activity. The major soil factors which influence the microbial population, distribution and their activity in the soil are:

• Soil fertility,



• Cultural practices,



• Soil moisture,

Environmental Microbiology—Soil



• Soil temperature,



• Soil aeration,



• Light,



• Soil pH (H-ion concentration)



• Organic matter.



• Food and energy supply,



• Nature of soil, and



• Microbial associations.

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All these factors play a great role in determining not only the number and type of organism but also their activities. Variations in any one or more of these factors may lead to the changes in the activity of the organisms which ultimately affect the soil fertility level. Brief account of all these factors influencing soil microflora/organisms and their activities is activities are as follows. Cultural practices (Tillage): Cultural practices viz. cultivation, crop rotation, application of manures and fertilizers, liming and gypsum application, pesticide/fungicide and weedicide application have their effect on soil organisms. Ploughing and tillage operations facilitate aeration in soil and exposure of soil to sunshine, and thereby, increase the biological activity of organisms, particularly, of bacteria. Crop rotation with legume maintains the favorable microbial population balance, particularly of N2. Fixing bacteria and thereby improve soil fertility. Liming of acid soils increases activity of bacteria and actinomycetes and lowers the fungal population. Fertilizers and manures applied to the soil for increased crop production, supply food and nutrition not only to the crops but also to microorganisms in soil and thereby proliferate the activity of microbes. Foliar or soil application of different chemicals (pesticides, fungicides, nematicides, etc.) in agriculture are either degraded by the soil organisms or are liable to leave toxic residues in soil which are hazardous to cause profound reduction in the  normal microbial activity in the soil. Soil fertility: Fertility level of the soil has a great influence on the microbial population and their activity in soil. The availability of N, P and K required for plants as well as microbes in soil determines the fertility level of soil. On the other hand, soil microflora has greater influence on the soil fertility level. Soil moisture: It is one of the important factors influencing the microbial population and their activity in soil. Water (soil moisture) is useful to the microorganisms in two ways i.e. it serve as source of nutrients and supplies hydrogen/oxygen to the organisms and it serve as solvent and carrier of other food nutrients to the microorganisms. Microbial activity & population proliferate best in the moisture range of 20% to 60%. Under excess moisture conditions/water logged conditions due to lack of soil aeration (Oxygen),

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anaerobic microflora become active and the aerobes get suppressed. While in the absence of adequate moisture in soil, some of microbes die out due to tissue dehydration others change their forms into resting stages spores or cysts and tide over adverse conditions. Therefore, optimum soil moisture (range 20 to 60 %) must be there for better population and activity of microbes in soil. Soil temperature: Next to moisture, temperature is the most important environmental factor influencing the biological, physical and chemical processes and, of microbes, microbial activity and their population in soil. Though microorganisms can tolerate extreme temperature (such as – 60° or + 60 u) conditions, but the optimum temperature range at which soil microorganisms can grow and function actively is rather narrow. Depending upon the temperature range at which microorganisms can grow and function, they are divided into three groups, namely:

• psychrophiles (growing at low temperature below 10°C)



• Mesophiles (growing well in the temp range of 20°C to 45°C) and



• thermopiles (can tolerate temperature above 45°C and optimum 45-60°C).

Most of the soil microorganisms are mesophilic (25 to 40°) and optimum temperature for most mesophiles is 37°C. True psychrophiles are almost absent in soil, and thermopiles, though present in soil, behave like mesophiles. True thermopiles are more abundant in decaying manure and compost heaps where high temperature prevails. Seasonal changes in soil temperature affects microbial population and their activity, especially in temperate regions. In winter, when temperature is low (below 50°C ), the number and activity of microorganisms falls down, and as the soils warms up in spring, they increases in number as well as activity. In general, population and activities of soil microorganisms are the highest in spring and lowest in winter season. Soil air (Aeration): For the growth of microorganisms, better aeration (oxygen, and sometimes, CO2) in the soil is essential. Microbes consume oxygen from soil air and gives out carbon dioxide. Activities of soil microbes is often measured in terms of the amount of oxygen absorbed or amount of CO2 evolved by the organisms in the soil environment. Under high soil moisture level/water logged conditions, gaseous exchange is hindered and the accumulation of CO4 occurs in soil air which is toxic to microbes. Depending upon oxygen requirements, soil microorganisms are grouped into categories, viz aerobic (require oxygen for life processes), anaerobic (do not require oxygen) and microaerophilic (requiring low concentration/level of oxygen). Light: Direct sunlight is highly injurious to most of the microorganisms except algae. Therefore, upper portion of the surface soil, a centimeter or less—is usually sterile or devoid of microorganisms. Effect of sunlight is due to heating and increase in temperature (More than 45°)

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Soil Reaction/Soil pH: Soil reaction has a definite influence/effect on quantitative and qualitative composite of soil microbes. Most of the soil bacteria, blue-green algae, diatoms and protozoa prefer a neutral or slightly alkaline reaction between pH 4.5 and 8.0 and fungi grow in acidic reaction between pH 4.5 and 6.5, while actinomycetes prefer slightly alkaline soil reactions. Soil reactions also influence the type of the bacteria present in soil. For example nitrifying bacteria (Nitrosomonas & Nitrobacter) and diazotrophs like Azotobacter are absent totally or inactive in acid soils, while diazotrophs like Beijerinckia, Derxia, and sulphur oxidizing bacteria like Thiobacillus thiooxidans are active in acidic soils. Soil Organic Matter: The organic matter in soil being the chief source of energy and food for most of the soil organisms, it has great influence on the microbial population. Organic matter influence directly or indirectly on the population and activity of soil microorganisms. It influences the structure and texture of soil and thereby activity of the microorganisms. Food and energy supply: Almost all microorganisms obtain their food and energy from the plant residues or organic matter/substances added to the soil. Energy is required for the metabolic activities of microorganisms. The heterotrophs utilize the energy liberated during the oxidation of complex organic compounds in soil, while autotrophs meet their energy requirement from oxidation of simple inorganic compounds (chemoautotroph) or from solar radiation (photoautotroph). Thus, the source of food and energy rich material is essential for the microbial activity in soil. The organic matter, therefore serves both as a source of food nutrients as well as energy required by the soil organisms. Nature of Soil: The physical, chemical and physico-chemical nature of soil and its nutrient status influence the microbial population, both quantitatively and qualitatively. The chemical nature of soil has considerable effect on microbial population in soil. The soils in good physical condition have better aeration and moisture content which is essential for optimum microbial activity. Similarly, nutrients (macro and micro) and organic constituents of humus are responsible for absence or presence of certain type of microorganisms and their activity. For example, activity and presence of nitrogen-fixing bacteria is greatly influenced by the availability of molybdenum, and absence of available phosphate restricts the growth of Azotobacter. Microbial associations /interactions: Microorganisms interact with each other giving rise to antagonistic or symbiotic interactions. The association existing between one organism and another, whether symbiotic or antagonistic, influences the population and activity of soil microbes to a great extent. The predatory habit of protozoa, and some mycobacteria which feed on bacteria, may suppress or eliminate certain bacteria. On the other hand, the activities of some of the microorganisms are beneficial to each other. For instance, organic

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acids liberated by fungi, increase in oxygen by the activity of algae, change in soil reaction, etc. favors the activity of bacteria and other organisms in soil. Root Exudates: In the soil where plants are growing, the root exudates also affects the distribution, density and activity of soil microorganism. Root exudates and sloughed off material of root surfaces provide an abundant source of energy and nutrients, and thus directly or indirectly, influence the quality as well as quantity of microorganisms in the rhizosphere region. Root exudates contain sugars, organic acids, amino acids, sterols, vitamins and other growth factors which have profound effect on soil microbes.

2.1.12 Rhizosphere Concept and It’s Historical Background The root system of higher plants is associated not only with soil environment composed of inorganic and organic matter, but also with a vast community of metabolically active microorganisms. As living plants create a unique habitat around the roots, the microbial population on and around the roots is considerably higher than that of root free soil environment and the differences may be both quantitative and qualitative.

• Rhizosphere: It is the zone/region of soil immediately surrounding the plant roots together with root surfaces, or it is the region where soil and plant roots make contact, or it is the soil region subjected to influence of plant roots and characterized by increased microbial.



• Rhizoplane: Root surface along with the closely adhering soil particles is termed as rhizoplane.



• Term “Rhizosphere” was introduced for the first time by the German scientist Hiltner (1904) to denote that region of soil which is subjected to the influence of plant roots. The concept of “Rhizosphere Phenomenon” which shows the mutual interaction of roots and microorganisms came into existence with the work of Starkey (1929), Clark (1939) and Rauath and Katznelson (1957).



• N. V. Krassinikov (1934) found that free-living nitrogen-fixing bacteria, Azotobacter were unable to grow in the wheat rhizosphere.



• Starkey (1938) examined the rhizosphere region of some plant species and demonstrated the effect of root exudates on the predominance of bacterial population, in particular, and other soil microorganisms, in general in the rhizosphere region. Thus, he put forth the concept of “Rhizosphere effect/phenomenon” for the first time.



• F E Clark (1949) introduced/coined the term “Rhizoplane” to denote the root surface together with the closely adhering soil particles.



• R. I. Perotti (1925) suggested the boundaries of the rhizosphere region and showed that it was bounded on one side by the general soil region (called as Edaphosphere), and on the other side, by the root tissues (called Histosphere).

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• G. Graf and S. Poschenrieder (1930) divided the rhizosphere region into two general areas, i.e. outer rhizosphere and inner rhizosphere, for the purpose of describing the same site of microbial action. • H. Katznelson (1946) suggested the R:S ratio, i.e. the ratio between the microbial population in the rhizosphere (R) and in the soil (S), to find out the degree or extent of plant roots effect on soil microorganisms. R:S ratio gives a good picture of the relative stimulation of the microorganisms in the rhizosphere of different plant species. • R:S ratio is defined as the ratio of microbial population per unit weight of rhizosphere soil (R), to the microbial population per unit weight of the adjacent non-rhizosphere soil (S). • A. G. Lochhead and H. Katznelson (1940) examined in detail the qualitative differences between the microflora of the rhizosphere and microflora of the non-rhizosphere region and reported that gramnegative, rod shaped and non-spore forming bacteria are abundant in the rhizosphere than in the non-rhizosphere soil. • C. Thom and H. Humfeld (1932) found that corn roots in acidic soils yielded predominantly Trichoderma, while roots from alkaline soils mainly contained Penicillium. • M.J. Timonin (1940) reported some differences in the fungal types and population in the rhizosphere of cereals and legumes. R: S ratio of fungal population was believed to be narrow in most of the plant species, usually not exceeding 10. • E. A. Peterson and others (1958) reported that the plant age and soil type influence the nature of fungal flora in the rhizosphere, and the number of fungal population gradually increases with the age of plant. • M. Adati (1932) studied many crops and found that though actinomycetes were relatively less stimulated than bacteria, but in some cases, the R:S ratio of actinomycetes was as high as 62. • R. Venkatesan and G. Rangaswami (1965) studied the rhizosphere effect in rice plant on bacteria, actinomycetes and fungi and reported that (i) for actinornycetes R: S was more (ranging from 0 to 25), depending on the age of plant roots and the dominant genera reported were Nocardia, (ii) R:S ratio reduced with the depth of soil. • E. A. Gonsalves and V. S. Yalavigi (1960) reported the presence of greater number of algae in the rhizosphere. • J. W. Rouatt reported positive rhizosphere effect on protozoa, but a negative effect on algae in wheat plants.

2.1.12.1 Microorganisms in the Rhizosphere and Rhizosphere Effect The rhizosphere region is a highly favorable habitat for the proliferation, activity and metabolism of numerous microorganisms. The rhizosphere

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microflora can be enumerated intensively by microscopic, cultural and biochemical techniques. Microscopic techniques reveal the types of organisms present and their physical association with the outer root tissue surface / root hairs. The cultural technique most commonly followed is “serial dilution and plate count method” which reveal the quantitative and qualitative population of microflora. At the same time, the cultural method shows the selective enhancement of certain categories of bacteria. The biochemical techniques used are designed to measure a specific change brought about by the plant or by the microflora. The rhizosphere effect is on most commonly found microorganisms viz. bacteria, actinomycetes, fungi, algae and protozoa. Bacteria: The greater rhizosphere effect is observed with bacteria (R:S values ranging from 10-20 or more) than with actinomycetes and fungi. Gramnegative, rod shaped, non-sporulating bacteria which respond to root exudates are predominant in the rhizosphere (Pseudomonas, Agrobacterium), while Gram-positive, rods, cocci and aerobic spore forming (Bacillus, Clostridium) are comparatively rare in the rhizosphere). The most common genera of bacteria are: Pseudomonas, Arthrobacter, Agrobacterium, Alcaligenes, Azotobacter, Mycobacterium, Flavobacter, Cellulomonas, Micrococcus and others have been reported to be either abundant or sparse in the rhizosphere. From the agronomic point of view, the abundance of nitrogen-fixing and phosphate-solubilizing bacteria in the rhizosphere assumes a great importance. The aerobic bacteria are relatively less in the rhizosphere because of the reduced oxygen levels due to root respiration. The bacterial population in the rhizosphere is enormous in the ranging form 10^8 to 10^9 per gram of rhizosphere soil. They cover about 4-10% of the total root area occurring profusely on the root hair region and rarely in the root tips. There is predominance of amino acids and growth factors required by bacteria, are readily provided by the root exudates in the region of rhizosphere. Fungi: In contrast to their effects on bacteria, plant roots do not alter/ enhance the total count of fungi in the rhizosphere. However, rhizosphere effect is selective and significant on specific fungal genera (Fusarium, Verticillium, Aspergillus and Penicillium), which are stimulated. The R:S ratio of fungal population is believed to be narrow in most of the plants, usually not exceeding to 10. The soil/serial dilution and plating technique used for the enumeration of rhizosphere fungi may often give erratic results as most of the spore formers produce abundant colonies in culture media giving a wrong picture/estimate (e.g., Aspergilli and Penicillia). In fact, the mycelial forms are more dominant in the field. The zoospore forming lower fungi such as Phytophthora, Pythium, Aphanomyces are strongly attracted to the roots in response to particular chemical compounds excreted by the roots and cause diseases under favorable conditions. Several fungi, e.g. Gibberella fujikurio produces phytohormones and influence the plant growth. Actinomycetes, Protozoa and Algae: Stimulation of actinomycetes in the rhizosphere has not been studied in much detail so far. It is generally

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understood that the actinomycetes are less stimulated in the rhizosphere than bacteria. However, when antagonistic actinomycetes increase in number they suppress bacteria. Actinomycetes may also increase in number when antibacterial agents are sprayed on the crop. Among the actinomycete, the phosphate solublizers (eg. Nocardia, Streptomyces) have a dominant role to play. As rule, actinomycetes, protozoa and algae are not significantly influenced by their proximity to the plant roots and their R:S ratios rarely exceed 2 to 3:1 and around roots of plants, R: S ratio for these microorganisms may go too high. Because of large bacterial community, an increase in the number or activity of protozoa is expected in the rhizosphere. Flagellates and amoebae are dominant and ciliates are rare in the region.

2.1.12.2 Factors affecting microbial flora of the Rhizosphere/ Rhizosphere Effect The most important factors which affect/influence the microbial flora of the rhizosphere or rhizosphere effect are: soil type & its moisture, soil amendments, soil pH, proximity of root with soil, plant species, and age of plant and root exudates.

• Soil type and its moisture: In general, microbial activity and population is high in the rhizosphere region of the plants grown in sandy soils and least in the high humus soils, and rhizosphere organisms are more when the soil moisture is low. Thus, the rhizosphere effect is more in the sandy soils with low moisture content.



• Soil amendments and fertilizers: Crop residues, animal manure and chemical fertilizers applied to the soil cause no appreciable effect on the quantitative or qualitative differences in the microflora of rhizosphere. In general, the character of vegetation is more important than the fertility level of the soil.



• Soil pH/Rhizosphere pH: Respiration by the rhizosphere microflora may lead to the change in soil rhizosphere pH. If the activity and population of the rhizosphere microflora is more, then the pH of rhizosphere region is lower than that of surrounding soil or nonrhizosphere soil. Rhizosphere effect for bacteria and protozoa is more in slightly alkaline, soil and for that of fungi, is more in acidic soils.



• Proximity of root with Soil: Soil samples taken progressively closer to the root system have increasingly greater population of bacteria, and actinomycetes and decreases with the distance and depth from the root system. Rhizosphere effect decline sharply with increasing distance between plant root and soil.



• Plant Species: Different plant species inhabit often variable microflora in the rhizosphere region. The qualitative and quantitative differences are attributed to variations in the rooting habits, tissue composition

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and excretion products. In general, legumes show/produce a more pronounced rhizosphere effect than grasses or cereals. Biennials, due to their long growth period, exert more prolonged stimulation on rhizosphere effect than annuals. • Age of Plant: The age of plant also alters the rhizosphere microflora and the stage of plant maturity controls the magnitude of rhizosphere effect and degree of response to specific microorganisms. The rhizosphere microflora increases in number with the age of the plant and reaches at peak during flowering which is the most active period of plant growth and metabolism. Hence, the rhizosphere effect was found to be more at the time of flowering than in the seedling or full maturity stage of the plants. The fungal flora (especially, Cellulolytic and Amylolytic) of the rhizosphere usually increases even after fruiting and the onset of senescence due to accumulation of moribund tissue and sloughed off root parts/tissues; whereas, bacterial flora of the rhizosphere decreases after the flowering period and fruit setting. • Root exudates/excretion: One of the most important factors responsible for rhizosphere effect is the availability of a great variety of organic substances at the root region by way of root exudates/excretions. The quantitative and qualitative differences in the microflora of the rhizosphere from that of general soil are mainly due to influences of root exudates. The spectrum of chemical composition of root exudates varies widely, and hence their influence on the microflora also varies widely. Root exudates are composed of the chemical substances like: S.N.

Root Exudates

Chemical Substances

1

Amino acids

All naturally occurring amino acids.

2

Organic acids

Acetic, butyric, citric, fumaric, lactic, malic, propionic, succinic, etc.

3

Carbohydrates/ sugars

Arabinose, fructose, galactose, glucose, maltose, mannose, oligosaccharides, raffinose, ribose, sucrose, xylose etc.

4

Nucleic acid derivatives

Adenine, cystidine, guanine, undine

5

Growth factors (phytohormones)

Biotin, choline, inositol, pyridoxine etc

6

Vitamins

Thiamine, nicotinic acid, biotin etc

7

Enzymes

Amylase, invertase, protease, phosphatase etc.

8

Other compounds Auxins, glutamine, glycosides, hydrocyanic acid peptides, UV-absorbing compounds, nematode attracting factors, spore germination stimulators, spore inhibitors etc.

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The nature and amount of chemical substances thus exuded are dependent on the species of plant, plant age, inorganic nutrients, temperature, light intensity, O2/CO2 level, root injury, etc. Another source of nutrients for the microorganisms in the rhizosphere region is the sloughed off root epidermis which exert selective stimulation effect on some specific groups of microorganisms. For instance, glucose and amino acids in the exudates readily attract Gram-negative rods which predominantly colonize the roots. Sugars and amino acids in the root exudates stimulate the germination of chlamydospores and other resting spores of fungi; stimulation effect of root exudates on plant pathogenic fungi, nematodes is also well known.

2.1.12.3. Alterations in Rhizosphere Microflora Foliar application of various chemicals leads to alterations in the rhizosphere microflora by changing the pattern of root exudates. The pattern of the rhizosphere microflora i.e. numbers and species composition can be changed/ altered by various factors, such as: • Soil amendments, • Foliar application of fertilizers/nutrients, fungicides, insecticides and hormones, and • Bacterization/microbial seed inoculants. Soil amendments: Soil amendments with inorganic and organic fertilizers can alter the rhizosphere microflora and an understanding of the type of changes in the microflora can be useful in the indirect control of pathogens. Dwivedi and Chaube (1985) showed that amendment of soil with neem-cake can stimulate the activity of actinomycetes which results into the reduction of propagules of Macrophomina phaseolina. It is also known to control phytopathogenic nematodes in soil by stimulating nematode-trapping fungi. Amendment of soil with castor and bean leaves stimulate the activity of Trichoderma viride and Penicillium in the rhizosphere leading to the control of Sclerotium rolfsii. Foliar application of fertilizers and agrochemicals: Translocation of photosynthate from leaves to roots takes place as a part of the normal metabolic activity in plants. Therefore, organic substances, including plant protection chemicals (fungicides, insecticides), growth regulators and plant nutrients applied to foliage/leaves get absorbed into the leaf tissue and further get translocated to roots along with photosynthates. Many workers have reported that foliar application with various chemicals cause marked alterations in the number and kind/qualities of microorganisms in the rhizosphere of several cereals and leguminous crop plants. Thus, such an approach can be used as a new tool in the biological control of root diseases, stimulation of activity of nitrogen-fixing bacteria and other beneficial microorganisms in the soil. Seed treatment with bio inoculants: Bio-inoculants such as Azotobacter, Beijerinckia, Azospirillum, Rhizobium or P-solubilizing microorganisms (eg.

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Bacillus polymyxa, Azotobacter croococcum, Aspergillus niger, Penicillium digitatum etc.) when applied to the seed/soil, help in the establishment of beneficial microorganisms in the rhizosphere region which will further benefit in plant growth, encourage inhibition of plant pathogenic organisms in the root vicinity and enrich the soil with added microbial biomass.

2.1.12.4 Associative and Antagonistic activities in the Rhizosphere In natural environments (eg. Soil, Air, Water, etc.) a number of relationships exist between individual microbes, microbial species and between individual cells. The composition of microflora of any habitat (soil/rhizosphere) is governed by the biological equilibrium created by the associations and interactions of all individuals found in the community. In soil and rhizosphere region, many microorganisms live in close proximity and their interactions with each other may be associative or antagonistic.

• Associative interactions/activities in rhizosphere: The dependence of one microorganism upon another for extra-cellular products (eg. amino acids & growth promoting substances) can be regarded as an associative activity/effect in rhizosphere. There is an increase in the exudation of amino acids, organic acids and monosaccharides by plant roots in the presence of microorganisms. Gibberellins and gibberellinlike substances are known to be produced by bacterial genera viz. Azotobacter, Arthrobacter, Pseudomonas, and Agrobacterium which are commonly found in the rhizosphere. Microorganisms also influence root hair development, mucilage secretion and lateral root development. Fungi inhabiting the root surface facilitate the absorption of nutrient by the roots. Mycorrhiza is one of the best known associative/symbiotic interactions which exist between the roots of higher plants and fungi. This mycorrhizal association has been found to improve plant growth through better uptake of phosphorus and zinc from soil, suppression of root pathogenic fungi and nematodes. Another example is association between the bacterium Rhizobium and roots of legumes and Azospirillum with cereal crops (wheat, rye, bajara, maize, etc.).



• Antagonistic interactions/activities in rhizosphere: The biochemical qualities of root exudates and the presence of antagonistic microorganisms, plays important role in encouraging or inhibiting the soil-borne plant pathogens in the rhizosphere region. Several mutualistic, communalistic, competitive and antagonistic interactions exist in the rhizosphere. The number and qualities of antagonistic microorganisms in the rhizosphere could be increased through artificial means such as fertilizer application, organic amendments, foliar spraying of chemicals, etc.

Antagonistic microorganisms in the rhizosphere play an important role in controlling some of the soil-borne plant pathogens. Stanier and group (1966)

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discovered the bacterial strain Pseudomonas fluorescens and the fluorescent pigments of this species in biological control of root pathogens. Strains of P. fluorescence are collectively called as “Fluorescent Pseudomonads”. They produce variety of biologically active compounds such as plant growth substances, cyanides, antibiotics and iron chelating substances called “Siderophores”. Rovira and Campbell (1975), showed that bacterial strains of P. fluorescens could lyse the hyphae of Gaumannomyces graminis var. Tritici, the causative agent of take-all disease of wheat. Fluorescent pseudomonads (P. fluorescens, P. putida) are known to produce iron chelating substances called Siderophores. These are low molecular weight, extra-cellular, iron-binding agents produced by pseudomonads in response to low iron stress or when Fe3+ is in short supply. Thus, iron stress triggers the formation of iron-binding ligands called siderophores. Siderophores contain the pigments Pyovirdin (Fluorescent) and Pyocyanin (non-Fluorescent) having iron chelating properties. Another pigment “Pseudobactin” is a fluorescent chelator of iron which is known to promote plant growth and inhibition of pathogenic bacteria in the rhizosphere. An antibiotic called “Pyrrolnitrin” reduces damping-off disease in cotton caused by Rhizoctonia solani. Several species of Bacillus are known to cause mycolysis in the rhizosphere. eg. Fusarium oxysporum hyphae are known to undergo lysis in soil due to these bacterial metabolites. The successful antagonists among fungi are Trichoderma sp (T. viride and T. harzianum, T. hamatum) and Gliocladium virens which parasitize, lyse or kill the phytopathogenic fungi in the soil. Antifungal and antibacterial actinomycetes in the rhizosphere play an important role in controlling pathogenic fungi and bacteria, for example, Micromonospora globosa is a potent antagonist of Fusarium udum causing wilt of pigeon pea. Amoebae are also known to play an antagonistic role in controlling soil fungi, eg. control of take-all disease of wheat caused by Gaumannomyces graminis through the use of Myxamoebae. There can also occur antagonisms between two fungi-producing metabolite and interfering the growth of the other fungus as in case of Peniophora antagonizing Heterobasidium.

2.1.12.5 Rhizosphere in relation to Plant Pathogens Plant root exudates influence pathogenic fungi, bacteria and nematodes in various ways. The effect may be in the form of attraction of fungal zoospores, or bacterial cells towards the roots; stimulation of germination of dormant spores and hatching of cysts of nematodes. Root exudates may contain inhibitory substances preventing the establishment of pathogens. The balance between the rhizosphere microflora and plant pathogens and soil microflora and plant pathogens is important in host-pathogenic relationship. In this context, the biochemical qualities of root exudates and the presence of antagonistic micro-organisms plays an important role in the proliferation and survival of root infecting pathogens in soil either through soil fungi stasis, inhibition or antibiosis of pathogens in the rhizosphere.

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Some of the most common interactions between plant roots and plant pathogenic microorganisms in the rhizosphere are as follows:

• Zoospore attraction: Amino acids, organic acids and sugars in the root exudates stimulate the movement and attraction of zoospores towards root of the plants. For example, attraction of zoospores has been reported in Phytophthora citrophthora (Citrus roots), P. parasitica (tobacco roots) and Pythium aphanidermatum (pea root).



• Spore germination: The spores or conidia of many pathogenic fungi such as Rhizoctonia, Fusarium, Sclerotium, Pythium, Phytophthora, etc. have been stimulated to germinate by the root exudates of susceptible cultivars of the host plants. There are some reports on the selective stimulation of Fusarium, Pseudomonas and root infecting nematodes in the rhizosphere region of the respective susceptible hosts. This stimulus to germination is especially important to those plant pathogens which are not vigorous competitors and remain in resting stage due to shortage of nutrients or fungistasis. As a rule, germination and subsequent hyphal development are promoted by non-host species and also by both susceptible and resistant cultivars of the host plants. The quantity and quality of microorganisms present in the rhizosphere of disease resistant crop varieties are significantly different from those of susceptible varieties.



• Changes in morphology and physiology of host plant: Changes in the physiology and morphology of host plant influence the rhizosphere microflora through root exudations. Hence, significant changes in the rhizosphere microflora of diseased plants were reported which are attributed to the nature and severity of the disease. Systemic virus diseases cause marked changes in the plant morphology and physiology to drastically alter the rhizosphere microflora.



• Increase in antagonists activity: Root exudates provide a food base for the growth of antagonistic organisms which plays an important role in controlling/suppressing some of the soil-born plant pathogens. Generally, rhizosphere of the resistant plant varieties harbour more number of Streptomyces and Trichoderma than that of susceptible varieties. For example, in the rhizosphere of pigeon pea varieties resistant to Fusarium udum, the population of Streptomyces was found more which inhibited the growth of the pathogen. High density of Trichoderma viride in the rhizosphere of tomato varieties resistant to Verticillium wilt has been reported with its ability to reduce the severity of wilt in susceptible plants.



• Inhibition of pathogen: Root exudates containing toxic substances such as glycosides and hydrocyanic acid may inhibit the growth of pathogens in the rhizosphere. It has been reported that root exudates from resistant varieties of Flax (eg. Bison) excrete a glucoside which on

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hydrolysis produces hydrocyanic acid that inhibits Fusarium oxysporum, the flax root pathogen. Exudates of resistant pea reduce the germination of spores of Fusarium oxysporum. In this light, the rhizosphere may be considered as a microbiological buffer zone in which the microflora serves to protect the plants against the attack of the pathogens.

• Attraction of bacteria and nematodes: Root exudates attracts phytopathogenic bacteria and fungi in the rhizosphere, for example, Agrobacterium tumefaciens have been reported to be attracted to the roots of the host plants like pea, maize, onion, tobacco, tomato and cucumber.

Host root exudates also influence phytopathogenic nematodes in two ways: (i) through stimulation of egg-hatching process and (ii) attraction of larvae towards plant roots.

2.1.13 Soil Microorganisms in Cycling of Elements or Plant Nutrient Soil microorganisms are the most important agents in the cycling/ transformation of various elements (N, P, K, S, Fe, etc.) in the biosphere; where the essential elements undergo cyclic alterations between the inorganic state as free elements in nature and the combined state in living organisms. Life on earth is dependent on the cycling of nutrient elements from their elemental states to inorganic compounds to organic compounds and back into their elemental states. The microbes through the process of biochemical reactions convert/breakdown complex organic compounds into simple inorganic compounds and finally into their constituent elements. This process is known as “Mineralization”. Mineralization of organic carbon, nitrogen, phosphorus, sulphur and iron by soil microorganisms makes these elements available for reuse by plants. In the following paragraphs, the cycling/transformations of some of the important elements are discussed. The four most important cycles are mention below:

• Nitrogen Cycle



• Sulphur Cycle/Sulphur Transformation



• Phosphorus Cycle/Transformation



• Iron Cycle/Transformation

2.1.13.1 Nitrogen Cycle Although molecular nitrogen (N2) is abundant (i.e 78-80 % by volume) in the earth’s atmosphere, but it is chemically inert and therefore, can not be utilized by most living organisms and plants. Plants, animals and most microorganisms, depend on a source of combined or fixed nitrogen (eg. ammonia, nitrate) or organic nitrogen compounds for their nutrition and growth. Plants require fixed nitrogen (ammonia, nitrate) provided by microorganisms, but about

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95 to 98 % soil nitrogen is in organic form (unavailable) which restricts the development of living organisms including plants and microorganisms. Therefore, cycling/transformation of nitrogen and nitrogenous compounds mediated by soil microorganisms is of paramount importance in supplying required forms of nitrogen to the plants and various nutritional classes of organisms in the biosphere. In nature, nitrogen exists in three different forms viz. gaseous/gas (78 to 80 % in atmosphere), organic (proteins and amino acids, chitins, nucleic acids and amino sugars) and inorganic (ammonia and nitrates). Biological N2 Fixation:

A. Symbiotic: Eg. Rhizobium (Eubacteria) legumes, Frankia (Actinomycete) and Anabaena (cyanobacteria) non-legumes. B. Non-symbiotic:

1. Free Living: e.g. Azobacter, Derxia, Bejerinkia, Rhodospirillum and BGA. 2. Associative: e.g. Azospirillum, Acetobacter, Herbaspirillim. Nutritional categories of N2 fixing Bacteria: A. Heterotrops B. Photoautotrophs

The sequence of biochemical changes from free atmospheric N2 to complex organic compounds in plant and animal tissues and further to simple inorganic compounds (ammonia, nitrate) and eventual release of molecular nitrogen (N2) back to the atmosphere is called “nitrogen cycle”. In this cycle, a part of atmospheric nitrogen (N2) is converted into ammonia and then to amino acids (by soil microorganisms and plant-microbe associations) which are used for the biosynthesis of complex nitrogencontaining organic compounds such as proteins, nucleic acids, amino sugars, etc. The proteins are then degraded to simpler organic compounds viz. peptones and peptides into amino acids, which are further degraded to inorganic nitrogen compounds like ammonia, nitrites and nitrates. The nitrate form of nitrogen is mostly used by plants or may be lost through leaching or reduced to gaseous nitrogen and subsequently goes into the atmosphere, thus completing the nitrogen cycle. Thus, the process of mineralization (conversion of organic form of nutrients to its mineral/inorganic form) and immobilization (process of conversion of mineral/inorganic form of nutrient elements into organic form) are continuously and simultaneously going on in the soil. Several biochemical steps involved in the nitrogen cycle are:

• Proteolysis



• Ammonification



• Nitrification

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• Nitrate reduction and



• Denitrification.

Proteolysis: Plants use the ammonia produced by symbiotic and nonsymbiotic nitrogen-fixation to make their amino acids and eventually, plant proteins. Animals eat the plants and convert plant proteins into animal proteins. Upon death, plant and animals undergo microbial decay in the soil and the nitrogen contained in their proteins is released. Thus, the process of enzymatic breakdown of proteins by the microorganisms with the help of proteolysis enzymes is known as “proteolysis”. The breakdown of proteins is completed in two stages. In first stage, proteins are converted into peptides or polypeptides by enzyme “proteinases” and, in the second stage, polypeptides/peptides are further broken down into amino acids by the enzyme “peptidases”. Proteins  → Peptides  → Amino Acids Proteinases

Peptidases

The amino acids produced may be utilized by other microorganisms for the synthesis of cellular components, absorbed by the plants through mycorrhiza or may be deanimated to yield ammonia. The most active microorganisms responsible for elaborating the proteolytic enzymes (Proteinases and Peptidases) are Pseudomonas, Bacillus, Proteus, Clostridium Histolyticum, Micrococcus, Alternaria, Penicillium, etc. Ammonification (Ammo acid degradation): Amino acids released during proteolysis undergo deamination in which nitrogen containing amino (–NH2) group is removed. Thus, process of deamination which leads to production of ammonia is termed as “ammonification”. The process of ammonification is mediated by several soil microorganisms. Ammonification usually occurs under aerobic conditions (known as oxidative deamination) with the liberation of ammonia (NH3) or ammonium ions (NH4) which are either released into the atmosphere or utilized by plants (paddy) and microorganisms, or still under favorable soil conditions, oxidized to form nitrites and then to nitrates. The processes of ammonification are commonly brought about by Clostridium sp. Micrococcus sp. Proteus sp., etc. and it is represented as follows.       

CH 3CHNH 2 COOH + 1 / 2O 2 de  → CH 3COCOOH + NH 3 min ase Alanine

Pyruvic acid

Ammonia

                                            

Nitrification: Ammonical nitrogen/ammonia released during ammonification are oxidized to nitrates and the process is called “nitrification”. Soil conditions such as well aerated soils rich in calcium carbonate, a temperature below 30°C, neutral pH, and less organic matter are favorable for nitrification in soil. Nitrification is a two stage process and each stage is performed by a different group of bacteria as follows.

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Stage I: Oxidation of ammonia of nitrite is brought about by ammonia oxidizing bacteria viz. Nitrosomnonas europaea, Nitrosococcus nitrosus, Nitrosospira briensis, Nitrosovibrio and Nitrocystis and the process is known as nitrosification. The reaction is presented as follows. 2 NH 3 + I / 2O 2 → NO 2 + 2H 2 + H 2 O Nitrite

Ammonia

Stage II: In the second step, nitrite is oxidized to nitrate by nitrite-oxidizing bacteria such as Nitrobacter winogradsky. Nitrospira gracilis, Nirosococcus mobiiis etc. and several fungi (e.g. Penicillium, Aspergillus) and actinomycetes (e.g. Streptomyces, Nocardia). NO(2− ) +

1 O 2 → NO(3− ) 2 Nitrate ions

Nitrite ions

The nitrate, thus formed, may be utilized by the microorganisms, assimilated by plants, reduced to nitrite and ammonia or nitrogen gas or lost through leaching depending on soil conditions. The nitrifying bacteria (ammonia oxidizer and nitrite oxidizer) are aerobic gram-negative and chemoautotrophic and are the common inhabitants of soil, sewage and aquatic environment. Nitrate Reduction: Several heterotrophic bacteria (E. coli, Azospirillum) are capable of converting nitrates to nitrites and nitrites to ammonia. Thus, the process of nitrification is reversed completely which is known as nitrate reduction. Nitrate reduction normally occurs under anaerobic soil conditions (waterlogged soils) and the overall process is as follows: Nitrate HNO 3 + 4H 2 Reductase  → NH 3 + 3H 2 O Nitrate

Ammonia

Nitrate reduction leading to production of ammonia is called “dissimilatory nitrate reduction” as some of the microorganisms assimilate ammonium for synthesis of proteins and amino acid. Denitrification: This is the reverse process of nitrification. During denitrification, nitrates are reduced to nitrites and then to nitrogen gas and ammonia. Thus, reduction of nitrates to gaseous nitrogen by microorganisms in a series of biochemical reactions is called “denitrification”. The process is wasteful, as available nitrogen in soil is lost to the atmosphere. The overall process of denitrification is as follows: NaR

NIR

NOR

Nitrate  → Nitrite  → Nitric Oxide → NoR

→ Nitrogen gas Nitrous Oxide  This process also called dissimilatory nitrate reduction as nitrate nitrogen is completely lost into atmospheric air. In the soils with high organic matter and anaerobic soil conditions (waterlogged or ill-drained), rate of denitrification is more. Thus, rice/paddy fields are more prone to denitrification.

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The most important denitrifying bacteria are Thiobacillus denitrificans, Micrococcus denitrificans, and species of Pseudomonas, Bacillus, Achromobacter, Serrtatia paracoccus, etc. Denitrification leads to the loss of nitrogen (nitrate nitrogen) from the soil which results into the depletion of an essential nutrient for plant growth and therefore, it is an undesirable process/reaction from the soil fertility and agricultural productivity. Although, denitrification is an undesirable reaction from agricultural productivity, but it is of major ecological importance since, without denitrification the supply of nitrogen including N2 of the atmosphere, would have not got depleted and NO3 (which are toxic) would have accumulated in the soil and water.

2.1.13.2 Sulphur Cycle/Sulphur Transformations Sulphur is the most abundant and widely distributed element in the nature and found both in free as well as combined states. Sulphur, like nitrogen is an essential element for all living systems. In the soil, sulphur is in the organic form (sulphur containing amino acids—cystine, methionine, proteins, polypeptides, biotin, thiamine, etc.) which is metabolized by soil microorganisms to make it available in an inorganic form (sulphur, sulphates, sulphite, thiosulphale, etc.) for plant nutrition. Of the total sulphur present is soil, only 10-15% is in the inorganic form (sulphate) and about 75-90% is in organic form. Cycling of sulphur is similar to that of nitrogen. Transformation/ cycling of sulphur between organic and elemental states and between oxidized and reduced states, is brought about by various microorganisms, especially bacteria. Thus, “the conversion of organically bound sulphur to the inorganic state by microorganisms is termed as mineralization of sulphur”. The sulphur/ sulphate, thus released are either absorbed by the plants or escape to the atmosphere in the form of oxides. Various transformations of the sulphur in soil result mainly due to microbial activity, although some chemical transformations are also possible (eg. oxidation of iron sulphide). The major types of transformations involved in the cycling of sulphur are: • Mineralization • Immobilization • Oxidation and • Reduction • Mineralization: The breakdown/decomposition of large organic sulphur compounds to smaller units and their conversion into inorganic compounds (sulphates) by the microorganisms. The rate of sulphur mineralization is about 1.0 to 10.0 percent/year. • Immobilization: Microbial conversion of inorganic sulphur compounds to organic sulphur compounds. • Oxidation: Oxidation of elemental sulphur and inorganic sulphur compounds (such as H2S, sulphite and thiosulphate) to sulphate (SO4) is brought about by chemoautotrophic and photosynthetic bacteria.

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When plant and animal proteins are degraded, the sulphur is released from the amino acids and accumulates in the soil which is then oxidized to sulphates in the presence of oxygen, and under anaerobic condition (water logged soils), organic sulphur is decomposed to produce hydrogen sulphide (H2S). H2S can also accumulate during the reduction of sulphates under anaerobic conditions which can be further oxidized to sulphates under aerobic conditions. Ionization

(a ) 2S + 3O 2 + 2H 2 O Light → 2H 2 SO 4  → 2H( + ) + SO 4 ( Aerobic) ( b) CO 2 + 2H 2 S Light →(CH 2 O) + H 2 O + 2S OR H 2 + S + 2CO 2 + H 2 O  → H 2 SO 4 + 2(CH 2 O)(anaerobic) The members of genus Thiobacillus (obligate chemolithotrophic, nonphotosynthetic) eg, T. ferrooxidans and T. thiooxidans are the main organisms involved in the oxidation of elemental sulphur to sulphates. These are aerobic, non-filamentous, chemosynthetic autotrophs. Other than Thiobacillus, heterotrophic bacteria (Bacillus, Pseudomonas, and Arthrobacter) and fungi (Aspergillus, Penicillium), some actinomycetes are also reported to oxidize sulphur compounds. Green and purple bacteria (Photolithotrophs) of genera Chlorbium, Chromatium, Rhodopseudomonas are also reported to oxidize sulphur in aquatic environment. Sulphuric acid produced during oxidation of sulphur and H:S is of great significance in reducing the pH of alkaline soils and in controlling potato scab and rot diseases caused by Streptomyces bacteria. The formation of sulphate/ Sulphuric acid is beneficial in agriculture in different ways : (i) as it is the anion of strong mineral acid (H2SO4) itcan render alkali soils fit for cultivation by correcting soil pH. (ii) solubilize inorganic salts containing plant nutrients and thereby increase the level of soluble phosphate, potassium, calcium, magnesium, etc. for plant nutrition. Reduction of Sulphate: Sulphate in the soil is assimilated by plants and microorganisms and incorporated into proteins. This is known as “assimilatory sulphate reduction”. Sulphate can be reduced to hydrogen sulphide (H2S) by sulphatereducing bacteria (eg. Desulfovibrio and Desulfatomaculum) and may diminish the availability of sulphur for plant nutrition. This is “dissimilatory sulphate reduction” which is not at all desirable from soil fertility and agricultural productivity viewpoint. Dissimilatory sulphate-reduction is favored by the alkaline and anaerobic conditions of soil and sulphates are reduced to hydrogen sulphide. For example, calcium sulphate is attacked under anaerobic condition by the members of the genus Desulfovibrio and Desulfatomaculum to release H2 S. CaSO 4 + 4H 2  → Ca(OH)2 + H 2 S + 2H 2 O

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Hydrogen sulphide produced by the reduction of sulphate and sulphur containing amino acids decomposition is further oxidized by some species of green and purple phototrophic bacteria (e.g. Chlorobium, Chromatium) to release elemental sulphur. Light

CO 2 + 2H 2 + H 2 S Enzyme  → (CH 2 O) + H 2 O + 2S Carbohydrate

The predominant sulphate-reducing bacterial, genera in soil are Desulfovibrio, Desulfatomaculum and Desulfomonas (all obligate anaerobes). Amongst these species, Desulfovibrio desulfuricans are most ubiquitous, nonspore forming, obligate anaerobes that reduce sulphates at rapid rate in waterlogged/flooded soils. While species of Desulfatomaculum are sporeforming, thermophilic obligate anaerobes reduce sulphates in dry land soils. All sulphate-reducing bacteria excrete an enzyme called “desulfurases” or “bisulphate Reductase”. Rate of sulphate reduction in nature is enhanced by increasing water levels (flooding), high organic matter content and increased temperature.

2.1.13.3  Phosphorus Cycle or Transformation Phosphorus is only second to nitrogen as a mineral nutrient required for plants, animals and microorganisms. It is a major constituent of nucleic acids in all living systems essential in the accumulation and release of energy during cellular metabolism. This element is added to the soil in the form of chemical fertilizers, or in the form of organic phosphates present in plant and animal residues. In cultivated soils, it is present in abundance (i.e. 1100 kg/ha), but most of this is not available to plants; only 15 % of total soil phosphorus is in available form. Both inorganic and organic phosphates exist in soil and occupy a critical position both in plant growth and in the biology of soil. Microorganisms are known to bring a number of transformations of phosphorus, these include:

• Altering the solubility of inorganic compounds of phosphorus,



• Mineralization of organic phosphate compounds into inorganic phosphates,



• Conversion of inorganic, available anion into cell components i.e. an immobilization process and



• Oxidation or reduction of inorganic phosphorus compounds. Of these, mineralization and immobilization are the most important reactions/ processes in phosphorus cycle.

Insoluble inorganic compounds of phosphorus are unavailable to plants, but many microorganisms can bring the phosphate into solution. Soil phosphates are rendered available either by plant roots or by soil microorganisms through secretion of organic acids (e.g. lactic, acetic, formic, fumaric, succinic

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acids, etc.). Thus, phosphate-dissolving/solubilizing soil microorganisms (e.g. species of Pseudomonas, Bacillus, Micrococcus, Mycobacterium, Flavobacterium, Penicillium, Aspergillus, Fusarium, etc.) play important role in correcting phosphorus deficiency of crop plants. They may also release soluble inorganic phosphate (H2PO4) into soil through decomposition of phosphaterich organic compounds. Solubilization of phosphate by plant roots and soil microorganisms is substantially influenced by various soil factors, such as pH, moisture and aeration. In neutral or alkaline soils, solubilization of phosphate is more as compared to acidic soils. Many phosphate-solubilizing microorganisms are found in close proximity of root surfaces and may appreciably enhance phosphate assimilation by higher plants. By their action, fungi, bacteria and actinomycetes make available the organically bound phosphorus in soil and organic matter and the process is known as mineralization. On the other hand, certain microorganisms, especially bacteria, assimilate soluble phosphate and use it for cell synthesis and on the death of bacteria, the phosphate is made available to plants. A fraction of phosphate is also lost in soil due to leaching. One of the ways to correct deficiency of phosphorus in plants is to inoculate seed or soil with commercial preparations (e.g. Phosphobacterin) containing phosphatesolubilizing microorganisms along with phosphatic fertilizers. Mineralization of phosphate is generally rapid and is more in virgin soils than cultivated land. Mineralization is favored by high temperatures (thermophilic range) and is more in acidic to neutral soils with high organic phosphorus content. The enzymes involved in mineralization (cleavage) of phosphate from organic phosphorus compound are collectively called as “Phospatases”. The commercially used species of phosphate-solubilizing bacteria and fungi are: Bacillus polymyxa, Bacillus megatherium, Pseudomonas strita, Aspergillus, Penicllium avamori and Mycorrhiza.

2.1.13.4 Iron Cycle or Transformation Iron exists in nature either as ferrous (Fe++) or ferric (Fe+++) ions. Ferrous iron is oxidized spontaneously to ferric state, forming highly insoluble ferric hydroxide. Plants as well as microorganisms require traces of iron, manganese copper, zinc, molybdenum, calcium, boron, cobalt etc. Iron is always abundant in terrestrial habitats, and it is oftenly in an unavailable form for utilization by plants and leads to the serious deficiency in plants. Soil microorganisms play important role in the transformations of iron in a number of distinctly different ways such as: A. Certain bacteria oxidize ferrous iron to ferric state which precipitate as ferric hydroxide around cells.

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B. Many heterotrophic species attack on insoluble organic iron salts and convert into inorganic salts. C. Oxidation-reduction potential decreases with microbial growth which leads to the formation of more soluble ferrous iron from highly insoluble ferric ions. D. Number of bacteria and fungi produce acids such as carbonic, nitric, sulphuric and organic acids which brings iron into solution. E. Under anaerobic conditions, the sulfides formed from sulphate and organic sulphur compounds, remove the iron from solution as ferrous sulfide. F. As microbes liberate organic acids and other carbonaceous products of metabolism, this results in the formation of soluble organic iron complex. Thus, iron may be precipitated in nature and immobilized by iron oxidizing bacteria under alkaline soil reaction and on the other hand, solubilization of iron may occur through acid formation. Some bacteria are capable of reducing ferric iron to ferrous, which lowers the oxidation-reduction potential of the environment (eg. Bacillus, Clostridium, Klebsiella, etc). However, some chemoautotrophic iron and sulphur bacteria such as ThiobacillusT. Ferroxiders and Ferrobacillus ferrooxidans can oxidize ferrous iron to ferric hydroxide which accumulates around the cells. Most of the aerobic microorganisms live in an environment where iron exists in the oxidized, insoluble ferric hydroxide form. They produce ironbinding compounds in order to take up ferric iron. The iron-binding or chelating compounds/ligands produced by microorganisms are called “Siderophores”. Bacterial siderophores may act as virulence factors in pathogenic bacteria and thus, bacteria that secrete siderophores are more virulent than nonsiderophores producers. Therefore, siderophore-producing bacteria can be used as biocontrol agents eg. Fluorescent pseudomonads are used to control Pythium, causing damping-off diseases in seedlings. Recently Vascular– Arbusecular–Mycorrhiza (VAM) has been reported to increase uptake of iron.

2.1.14 Organic Matter Soil organic matter plays important role in the maintenance and improvement of soil properties. It is a dynamic material and is one of the major sources of nutrient elements for plants. Soil organic matter is derived to a large extent from residues and remains of the plants together with the small quantities of animal remains, excreta, and microbial tissues. Soil organic matter is composed of three major components i.e. plants residues, animal remains and dead remains of microorganisms. Various organic compounds are made up of complex carbohydrates (cellulose, hemicellulose, starch), simple sugars, lignins, pectins, gums, mucilages, proteins, fats, oils, waxes, resins, alcohols, organic acids, phenols, etc. and other products. All these compounds constituting the soil organic matter can be categorized in the following way.

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Organic Matter (Undecomposed) A. Organic: • Nitrogenous: 1. Water Soluble e.g. nitrates, ammonical compounds, amides, amino acids, etc. 2. Insoluble e.g. proteins, nucleoproteins, peptides, alkaloids purines, pyridines, chitin, etc. • Non Nitrogenous: — Carbohydrates e.g. sugars, starch, hemicellulose, gums, mucilage, pectins, etc. — Micellaneous: e.g. lignin, tannins, organic acids, etc. — Ether solube: e.g. fats, oils, waxes etc. B. Inorganic   The organic complex/matter in the soil is, therefore made up of a large number of substances of widely different chemical composition and the amount of each substance varies with the type, nature and age of plants. For example, cellulose in a young plant is only half of the mature plants; watersoluble organic substances in young plants are nearly double to that of older plants. Among the plant residues, leguminous plants are rich in proteins than the non-leguminous plants. Grasses and cereal-straws contain greater amount of cellulose, lignin, hemicelluloses than the legumes, and as the plant gets older, the proportion of cellulose, hemicelluloses and lignin gets increased. Plant residues contain 15-60% cellulose, 10-30 % hemicelluslose, 5-30% lignin, 2-15 % protein and 10% sugars, amino acids and organic acids. These differences in composition of various plant and animal residues have great significance on the rate of organic matter decomposition, in general, and of nitrification and humification (humus formation), in particular. The end products of decomposition are CO2, H2O, NO3, SO4, CH4, NH4, and H2S depending on the availability of air.

Factors Influencing rate of Organic Matter Decomposition In addition to the composition of organic matter, nature and abundance of microorganisms in soil, the extent of C, N, P and K, moisture content of the soil and its temperature, pH, aeration, C:N ratio of plant residues and presence/ absence of inhibitory substances (e.g. tannins), etc. are some of the major factors which influence the rate of organic matter decomposition. As soon as plant and animal residues are added to the soil, there is a rapid increase in the activity of microorganisms. These are not true soil organisms, but they continue their activity by taking part in the decomposition of organic matter, and thereby release plant nutrients in the soil. Bacteria are the most abundant organisms playing important role in the decomposition of organic matter. Majority of bacteria involved in decomposition of organic matter are

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heterotrophs and autotrophs are least in proportion which are not directly involved in organic matter decomposition. Actinomycetes and fungi are also found to play important role in the decomposition of organic matter. Soil algae may contribute a small amount of organic matter through their biomass but they do not have any active role in organic matter decomposition. The various microorganisms involved in the decomposition of organic matter are listed in the following table. List of Microorganisms involved in organic matter decomposition Constituents

Microorganisms Bacteria

Fungi

Actinomycetes

Cellulose

Achromobacter, Bacillus,  Cellulomonas, Cellvibrio, Clostridium, Cytophaga, Vibrio Pseudomonas, Sporocytophaga, etc.

Aspergillus, Chaetomium, Fusarium, Pencillium Rhizoctonia, Rhizopus, Trichoderma, Verticilltttm

Micromonospora, Nocardia, Streptomyces, Thermonospora

Hemicellulose

Bacillus, Achromobacter, Cytophaga Pseudomonas, Erwinia, Vibrio, Lactobacillus

Aspergillus, Fusarium, Chaetomium, Penicillium, Trichoderma, Humicola

Streptomyces, Actinomycetes

Lignin

Flavobacterium, Pseudomonas, Micrococcus, Arthorbacter, Xanthomonas

Humicola, Fusarium Fames, Pencillium, Aspergillus, Ganoderma

Streptomyces, Nocardia

Starch

Achromobacter, Bacillus, Clostridium

Ftisarium, Fomes, Aspergillus, Rhizopus

Micromonospora, Nocardia, Streptomyces

Pectin

Bacillus, Clostridium, Pseudomonas

Ftisarium, Verticillum

 

Chitin

Bacillus, Achromobacter, Cytophaga, Pseudomonas

Mucor, Fusarium, Aspergillus, Trichoderma

Streptomyces, Nocardia, Micromanospora

Proteins & Nucleic acids

Bacillus, Pseudomonas, Clostriddum, Serratia, Micrococcus

Penicillium, Rhodotorula

Streptomyces

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Aeration: Good aeration is necessary for the proper activity of the microorganisms involved in the decomposition of organic matter. Under anaerobic conditions, fungi and actinomycetes are almost suppressed and only a few bacteria (Clostridium) take part in anaerobic decomposition. The rate of decomposition is markedly retarded. It was found that under aerobic conditions, 65 per cent of the total organic matter decomposes during six months, while under anaerobic conditions only 47 per cent organic matter can be decomposed during the same period. Anaerobic decomposition of organic matter results into production of large quantity of organic acids and evolution of gases like methane (CH4) hydrogen (H2) and carbon dioxide (CO2). Temperature: The rate of decomposition is more rapid in the temperature range of 30° to 40°. At temperatures below or above this range, the rate of decomposition is markedly retarded. Appreciable organic matter decomposition occurs at 25°C and further fluctuation in the soil temperature has little effect on decomposition. Moisture: Adequate soil moisture i.e. about 60 to 80 per cent of the water-holding capacity of the soil is must for the proper decomposition of organic matter. Too much moisture leads to insufficient aeration which results in the reduced activity of microorganisms and thereby, checks the rate of decomposition. Soil pH/soil reaction: Soil pH affects directly the kind, density and the activity of fungi, bacteria and actinomycetes involved in the process of decomposition and thereby rate of decomposition of organic matter. The rate of decomposition is more in neutral soils than that of acidic soils. Therefore, treatment of acid soils with lime can accelerate the rate of organic matter decomposition. C:N ratio: C:N ration of organic matter has great influence on the rate of decomposition. Organic matter from diverse plant-tissues varies widely in their C:N ratio (app. 8-10 %). The optimum C:N ratio in the range of 20-25 is ideal for maximum decomposition, since a favorable soil environment is created to bring about equilibrium between mineralization and immobilization processes. Thus, a low nitrogen content or wide C:N ratio results into slow decomposition. Protein rich, young and succulent plant tissues are decomposed more rapidly than the protein-poor, mature and hard plant tissues. Therefore, C:N ratio of organic matter as well as soil should be narrow/less for better and rapid decomposition. Thus, high aeration, mesophilic temperature range, optimum moisture, neutral/alkaline soil reaction and narrow C:N ratio of soil and organic matter are required for rapid and better decomposition of organic matter.

2.1.14.1 Microbiology of decomposition of various constituents in organic matter When plant and animal residues are added to the soil, the various constituents of the soil organic matter are decomposed simultaneously by the activity of

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microorganisms and carbon is released as CO2, and nitrogen as NH4 →NO3 , for the use by plants. Other nutrients are also converted into plant usable forms. This process of release of nutrients from organic matter is called mineralization. The insoluble plant residues constitute the part of humus and soil organic matter complex. The final product of aerobic decomposition is CO2 and that of anaerobic decomposition are hydrogen, ethyl alcohol (C2H5OH), various organic acids and carbon dioxide (CO2). Soil organisms use organic matter as a source of energy and food. The process of decomposition is initially fast, but slows down considerably as the supply of readily decomposable organic matter gets exhausted. Sugars, water-soluble nitrogenous compounds, amino acids, lipids, starches and some of the hemicellulases are decomposed first at rapid rate, while insoluble compounds, such as cellulose, hemicellulose, lignin, proteins, etc., which form the major portion of organic matter are decomposed later, slowly. Thus, the organic matter added to the soil is converted by oxidative decomposition to simpler substances which are made available in stages for plant growth and the residue is transformed into humus. The microbiology of decomposition/degradation of some of the major constituents (viz. cellulose, hemicellulose, lignin, proteins, etc.) of soil organic matter/plant residues are discussed in brief in the following paragraphs. Decomposition of Cellulose:  Cellulose is the most abundant carbohydrate present in plant residues/organic matter in nature. When cellulose is associated with pentosans (eg. xylans & mannans), it undergoes rapid decomposition, but when associated with lignin, the rate of decomposition is very slow. The decomposition of cellulose occurs in two stages: (i) in the first stage, the long chain of cellulase is broken down into cellobiose and then into glucose by the process of hydrolysis in the presence of enzymes cellulase and cellobiase, and (ii) in the second stage into glucose is oxidized and converted CO2 and water. Cellulose

Cellobiase

1. Cellulose  → Cellobiose  → Glucose Hydrolysis Hydrolysis Oxidation

Oxidation

2. Glucose → Organic Acids → CO 2 + H 2 O The intermediate products formed/released during enzymatic hydrolysis of cellulose (eg. cellobiose and glucose) are utilized by the cellulosedecomposing organisms or by other organisms as source of energy for biosynthetic processes. The cellulolytic microorganisms responsible for degradation of cellulose through the excretion of enzymes (cellulase & cellobiase) are fungi, bacteria and actinomycetes. Decomposition of Hemicelluloses: Hemicelluloses are water-soluble polysaccharides and consist of hexoses, pentoses, and uronic acids and are the major plant constituents second only in quantity to cellulose, and sources of energy and nutrients for soil microflora. When subjected to microbial decomposition, hemicelluloses degrade initially at faster rate and are first hydrolyzed to their component sugars and

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uronic acids. The hydrolysis is brought about by number of hemicellulolytic enzymes known as “hemicellulases” excreted by the microorganisms. On hydrolysis, hemicelluloses are converted into soluble monosaccharides/sugars (e.g. xylose, arabinose, galactose and mannose) which are further converted to organic acids, alcohols, CO2 and H2O and uronic acids are broken down to pentoses and CO2. Various microorganisms including fungi, bacteria and actinomycetes, both aerobic and anaerobic, are involved in the decomposition of hemicelluloses. Lignin Decomposition: Lignin is the third most abundant constituent of plant tissues, and accounts about 10-30 per cent of the dry matter of mature plant materials. Lignin content of young plants is low and gradually increases as the plant grows old. It is one of the most resistant organic substances for the microorganisms to degrade, however, certain Basidiomycetous fungi are known to degrade lignin at slow rates. Complete oxidation of lignin result in the formation of aromatic compounds such as syringaldehydes, vanillin and ferulic acid. The final cleavages of these aromatic compounds yield organic acids, carbon dioxide, methane and water. Protein Decomposition: Proteins are complex organic substances containing nitrogen, sulphur, and sometimes phosphorus in addition to carbon, hydrogen and oxygen. During the course of decomposition of organic matter, proteins are first hydrolyzed to a number of intermediate products, e.g. proteases, peptides etc. collectively known as polypeptides The intermediate products so formed are then hydrolyzed and broken down ultimately to individual amino acids, or ammonia and amides. The process of hydrolysis of proteins to amino acids is known as “aminization or ammonification”, which is brought about by certain enzymes, collectively known as “proteases” or “proteolytic” enzymes secreted by various microorganisms. Amino acids and amines are further decomposed and converted into ammonia. During the course of ammonification, various organic acids, alcohols, aldehydes, etc. are produced which are further decomposed finally to produce carbon dioxide and water. All types of microorganisms, bacteria, fungi, and actinomycetes are able to bring about decomposition of proteins. In acid soils, fungi are predominant, while in neutral and alkaline soils, bacteria are dominant decomposers of proteins.

2.1.15 Notable contributions made by several scientists in the field of soil microbiology There is enough evidence in the literature to believe that microorganisms were the earliest of the living things that existed on this planet. Man depends on crop plants for his existence and crop plants in turn depend on soil and soil microorganisms for their nutrition. Scientists form the beginning studied the microorganisms from water, air, soil, etc. and recognized the

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role of microorganisms in natural processes and realized the importance of soil microorganisms in growth and development of plants. Thus, we see that microorganisms have been playing a significant role, long before they were discovered by man. Today, soil is considered to be the main source of scavenging the organic wastes through microbial action and is also a rich storehouse for industrial microflora of great economic importance. Unlike soil science whose origin can be traced back to Roman and Aryan times, soil microbiology emerged as a distinct branch of soil science during the first half of the 19th century. Some of the notable contributions made by several scientists in the field of soil microbiology are highlighted in the following paragraphs.

• V. Leeuwenhock (1673) discovered and described microorganisms through his self-made first simple microscope with magnification of 200 to 300 times. He observed minute, moving objects which he called “animalcules” (small animals) which are now known as protozoa, fungi and bacteria. He, for the first time, made the authentic drawings of microorganisms (protozoa, bacteria, fungi).



• Robert Hook (1635-1703) developed a compound microscope with multiple lenses and described the fascinating world of the microbes.



• J. B. Boussingault (1838) showed that leguminous plants can fix atmospheric nitrogen and increase nitrogen content in the soil.



• J. Von Liebig (1856) showed that nitrates were formed in soil due to addition of nitrogenous fertilizers in soil.



• S. N. Winogradsky discovered the autotrophic mode of life among bacteria and established the microbiological transformation of nitrogen and sulphur. He isolated for the first time nitrifying bacteria and demonstrated role of these bacteria in nitrification (l890); further he demonstrated that free-living Clostridium pasteuriamum could fix atmospheric nitrogen (1893). Therefore, he is considered as “Father of soil microbiology”.



• W. B. Leismaan (1858) and M. S. Woronin (1866) demonstrated that root nodules in legumes were formed by a specific group of bacteria.



• Jodin (1862, France) gave the first experimental evidence of elemental nitrogen fixation by microorganisms.



• Robert Koch (1882) developed gelatin plate/streak plate technique for isolation of specific type of bacteria in soil, formulated Koch’s postulates to establish causal relationship between host-pathogen and disease.



• R. Warington (1878) showed that nitrification in soil was a microbial process.



• B. Frank (i) discovered (1880) an actinomycetes “Frankia” (Actinorhizal symbiosis) inducing root nodules in non-legumes tress of genera Alnus sp. and Casurina growing in temperate forests, (ii) coined (1885) the term

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“ Mycorrhiza” to denote association of certain fungal symbionts with plant roots (Mycorrhiza - A symbiotic association between a fungus and roots of higher plants. Renamed the genus Bacillus as Rhizobium (1889). • H. Hellriegel and H. Wilfarth (1886) showed that the growth of nonlegume plants was directly proportional to the amount of nitrogen supplied, whereas, in legumes there was no relationship between the quantity of nitrogen supplied and extent of plant growth. They also suggested that bacteria in the root nodules of legumes accumulate atmospheric nitrogen and make it available to plants. Showed that a mutually beneficial association exists between bacteria (Rhizobia) and legume root, and legumes could utilize atmospheric nitrogen (1988). • M. W. Beijerinck (1888) isolated root nodule bacteria in pure culture from nodules in legumes and named them as Bacillus radicola. Considered as father of “Microbial ecology”. He was the first Director of the Delft School of Microbiology (Netherland). • Beijerinck and Winogradsky (1890) developed the enrichment culture technique for isolation of soil organisms, proved independently that transformation of nitrogen in nature is largely due to the activities of various groups of soil microorganisms (1891). Therefore, they are considered as “Pioneers in soil bacteriology”. • S. N. Winogadsky (1891) demonstrated the role of bacteria in nitrification, and further in fill 1983 demonstrated that free living Clostridium pasteurianum could fix atmospheric nitrogen. • Omeliansky (1902) found the anaerobic degradation of cellulose by soil bacteria. • J. G. Lipman and P. E. Brown (1903, USA) studied ammonification of organic nitrogenous substances by soil microorganisms and developed the Tumbler or Beaker for studying different types of transformation in soil. • Hiltner (Germany, 1904) coined the term “Rhizosphere” to denote that region of soil which is subjected to the influence of plant roots. Rhizosphere is the region where soil and plant roots make contact. • Russel and Hutchinson (1909, England) proved the importance of protozoa controlling/maintaining bacterial population and their activity in soil. • Conn (1918) developed “Direct soil examination” technique for studying soil microorganisms. • Rayner (192I) and Melin (1927) carried out intensive study on Mycorrhiza. • S. A. Waksman published the book “Principles of soil Microbiology” and thereby encouraged research in soil microbiology (1927). Studied

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the role of soil as the source of antagonistic organisms with special reference to soil actinomycetes (1942) and discovered the antibiotic “Streptomycin” produced by Streptomyces griseus, a soil actinomycets (1944). • Rossi (1929) and Cholondy (1930) developed “Contact Slide/Buried Slide” technique for studying soil microflora. • Van Niel (1931) studied chemoautotrophic bacteria and bacterial photosynthesis. • Bortels (1936) demonstrated the importance of molybdenum in accelerating nitrogen fixation by nodulating legumes. • Garrett (1936) established school in UK on “Soil fungi and ecological classification”. • Kubo (1939, Japan) showed/proved the role and importance of “leghaemoglobin” (Red pigment) present in root nodules of legumes in nitrogen fixation. • Ruinen (1956), Dutch microbiologist, coined the term “Phyllosphere” to denote the region of leaf influenced by microorganisms. • Alien (1980) suggested that, VAM fungi stimulate plant growth by physiological effects other than by enhancement of nutrient uptake. • Jensen (1942) developed the method of studying nodulation on agar media in test tubes. • Barbara Mosse and J. W. Gerdemann (1944) reported occurrence of VAM (vesicular-arbuscular Mycorrhiza) fungi (Glomus, Aculopora genera) in the roots of agricultural crop plants which help in the mobilization of phosphate. • Starkey (1945) studied role of bacteria (Bacillus and Clostridium) in the transformation of iron. • Barker (1945) studied anaerobic fermentation by methane bacteria (Methanococcus, Methanosarcina). • Thornton (1947) studied root nodule bacteria forming clovers. • Virtanen (1947) studied chemistry and mechanism of leghaemoglobin in nitrogen fixation. • Nutman (1948, England) studied hereditary mechanism of root nodulation in legumes. • Burris and Wilson (1957) developed the “Isotope technique” to quantify the amount of nitrogen fixed and further isolated and characterized the enzyme “Nitrogenase”. • Bergersen (1957, Australia) elaborated the biochemistry of nitrogen fixation in legume root nodules. • Carnham (1960, USA) discovered nitrogen fixation by cell-free extract of Clostridium pasteurianum.

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• Alexander Fleming started the “School of soil microbiology” at Cornell University to study microbial aspects of pesticides degradation (1961) and developed the antibiotic “Penicillin” from the fungus Penicillium notatum (1929).



• Date, Brockwell and Roughley (1962, Australia) developed the technique of bio-inoculants production & seed application.



• Hardy & Associates (1968, USA) developed the technique of measurement of nitrogenase activity by acetylene-reduction test coupled with gas chromatography and thereby, estimation of biological nitrogen fixation.



• R J Swaby (1970, Australia) developed “Biosuper”, containing rock phosphate sulphur, and Thiobacillus which was used to enhance the phosphorus nutrition of plants.



• Foog and Stewart (1970, UK) intensified the work on N2 fixing bluegreen algae.



• Trinick (1973, Australia) isolated Rhizobia from root nodule of genus Trema (Parasponia) which was an unique association of Rhizobium with non-leguminous plants causing root nodulation.



• Dobereiner and associates (1975, Brazil) studied nitrogen fixing potential of Azospirillum in some tropical forage grasses like Digitaria, Panicum and some cereals like maize, sorghum, wheat, rye etc. in their roots. He reported four species of Azospirillum viz. A. lipoferum, A. brasilense, A. amazonense and A. serpedica. He coined the term “Associative Symbiosis” to denote the association between nitrogen fixing Azospirillum and cereal roots. Recently this terminology has been changed and renamed as “Diazotrophic Biocoenocis”.



• Challham and Associates (1978) isolated an actinomycetous endophyte Frankia sp. from root nodules of Camptonia peregrina which is again an example of non-leguminous root nodulation.



• Dommergues & associates (France and Senegal) had discovered/ reported nodules on stem of Sesbania rostrata which could fix nitrogen, and therefore, this legume can be used as an excellent green manure crop in low land rice cultivation. Similarly they also discovered N2 fixing stem nodules on Casurina sp. caused by Frankia, an actinomycete.



• Louis Pasteur proved the role of soil microorganisms in biochemical changes of elements. He also showed that decomposition of organic residues in soil was dependent on the nature of organic matter and environmental conditions.



• Brefeld introduced the practice of isolating soil fungi by “Single Cell” technique and cultivating/growing them on solid media. He used gelatin (first solidifying agent) in culture media as solidifying agent.

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• Gerretsen & Mulder (Holland) studied “Phosphate mobilization” by soil microorganisms and showed the importance of molybdenum in nitrogen metabolism by microorganisms. • Fritch, Fogg & Stewart (UK) and lyengar (India) studied fixation by algae, in general and micro algae, in particular. They also intensified the work on N2 fixing BGA. • James Trappe and Don Marx worked on ectomycorrhiza, colonizing the roots of forest trees. • W. S. Cook, G. C. Papavizas, J. Baker and N.S. Kerr contributed to the field of biological control of plant pathogens using antagonistic organisms from soil. From the beginning of 20th century, emphasis was given to the study of microorganisms in soil in relation to their physiology, ecology, interrelationship, role in soil processes and soil fertility. Further role of fungi and actinomycetes in cellulosedecomposition was better understood and cellulose decomposing, sulphur-oxidizing, iron bacteria, etc. were isolated from soil and studied in detail.

2.1.16

Notable contributions made by Indian scientists in the field of soil microbiology During the last few decades, greater emphasis has been given on some of the important aspects in soil microbiology in India, which are: • Characterization of N2–fixing Azotobacter, Rhizobium, Beijerinckia, BGA, etc. • Studieson P-solubilizing bacteria and fungi, celluloytic microorganisms, silage production role of humic acid, etc. • Establishment (1979) of All India Coordinated Project (AICP) on BNF at IARI and field-oriented work on BNF. • Standardization of methods of bio-inoculants application to seed and soil. • Seed bacterization and response of crops to bio-inoculants.  Some of the most important contributions made on the different aspects in the field of soil microbiology by the scientists and research institutes in the country are as follows: • C. N. Acharya (1940) contributed towards the better utilization of agricultural wastes for the production of biogas & compost. • Sundara Rao (1962) established the “Division of Microbiology” at IARI, New Delhi. • Madhok (Punjab) introduced the practice of using bacterial cultures for berseem. • Sanyasi Raju & Rajagopalan (Coimbatore) initiated the research work on root nodulation in legumes at Madras, Agil. College.

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• P. K. Dey (West Bengal) worked on free-living N2 fixing organisms viz Azotobacter, Beijerinckia and BGA in rice fields and discovered N2 fixation by BGA in paddy. • M.O.P. lyengar (Madras Univ.) laid foundation stone for algal research in India. • Sadasivan (Madras) and Saxena (Allahabad) studied ecology and physiology of soil fungi along with rhizosphere phenomenon. • Singh B.N. did pioneering research on soil protozoa in India. • Bhar J. V. (Bangalore) initiated work on the role of earthworms in the maintenance of soil fertility, biological nitrogen fixation and microbiology of phyllosphere. • Thirumalacher (Hindustan Antibiotics, Pune) developed antifungal antibiotics like Haymycin and Aureofungin. • Nandi (Bose Res. Institute, Calcutta) worked on production technology of antibiotics and bacterial fertilizers (Biofertilizers). • Desikachray (Madras) studied taxonomy of BGA in India. • Thomas (BARC, Mumbai) studied physiology of algae in India. • Raja Rammohan Rao (CRRI, Cuttak) studied on rhizosphere nitrogen fixation phenomenon. • Bhagyaraj (GKVK, Bangalore) studied Mycorrhiza and N2 fixation interactions. • Verma (JNU, Delhi) studied/worked on sulphur metabolism. • Subramaniam & Mahadevan (Univ. Madras) studied fundamental aspects of N2 fixation. • Modi, Sushil Kumar, Das and Thomas carried research on “Genetics of “Nif” gene in relation to BNF by Rhizobium, Azospirillum and Kelbsiella. • Bharadwaj (Palampur) studied/worked on microbiology of organic matter decomposition & role of celluloytic microorganisms. • Gaur (IARI) and Mishra (Hissar) studied the role of celluloytic microorganisms in accelerating the process of composting and compost-making. • Karla and Garcha (Ludhiana) studied the phenomenon of cellulose degradation and legume bacteriology. • Ranganathan & Nellakantan (NDRI, Karnal) worked on silage microbiology and process of anaerobic decomposition in biogas production. • Vadher, Gupta, Sethunatathan and Raghu studied role of soil enzymes and microbiology of pesticide degradation in soil. • Dart & Wani (non-symbiotic N2 fixation), Thomas, Kumar Rao, Nambiar and Rupela (symbiotic N2 fixation) and Krishna (VAM

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fungi). These scientists at ICRISAT, Hyderabad work on symbiotic and non-symbiotic N2 fixation in gram, groundnut, arhar, sorghum and millets. • N. V. Joshi (1920) reported first isolation and identification of Rhizobium from different cultivated legumes • Gangulee and Madhok studied physiology of Rhizobium and production of Rhizobium inoculants. • Sen and Pal (1957) studied solubilization of phosphate by soil microorganisms. • A. Sankaran (1958) standardized quality of legume inoculants for first time in India. • P. K. Dey and R. Bhattacharya isolated for the first time a new, nonsymbiotic N2 fixing bacterium Derixa gummosa in the world. • V. Iswaran (1959) reported the use of Indian peat as carrier for Biofertilizers production. • Dube J. N. (1975) reported coal (wood coal), an alternative to peat, as carrier material for biofertilizer production.

CHAPTER

3 3.1

Environmental Microbiology— Water and Air

WATER AND WATER MICROORGANISMS

Water is an essential element that makes life on earth possible. Without water, there would be no life. Although 71% of the Earth’s surface is covered by

Schematic representation of the Water cycle

water, only a tiny fraction of this water is available to us as fresh water. About 97% of the total water available on Earth is found in the ocean and is too salty for drinking or irrigation. The remaining 3% is fresh water. Of this, 2.997% is locked in ice caps or glaciers. Thus only 0.003% of the Earth’s total volume

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of water is easily available to us as soil moisture, groundwater, water vapor and the water in lakes, streams, rivers and wetlands. This makes water a very precious source. Clean water is a colourless, tasteless and odourless liquid that has a boiling point at 100°C and freezes at 0°C (under the pressure of 760 mm Hg). Water occurring in nature contains dissolved salts and gases, especially sea and mineral waters. Water covers 70% of the earth’s surface, and thus, it is the most essential habitat of life. The overall volume of inland waters is estimated at 7.5 × 105 km3, of seas and oceans at 1.4 × 109 km3, and of glaciers and continental glaciers at 1.8 × 107 km3. Water makes up the most crucial component of living organisms (70–90% of cell mass) and fulfils a purpose in taking part in various biological reactions and processes.

3.1.1 Types of microorganisms in water The biotopes of water microorganisms may be underground and/or surface waters as well as bottom sediments.

• The underground waters (mineral and thermal springs, ground waters) due to their oligotrophic character (nutrient-deficient) are usually inhabited by a sparse microflora that is represented by a low number of species with almost a complete lack of higher plants or animals.



• The surfacewaters such as streams, rivers, lakes and seawaters are inhabited by diverse flora and fauna. Microorganisms in those waters are a largely varied group. Next to the typical water species, other microorganisms from soil habitats and sewage derived from living and industrial pollution occur.



• Bottom sediments are a transient type of habitat i.e. the soil water habitat that is almost always, typically, oxygen-free in which the processes of anaerobic decomposition by microorganisms cause the release of hydrogen sulphide and methane into water. In the bottom sediment, anaerobic putrefying microflora, cellulolytic bacteria and the anaerobic chemoautotrophs develop.

3.1.1.1 Groups of water organisms Microorganisms occupy surfacewaters in all of the zones; they may be suspended in water (plankton) cover stationary underwater objects, plants etc. (periphyton), or live in bottom sediments (benthos). Plankton: The group of organisms that passively float in water, not being able to resist the movement and the flow of water mass, is called plankton or bioseston. We differentiate:

• phytoplankton (plant plankton) • zooplankton (animal plankton)

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• protozoa plankton • bacterioplankton (bacteria plankton)



• virus plankton



• Phytoplankton are mainly microscopic algae and blue-green algae. It is a varied community in terms of the systematics and mainly composed of forms smaller than 50 mm. Sea phytoplankton are dominated by diatoms and dinophyta, whereas freshwater phytoplankton are dominated by cryptophytes, diatoms, green algae, and blue-green algae.



• Zooplankton are small water animals that occur in plankton. There are three systematic groups that occur in fresh waters: rotifers, branchiopods and copepods. The sea water plankton is composed of copepods, ctenophores, urochordata, arrow-worms as well as some species of snails. Most of them are filtrators (condensed suspended particles) or predators.



• Protozoa plankton consist of protozoa which occupy the open water zones like flagellates and ciliates. They are the main consumers of bacteria. Moreover, most ciliates feed upon flagellates, algae and smaller ciliates. The protozoa itself feeds upon the zooplankton.



• The heterotrophic bacteria plankton occupy waters which are abundant in organic compounds. The amount of bacteria in open waters varies between 105-107 cells in 1 ml.



• Virus plankton is composed of viruses which are the smallest element of plankton. Their numbers may be very high (108 in 1 ml) in various fresh and sea water habitats. Viruses are, next to the protozoa, a crucial factor in bacteria mortality.

Distribution of plankton: The ability to hover in mid-water is possible due to the presence of mucous membranes around the cell, gas vacuoles or lipids contained inside the cells. The distribution of species and numbers of water organisms differs greatly since the biotic and abiotic factors vary in particular water basins. The distribution in lotic waters such as rivers, springs and streams is more or less the same. Especially high numbers are found in the mid course of the river where the bottom and the main-stream speed are favourable for development of lakes. Where the flow of water is limited, a closely related vertical distribution of mainly phytoplankton to stratification has been observed. During calm, quiet weather where the air meets the water, neuston appears upon the surface of the water. This is composed of bacteria, algae and pleuston which is composed of larger organisms.

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Group of organisms living in lakes

Periphyton: Periphyton occupy the shoreline zones. They are a group of organisms that create outgrowths upon various objects and underwater plants. Most of the time they usually consist of small algae—diatoms, green algae and bacteria. Moreover, various settled or semi-settled protozoa, eelworms, oligochaetes, insect larva, and even crustaceans, make up the periphyton biocenosis. Periphyton has a characteristic complex biocenosis and many ecological relationships can be observed between its components. Benthos: The bottom habitat is occupied by a group of organisms called the benthos. The muddy bottom contains an abundance of organic compounds that are created as a result of dead matter decomposition (fallen parts of plants and animals). At great depths, the bottom is free from any plants which, due to a lack of light, can not grow. However, the absence of oxygen supports the development of, among others, an oxygen-free putrid microflora. Among the benthos microflora, the most numerous are bacteria and fungi (decomposers) as well as some animals (detritophages). Both of the above groups are responsible for decomposition of the organic matter. Benthos of shallow reservoirs may also contain some algae.

3.1.2 Factors limiting growth of microorganisms in water The development of microorganisms in water is influenced by a large number of chemical and physical factors which, in various ways, interact or oppose each other. They have an influence on the size and the species composition

Environmental Microbiology—Water and Air

3.5

of the microbial biocenosis as well as on their appearance and life processes. Within water ecosystems, two groups of factors that have a crucial influence on the quantitative and qualitative relationships between microorganisms may be distinguished:

• abiotic factors—light and thermal energy, water reaction, water flow, climate and the compounds dissolved and suspended in water (dead organic matter, non-organic compounds and gasses such as oxygen, carbon dioxide, methan and others).



• biotic factors—all water living organisms such as plants, animals, microorganisms and the relationship between them.

3.1.3 Characterization of water microorganisms Bacteria: In terms of morphology, most water bacteria resemble soil bacteria– their cells are round, cylindrical or screw like. There are also thread-like and stem-like shapes. The threads may be or not be branched, single or in groups. Various water bacteria may create clusters made up of various numbers of cells in the following shapes: spherical, star-shaped, lamellar, and filiform. Most water bacteria are active and mobile using cilia or flagella (e.g. Vibrio, Pseudomonas). Bacteria in water may swim slowly (plankton) or occupy a fixed substrate. Oligotrophic water bacteria in clean waters occur as microforms with cells smaller then 1 mm, usually 0.4 mm. Water oligotrophs rarely multiply, their generation cycle lasts from a few dozen to two hundred hours. Polluted waters are predominantly occupied by bacteria of the gram-negative rods group. The ratio of rods to cocci is about 90:1. Clean waters (rivers and streams) contain a sparse microflora and the ratio of rods to cocci is 1:1.5, which indicates the dominance of cocci. The number of bacteria in water depends mainly on the organic matter content. In clean waters, they occur in low numbers whereas polluted waters contain up to several million cells per 1 ml of water. Bacteria that occur in water habitats may be divided into the following: • autochthonous (native), constantly occupying water habitats. • allochthonous (foreign), finding their way from the soil or the air as well as microorganisms that get into the water basins along with municipal and industrial sewage. Autochthonous bacteria: We can distinguish photoautotrophs, chemoautotrophs and chemoorganoautotrophs.

Photosynthesizing bacteria (photoautotrophs) Purple and green bacteria are among the photosynthesizing autotrophs. Due to their metabolism, these bacteria can be divided into the following groups:

• Filiform green bacteria (Chloroflexaceae),



• Sulfuric green bacteria (Chlorobiaceae),

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• Sulfuric purple bacteria (Chromatiaceae and Ectothiorhodaceae),



• Non-sulfuric purple bacteria (Rhodospirillaceae),



• Heliobacteria (Heliobacteriaceae) The photosynthesis of bacteria is carried out slightly differently from that of plants. Most importantly, it is an oxygen-free process which requires the presence of reduced mineral compounds and it is not accompanied by a release of oxygen but by a production of oxidized non-organic or organic compounds. The assimilating pigments of bacteria are categorized by the ability to absorb infrared light that is not absorbed by green plants. The photosynthesis in surface waters is conducted mainly by algae and plants and the role of the bacterial photosynthesis is less important.

Chemosynthesizing bacteria (chemoautotrophs) Chemoautotrophs get energy from the oxidation processes of non-organic compounds. Depending on the nature of the oxidized substrate, the following can be distinguished: nitrifying, ferruginous, sulfuric and hydrogen bacteria. • The role of the nitrifying bacteria in surface waters is the oxidation of ammonia and nitrite to nitrate. In greater concentrations, the above compounds may be harmful to water organisms as well as to humans (in cases when such water is utilized for water supply systems). Moreover, the production of nitrate is a fundamental process that supplies water plants with a source of nitrogen. • Ferruginous bacteria grow in waters when the content of bivalent iron ranges between 0.15-8.5 mg/dm3. Their negative influence includes corrosion and fouling of plumbing, sewage systems and different metal constructions. The most common ferruginous bacteria are the Leptothrix ochracea and Crenothrix polyspora and they belong to the filamentous bacteria which are categorized by the fact that the single cells form thread-like forms surrounded by a gelatinous sheath of varying thickness. Stored ferruginous substances in cells change the coloration of cell threads into a yellow or dark-brown shade. The ferruginous bacteria are very common in fresh bodies of water especially in waters from wells and springs, where it is possible to observe their clusters with naked eye. Moreover, they occur abundantly in muddy streams, marshes and ponds. • Sulfuric bacteria occur mainly in waters containing hydrogen sulfide which is toxic for most microorganisms, whereas for this group, it is one of the crucial compounds for survival. These bacteria can be found in mineral springs that contain hydrogen sulfide of geological origin as well as in highly polluted waters where it is produced as a result of oxygen-free protein decomposition or desulfurication processes. The typical representatives of the sulfuric bacteria are: bacteria that move in sliding motions Beggiatoa alba and fixed to the bottom Thiothrix nivea.

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3.7

The forms of individual sulfuric bacteria are: (i) Thiobacillus thioparus—stores sulfur derived from oxidation of thiosulfate. (ii) Thiobacillus thiooxidans—grows in acidic habitats of pH 1.0 – 4.0. (iii) Thiobacillus ferroxidans—besides thiosulfates and tetrationans, it possesses the ability to decompose ferruginous salts. (iv) Thiobacillus denitrificans—is a relative anaerobe and it has an ability to utilize nitrates as the electron acceptor during the oxidation of hydrogen sulfide. In aerobic conditions, the above function is performed by the oxygen.

• Hydrogen bacteria posses an ability to oxidize hydrogen using oxygen as a final acceptor of electrons. Most often, they feed heterotrophically and switch to autotrophic feeding when hydrogen is present in the habitat. The most widespread species belong to the genus Hydrogenomonas. Micrococcus denitrificans belongs to a group of the hydrogen bacteria and they conduct the oxidation of hydrogen while simultaneously reducing nitrate down to molecular nitrogen. Desulfovibrio desulfuricans also oxidizes hydrogen while reducing sulphate down to hydrogen sulphide.

Heterotrophic bacteria (chemoorganotrophs) A predominant part of autochthonous bacteria which occur in water basins are the chemoorganotrophic bacteria which belong to a group of saprophytes that feed upon dead plant and animal organic matter. Typical bacteria plankton that occupy an entire water mass are the cilliated gram-negative rods and they represent the following genera: Pseudomonas, Achromobacter, Alcaligenes, Vibro and Aeromonas, as well as the gram-positive cocci that belong to the Micrococcus genus, treponema and spiral bacteria of the Spirillum genus. The underwater parts of higher plants and the underwater fixed particles are colonized by numerous stem-like bacteria (e.g. Caulobacter), sheathed, filiform, and gemmating bacteria (e.g. Hyphomicrobium), which are one of the microorganisms forming the periphyton. Organisms which usually grow in bottom sediments are oxygen-free putrefactive bacteria, then oxygenfree cellulolytic bacteria and finally, oxygen-free chemoorganotrophs such as Desulfovibrio genus that reduce sulfate down to the hydrogen sulphide. In addition, there are some less numerous oxygen-free methanegenerating bacteria which reduce organic compounds down to methane.

Allochthonous bacteria Waters of high fertility and also highly polluted surface waters are abundant in saprophytes and parasitic bacteria from among which, the following are predominant: gram-negative intestinal rods of Escherichia coli as well as

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Environmental Biotechnology

the Proteus genus, Klebsiella and Enterobacter, and also rods of Pseudomonas aeruginosa and of the Arthrobacter genus. Moreover, gram-positive rods (bacilli) of the Bacillus, Corynebacterium and Clostridium genera, which are washed out from the soil and get into the bodies of water during heavy rainfalls, also belong to the allochthonous bacteria. Municipal wastes are the main source of pathogenic bacteria. Moreover, during the infiltration processes and surface run-offs, soil bacteria find their way into the waters as well. The role of air in water contamination is significant in densely populated areas of cities and industrial regions. Water fungi: In contrast to bacteria which grow best in waters of pH between 6-8, fungi occur only in waters below pH 6.0. Usually, fungi occur in shallow waters, right on or just below the surface, which is closely connected to the fact that the organisms require significant amounts of oxygen. The predominant fungi in water environments are represented by mold fungi which belong to the Oomycota class (Leptomitus, Phytophthora) and to the class of Zygomycota (Mucor and Rhizopus). Relatively frequently, fungi belonging to Ascomycota as well as the Deuteromycota, are found in surfacewaters. Almost all fungi are heterotrophs that decompose organic matter; waters are occupied by both saprophytes and parasites which colonize water plants and animals. They have more diverse shapes than bacteria and they differentiate into larger cells and more complicated structures. In addition to unicellular ones, there are also multi-cellular fungi with large mycelium. Fungi usually, do not occur in clean waters. They grow in abundance on the bottom of waters polluted by sewage (e.g. Leptomitus lacteus). Blue-green algae: Blue-green algae are a group of organisms previously considered to be algae. Currently they are classified to the Procaryota kingdom and the sub-kingdom of Eubacteria. There are unicellular, colonial (loose cells connected with a single mucus envelope) and filamentous forms. The prokaryotic organisms contain a nucleoid instead of an isolated nucleus. In contrast to other bacteria, they are capable of conducting oxygen photosynthesis. They contain chlorophyll and sometimes disguise it in other photosynthesizing pigments: ficocyanine and alloficocyanine. Characteristically, the blue-green colouring of blue-green algae comes from the combination of chlorophyll and ficocyanine. Blue-green algae reproduce mainly through proliferation by cell fission. Their characteristic trait is that they possess gaseous vacuoles which allow movement in water to places of better illumination. Some (Anabaena) are capable of binding atmospheric nitrogen in structures called heterocysts. Due to their resistance to extreme environmental conditions, they are ubiquitous. They can be found in deserts and in hot springs. Blue-green algae can cause blooming in lakes and other water reservoirs. Some of them produce toxic metabolites. Algae: Algae are the simplest autotrophic eukaryotes that incorporate over 20 thousand species. Algae occur in fresh and seawaters. They are important

Environmental Microbiology—Water and Air

3.9

producers of organic matter and oxygen. Algae live in the form of single cells or they create multicellular body of various shapes called thallus (threads, spheres, multilayer clusters). The composition of algae community changes significantly with respect to quality and quantity, depending on the content of the mineral salts in any given reservoir as well as on the characteristics of the substances that make up the main pollutant. The following are the characteristic algae that occur in oligotrophic waters: diatoms of the following genera: Asterionella, Tabellaria, Melosira and some other algae (Dinobrion). In eutrophic waters, the content of algae is completely different. Most of all, such waters contain only a vestigial number of diatoms, and instead of them, the algae from the Dinophyta class as well as the Spirogyra genus, appear. Algae are subdivided into the following classes:

• Chlorophyta—green algae that contain chlorophyll a and b types, a cellulosic cell wall and starch as reserve material. They have a diverse constitution both unicellular and multi-cellular forms exist usually as thread-like structures. Cells may be motile; (then they are equipped with flagella), or non-motile. Chromatophors of various shapes have a green coloration. They reproduce vegetatively or sexually. Vegetative reproduction consists of the division of cells and the fragmentation of thread-like forms.



• Chrysophyta—this group involves diatoms important for the water environment. They are common algae and occur in fresh and sea waters, bottom sediments and soil. Chrysophyta contain a and c types of chlorophyll. Their cell wall is enriched in silica. They produce lipids as reserve material.



• Euglenophyta—Euglenoids usually have an elongated shape. Their cells are equipped with flagella that allow movement in water or they move by crawling along the bottom. The cells are surrounded by a soft envelope called the pellicle. Chromatophores contain chlorophyll, carotenes and xanthophylls. Within the cell there is a clearly visible nucleus and the eyespot called stigma, sensitive to a light stimulus. Euglenoid cells, create cysts, which make survival in unfavorable conditions possible. They grow in waters containing high levels of organic compounds. There are also parasitic forms.



• Pyrrophyta—occur usually individually. Some cells are surrounded by a cellulose wall whereas others are deprived of any cell walls. They usually possess two flagella that allow their movement. Within the protoplasm, there is an isolated cell nucleus and yellow-green or yellow-brown chromatophores. They reproduce by division and some have been observed to reproduce sexually. Pyrrophyta occur in slightly salty or sea waters and only selected species live in fresh waters. In lakes, there is Ceratium hirundinella, a species that sometimes appears in large masses.

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• Rhodophyta—contain chlorophyll type a and b as well as other pigments such as carotene, xanthophylls and phycobilin pigments: phycoerythrin, phycocyanin. They store starch as a storage product. Their cell wall has two layers: the inner one is made of cellulose whereas the outer one is made of pectin. They reproduce asexually, through fragmentation of the thallus, and sexually, through oogamy.



• Phaeophyta—brown algae, containing chlorophyll a and c as well as carotenoids (fucoxanthin). Their reserve material is laminarin (â-1, 3-glycan) and chrisolaminarin, mannitol and lipids. Their cell wall is also a bi-layer: the inner wall is made of cellulose whereas the outer wall is made of pectin. Brown algae are multi-cellular organisms that possess the highest level of specialization of the thallus of all algae with high anatomic and morphological variation. They reproduce by zoospores, and also sexually, by gametes.

Water protozoa: Protozoa live in all types of waters, from small puddles, to inland waters, to the seas. They feed heterotrophically, absorbing the dissolved organic compounds or feeding upon bacteria. They are most numerous in highly polluted waters and are the element of activated sludge. When the pollution level is not too high, ciliates become predominant, and that concerns both the free-swimming ones (e.g. Paramecium) and the settled ones (e.g. Vorticella). Protozoa can be sub-divided into four classes:

• Flagellata – flagellates. These move utilizing long flagella. They feed heterotrophically and occur in polluted waters or in inefficiently functioning activated sludge. Besides dissolved substances, they may also absorb bacteria or unicellular algae. Flagellates live individually or in colonies. There are parasitic forms among them too. This is exemplified by a human parasite Giardia lamblia and the Trypanosoma gambiense which is transferred on to humans by the Tsetse fly causing African sleeping-sickness and neurological disturbances.



• Rhizopoda amoebae. The cells move around utilizing the plasmatic pseudopodia which are used for locomotion and for capture of food. Some amoebae have a changeable shape others; however, have a constant shape as they are equipped with a mini-skeleton or an outer shell. Some amoebae lead a parasitic life (Entamoeba histolytica).



• Ciliata – ciliates. Most of the representatives lead a free-swimming life style (Paramaecium, Euplotes); others crawl or are attached to the bottom. They feed upon bacteria, algae and organic substances. Ciliates occur in large numbers in polluted waters and in activated sludge. Some, such as Balantidum coli which causes dysentery, are parasites of animals and humans.

Environmental Microbiology—Water and Air



3.11

• Sporozoa. Only parasites belong to this class and representatives are Cryptosporidium parvum, causing intestinal diseases and Plasmodium malariae, causing the malaria. The second parasite attacks the red blood cells. This pathogen is carried by the Anopheles mosquito.

3.1.4 Sources and types of pollutants Waters become polluted as a result of domestic and industrial sewage disposal into surfacewaters, which contain huge amounts of various compounds that affect the biocenosis of water reservoirs. Besides sewage, pollution is also caused by rain run-offs which wash away different fertilizers and crop protection products. Moreover, the pollutants also transfer into waters from the surrounding air. This usually results from industrial dust which falls directly into the water or is washed away from the ground surface by rain. Important gases are: sulphur dioxide, nitrogen oxides, carbon oxides and dioxides which get into the waters mainly in highly industrial areas. Some of the above compounds undergo microbiological decomposition relatively easily, becoming food for heterotrophic microorganisms, others are resistant to such decomposition and are harmful or toxic to microorganisms. Examples of these are the following: cyclic compounds, engine oil, lubricants, chlorinated hydrocarbons, pesticides, and among the mineral pollutants— heavy metal salts.

3.1.5 Self-purification of surfacewaters Self-purification encompasses complex co-operation between physical and biochemical factors such as: sedimentation (settling), oxidation, an exchange of volatile substances between the atmosphere and water, and the release of gaseous products of metabolism into the atmosphere. However, the critical role is played by the biological factors. A wide range of microorganisms and higher organisms participate in self-purification processes. Bacteria and

Succession of microorganism during the self-purification process

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fungi are the most crucial as they are capable of mineralising various mineral components. Proteins, simple and complex sugars, fats, cellulose, lignin, wax and others undergo degradation during the process of self-purification. As a result of mineralization, the following compounds are created: H2O, CO2, NO3-, SO42-, PO43-, and other simple compounds. With the progression of selfpurification the populations of microorganisms that act in the environment, change. The self-purification process utilizes large amounts of oxygen during the biochemical processes. The amount of oxygen that is used up in any specified time by water microorganisms is called the biochemical oxygen demand, BOD. By analysing the BOD, it is possible to determine the concentration of the organic compounds dissolved in water which are susceptible to biological oxidation. The discharge of impurities into the water reservoir creates a sudden change in chemical, biological and physical conditions. Simultaneously, right below the area of the discharge, the process of self-purification begins. The process leads to the formation of zones containing, characteristically, gradually decreasing levels of pollution.

Saprobic zones. Self-purification of lotic waters Zones of various levels of organic pollution are called saprobic. In particular zones, the content of biocenoses is different and altered to fit the existing conditions. Species, which clearly dominate other species and have adapted to the existing conditions, are found there. Zones differ in the dynamics of dissimilation processes, the intensity of oxygen intake, and the appearance of the water. There are three different saprobic zones: poli-, mezo- and oligosaprobic. Polisaprobic zone is the zone of highest concentration of pollutants with cloudy, dirty-grey, fetid odorous waters. The high concentration of various organic compounds ensures development of selected heterotrophic microflora, which, while conducting biodegradation, use up large amounts of oxygen leading to its deficit. In anaerobic conditions, the following gases are formed: H2S, NH3, CH4, N2 and others. There is a lack of green plants in this zone. Among the organisms which are capable of surviving in such conditions, the dominant ones are Zooglea ramigera and Sphaerotilus natans bacteria. There are also sulphur bacteria (in the presence of hydrogen sulphide), especially of the Beggiatoa and Thiothrix genera; protozoa are also numerous. Reducers (decomposers) are the most frequently occurring organisms in this zone. In the mezosaprobic zone there is further intensive breakdown of the organic compounds but the amount of oxygen is sufficient to sustain a full demand. The water becomes clear, often of green coloration, due to the abundantly flourishing algae. The number of reducers decreases. Besides the microorganisms mentioned above, sewage fungi Leptomitus lacteus, bluegreen algae, sparse diatoms and green algae appear. The mineralization of the organic compounds is finished within the zone, excluding, humus compounds

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which are difficult to decompose. The mezosaprobic zone is divided into a-and b-zones. The a–mezosaprobic zone is a heterotrophic one in comparison to the b-mezosaprobic zone which is rather autotrophic. The b–mezosaprobic zone is cleaner and higher numbers of algae species occur here. Oligosaprobic zone is a section where the inflow of impurities ends and water returns to its previous state of natural water. Water is clear, odorless and well oxidized. The zone is mainly inhabited by ferruginous and nitrifying bacteria (the chemosynthesizing bacteria); sparse blue-green algae, many diatoms and green algae, and few protozoa, occur in the biocenose.

Self-purification of lentic waters A self-purification process takes a different course in lentic waters; the saprobic zones do not evolve here even though the purifying mechanism remains the same. The impurities introduced into water fall down to the bottom (their density is greater than that of water) where they are decomposed.

Microbiological processes within bottom sediments There are complicated chemical, physical, and biological processes taking place between water and the bottom sediments, which are important for the reservoir as a whole. The water/sediment arrangement, the microorganisms quality-quantity ratio and the direction of bio-chemical changes have a significant influence upon the level of biogenes (nitrogen, phosphorus, sulphur compounds) within the reservoir (its fertilization). In the bottom sediments, the aerobic decomposition of organic constituents takes place in the upper layers (from a few to several mm) and are a source of soluble mineral salts. Whereas, the anaerobic biodegradation, which takes place below, causes a release of substances, often poisonous, to the water habitat (e.g. H2S, CH4).

Bottom sediments play an important role in lentic water self-purifying process where the organic suspended matter falls to the bottom as a result of lack of water movement. They have a significant influence upon the conversion of the biogenic compounds which affect the quality of water. The most crucial factor regulating the speed of nitrogen and phosphorus penetration (also Fe and Mg) from within the sediments into water is the content of the dissolved oxygen within the layer near the bottom. Active diffusion of phosphates into water begins when the content of the dissolved oxygen falls below 1mg O2/ dm3. Moreover, the following also have an influence upon the release of phosphates: temperature, organic compound decomposition, water pH, redox potential. In bottom sediments of various surface water reservoirs, the following microorganisms are the most common: aerobic cellulolytic bacteria (of the Sporocytophaga, Cytophaga, Pseudomonas, Achromobacter genera) as well as anaerobes such as the Clostridium genus. The latter takes part in the decomposition process of hemicellulose. In oxygen-free conditions, numerous putrefying bacteria (releasing H2S from proteins), SO42- reducing bacteria,

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denitrifying bacteria (NO3– reducing), methane generating (CH4 releasing) and hydrogen bacteria grow. Moreover, ammonificating bacteria are abundant in bottom sediments. Nitrifying bacteria usually occur in small numbers in upper layers of sediments as they are obligate aerobes. The presence of CH4-oxidizing aerobic bacteria in bottom sediments also depends upon the concentration of oxygen and iron.

3.1.6 Water-transmitted pathogenic microorganisms Bacteria: The group of obligate pathogenic bacteria, which occur in polluted surface waters, contain rods causing typhoid fever (Salmonella typhi), as well as other gram-negative bacteria of the Salmonella genus, which are the cause of various infections of the digestive tract. Bacterial dysentery caused by Gramnegative rods of the Shigella genus are not as common as the above. In surface waters of tropical countries, bacteria of the Vibrio cholerae genus (cholera), frequently occur. Moreover, Mycobacterium tuberculosis causing tuberculosis and treponema of the Leptospira, can be also found in polluted waters. The latter bacteria cause bacterial jaundice. Beside the obligate pathogenic bacteria, there are also numerous gram-negative bacteria in surface waters which are described as opportunistic microorganisms (facultatively, pathogenic). These belong to the Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acinetobacter, Proteus and Providencia genera. All of the rods are part of the usual flora of the intestine and are not typically pathogenic for as long as they occur in human or animal digestive tracts. In some cases though, these bacteria find their way into other organs becoming a potential cause of different illnesses such as inflammation of urinary and respiratory systems and also sepsis, which is a general infection of all internal organs. Waterborne Bacterial Infections Disease type

Species or genera of bacteria

Typhoid

Salmonella typhi

Paratyphoid

Salmonella paratyphi

Animal salmonellosis

Salmonella sp.

Bacterial dysentery

Shigella sp.

Cholera

Vibrio cholerae, Vibrio cholerae type eltor

Stomach and intestine catarrhs

Enteropatogenic Escherichia coli, Klebsiella pneumoniae,Aeromonas hydrophila, Plesiomonas shigelloides, Pseudomonasaeruginosa, Vibrio parahaemolyticus, Campylobacter (Vibrio)fetus subsp. jejuni, Clostridium perfringens, Bacillus cereus

Yersiniosis

Yersinia enterocolitica

Tularemia

Pasteurella (Francisella) tularensis

Leptospirosis

Leptospira sp.

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Skin infections

Pseudomonas aeruginisa, Mycobacterium (M. balnei, M. phlei,M. marinum, M. kansasii, M. fortuitum, M. cholonei, M.Gorgonae

Bacteremia conjunctivitis, ear and upper-respiratory system infection

Psudomonas aeruginosa, Pseudomonas cepacia

Fever (pyrogens)

Gram-negative water rods (Pseudomonas, Achromobacter,Xantomonas, Moraxella, Acinetobacter)

Legionnaires disease

Legionella pneumophila

Viruses

Besides pathogenic bacteria of surface waters, into which municipal and industrial sewage is disposed, the waters also contain significant amounts of other pathogenic microorganisms such as the Polio virus. They are responsible for causing the Heine Medina disease (polio). Enteroviruses, which cause intestinal infections, occur even in slightly polluted rivers. Intestinal viruses which may be transmitted by water and diseases caused by them Viruses

Number of types

Diseases

Poliovirus

3

Palsies, meningitis, fever

ECHO

34

Meningitis, respiratory system diseases, rash, diarrhea, fever

Coxsackie A

23

Herpangina, respiratory system diseases, meningitis, fever

Coxsackie B

6

Cardiac muscle inflammation, innate heart defects, rash, fever, meningitis, respiratory system diseases, pleurodinia

Enteroviruses

4

Meningitis, encephalitis, respiratory system diseases, acute hemorrhage conjunctivitis, fever

Hepatitis virus, type A

1

Hepatitis type A

Norwalk virus

1

Epidemic diarrhea, fever

Parvovirus

3

Accompany the system diseases

Adenoviruses

41

Respiratory system disease, eye infections, diarrhea

Rotaviruses

4

Epidemic diarrheas among children)

Reoviruses

3

Respiratory system diseases

respiratory

(mainly

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Protozoa Infections of the digestive tract caused by protozoa may come from contaminated water. Most parasitic protozoa produce cysts which are able to survive inside their host in unfavorable conditions. When the conditions improve, cysts transform into so called trophozoits, the vegetative form occurring in humans. Waterborne diseases caused by protozoans: Intestinal viruses which may be transmitted by water and diseases caused by them Pathogenic protozoa

Disease

Symptoms

Giardia lamblia (flagellates)

giardiosis

Chronic diarrhea, stomach cramps, flatulence, weight loss, fatigue

Cryptosporidium parvum (sporozoa)

cryptosporidiosis

Stomach aches, loss of appetite, watery diarrhea, weight loss

Entamoeba histolytica (amoebae)

amoebiosis (amoebicdysentery)

Anywhere from slight to acute diarrhea, fever with shivers

Acanthamoeba castellani (amoebae)

amoebicmeningoencephalitis

Symptoms from the central nervous system

Naegleria gruberi (amoebae)

amoebicmeningoencephalitis

Gets into the brain of swimmers through the nose, causes acute symptoms of meningitis and encephalitis ending in death

Balantidium coli (ciliates)

Balantidial dysentery

Hemorrhage diarrhea caused by an ulceration of the large intestine

Parasitic fungi In polluted surface waters, parasitic fungi can also occur, for example, Microsporum sp., Trichophyton sp. and Epidermophyton sp. They are dermatophytes, causing ringworm and other cutaneous infections.

Parasitic worms Human parasites are not usually included in the scope of microbiological research, however, along with other pathogens (viruses, bacteria, protozoa) they pose a serious threat to human health. They occur in sewage and may find their way into waters from soils as a result of infiltration and surface runoffs. The infectious forms of the parasitic worms are their eggs. The eggs are

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excreted in great numbers outside the hosts’ body along with faeces and spread through sewage, soil or food. The worms’ eggs are very resistant to external factors and thus, are difficult to eliminate from sewage by chlorination. Parasitical worms in the human body Parasite

Symptoms

Human ascarid— Ascarislumbricoides (Nematoda)

Ascariasis. Nematodes’ larva causes inflammation reactions in various parts of the body. Sometimes, it breaks up the pulmonary alveolus. If the intestines contain a lot of ascarids, it may cause intestinal obstruction or a puncture causing damage to the abdomen lining.

Whipworm— Trichiuristrichura (Nematoda)

Trichomoniasis disease is caused by nematodes living in human caecum and the large intestine. It creates changes in the mucosa, and at high infestation, a serious loss of mucous membrane. Sometimes appendicitis may occur.

Spiny—headed worms (Acanthocephala)

Ascanthocephaliasis disease is caused by invertebrates which incorporate only parasitic forms. The parasites live in the intestines of all vertebrate representatives. In water environment, their hosts are usually crustaceans. The disease manifests itself by inflammation of the digestive system and its physical damage.

Tapeworms— Taeniasaginata, T. solium (Cestodes)

Parasites develop inside the intermediate host until reaching the larva stage called the cysticercus and infect humans who are its final host. Tapeworms live in the small intestine causing nausea, chronic dyspepsia, stomachaches and weight loss.

Flukes— Schistosomamansoni (Trematodes)

Schistosomatosis—caused by Schistosoma mansoni, manifests itself by the ailment of the digestive system, intestine mucosa inflammation and cirrhosis of the liver.

3.1.7 Sanitary quality of water The possibility of infection by water imposes a constant need to control the hygienic-sanitary quality of not only drinking water, but also that of swimming pools and surface waters. Water gets infected by pathogenic bacteria excreted by ill people and carriers (people who keep excreting pathogens with faeces

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long after they have suffered from an illness). Pathogenic microorganisms are present in sewage and surface waters in lower numbers than other microorganisms. Therefore, they are more difficult to detect than the plentiful saprophytic bacteria. Consequently, much more complex diagnostic methods need to be used in order to detect them.

Indicator microorganisms Current norms are based on indirect inference about the presence of pathogenic microorganisms relying on the number of indicator microorganisms, which permanently live in human and animal digestive tracts as saprophytes. Their presence indicates that the water is polluted with faecal matter and, consequently, there is danger of contamination with pathogenic microorganisms. Bacteria, which serve as sanitary indicators, should meet the following conditions: 1. They must be constantly present in the human digestive tract so that they allow the detection of the water’s contamination with faecal matter. 2. The number of indicator bacteria within the intestine and faeces should be high. 3. Among them, there should be non-spore-forming bacteria as it enables the detection of ‘fresh’ faecal-matter water pollution. 4. Their identification must be possible with readily available methods. 5. Their life span in the external environment should be longer than that of pathogenic bacteria. 6. They should not be able to reproduce in a water environment under natural conditions.

Types of indicator bacteria utilized to assess the health quality of water In routine laboratory work, which conducts sanitary-epidemiological supervision, it is impossible to constantly monitor water for all pathogenic and potentially pathogenic microorganisms, which may be found in water. Therefore, routine monitoring concentrates mainly on detecting bacteria that indicate faecal contamination of water. The sanitary quality of water may be checked by utilizing the saprophytic microflora that occupy the human large intestine. The following indicators of water contamination have been adopted: • Coliforms • Faecal coliforms • Faecal streptococci • Bacilli of Clostridium genus, sulphite-reducing bacteria and in some instances: staphylococci–coagulase positive • Pseudomonas aeruginosa

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Coliforms: Bacteria of the coli group are mainly made up of strains of Escherichia coli as well as the genera: Enterobacter, Citrobacter and Klebsiella. They are detected on media containing lactose at 37°C. Faecal coliforms (thermotolerant) are mainly strains of Escherichia coli and only some of the strains of Enterobacter, Citrobacter and Klebsiella, which have an ability to ferment, lactose at 44 oC. The presence of coliforms or faecal coliforms in a water sample indicates relatively recent contamination of water with faecal matter, sewage, soil or with decaying plants. For most types of waters, a quantitative determination of both groups of coliforms is recommended.

Faecal streptococci While in a water environment, faecal streptococci are characterized by a slightly longer period of survival and resistance to most disinfecting products than the coliforms. Faecal streptococci include microorganisms of Enterococcus and Streptococcus genera, which belong to the serological group of Lancefield D. Detection of faecal streptococci in a test sample, significantly exceeding the coli group bacteria, may suggest water contamination with animal faecal matter or sewage from animal farms.

Bacilli of Clostridium genus The detection of sulphite reducing bacteria (mainly strains of Clostridium perfringens) may suggest less recent contamination with faecal matter; their endospores are able to survive for many years in unfavourable conditions. Sulphite–reducing clostridia are a good indicator of properly conducted water treatment processes—coagulation, sedimentation, and filtration. Endospores of these bacteria as well as the cysts of parasitic protozoa (Cryptosporidium parvum, Giardia lamblia) ought to be eliminated in those stages of water treatment, because they are especially resistant to the disinfecting agents. Conducting analysis of a water sample, in order to detect bacteria of the Clostridium genus, is technically less complicated than searching for parasitic protozoa and it ensures that the treated water is free from protozoa and from the eggs of pathogenic worms (Helminthes).

Pseudomonas aeruginosa Currently, detection of Pseudomonas aeruginosa bacteria in drinking water, running water, swimming pools and surface waters is recommended in addition to the above elements of sanitary analysis. They are gram-negative rods that do not produce spores. Their characteristic trait is the ability to produce a blue-green pigment—pyocyanin as well as a fluorescent pigment— fluorescein. Representatives of this species were isolated from human faeces, and in cases of infection—from urinary tracts, inner ear, suppurating wounds, etc. These bacteria pose a potential pathogenic danger for both humans and

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animals. In addition, they are widely distributed in surface waters and soil. It is also important that the species may live in chlorinated water because it is, to some extent, resistant to disinfection.

Staphylococci The Staphylococcus genus is mainly used to assess sanitary quality of swimming pools. Recreation waters are the cause of infections of respiratory tracts, skin and eyes. For this reason, microbiological analysis based on standard indicators (coliforms) is insufficient. Some researchers have recommended Staphylococcus aureus to be used as an additional indicator of sanitary quality of recreational waters, because its presence is associated with human activity in these waters.

Total number of bacteria In routine analysis, the total number of bacteria present in 1 ml of water is also determined by an agar plate method. One set of plates is incubated at 37°C for 48 h (mesophilic bacteria). Another set of plates is incubated at 22°C for 72 h (psychrophilic bacteria). After incubation, the colonies are counted and the amount of cfu/ml (colony forming units) can be calculated.

The total number of psychrophilic bacteria Non-pathogenic water bacteria grow mainly at lower temperatures. It is important that gram-negative bacteria in water produce lipopolysaccharides in their cell wall which can be toxic – like endotoxins of pathogenic bacteria. Because of this, their numbers in water should be constantly monitored. A large increase in their numbers is evidence of the presence of easily available organic compounds in the water. Theoretically, the presence of 0.1 mg organic carbon in water can result in an increase of bacteria up to 108 cfu in 1 ml. Phosphorus is also a factor which stimulates the growth of microorganisms. Adding even small amounts of this element (< 50 mg/l) causes 10 times the acceleration of bacterial growth in a water treatment plant.

The total number of mesophilic bacteria More dangerous are high numbers of bacteria growing at 37 °C, because among this high population, pathogenic forms may be found which are dangerous for human health. High number of bacteria in samples of water can prove that water treatment processes proceed badly or that polluted water is siphoned.

Reasons of increasing levels of total number of bacteria in water An increase in the total number of bacteria in water samples can also be proof of development of microorganisms on inner surfaces, especially on pipe-joints, seals and of the creation of the layer called a biofilm. Biofilms of microorganisms are of concern because of the potential protection of pathogens from the action of residual disinfectant in the water and the regrowth of indicator bacteria

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such as coliforms. High number of total bacteria is an indicator of potential pathogens and one should start looking for the source of pollution and taking proper actions. Sometimes, additional chlorination is needed e.g. for drinking water over 0.2 mg Cl2/l. In some cases, changes to the construction of the water supply system and removal of the biofilm are effective protection against the excessive level of microorganisms in water.

3.1.8 Wastewater treatment Wastewater (sewage) is polluted water which includes all harmful liquid, solid or gaseous substances introduced into waters or soil that may lead to a contamination of surface or underground waters. Sewage also includes: usedup liquids, solutions, colloids, suspensions, radio-contaminated waters, saline waters, heated cooling waters, precipitation waters or waters which contain various impurities from urban and rural areas. A. Classification based on origin

• domestic sewage contains large amounts of faecal matter, plant and animal wastes, surface-active agents, urea. The sewage comes from households, public lavatories and industrial facilities posing a serious hygienic and epidemiological threat,



• industrial (technological), evolve during all types of industrial processes (manufacturing and processing),



• precipitation (rain and meltwaters) contain various atmospheric impurities (dusts, microorganisms, gaseous substances), surface runoffs, streets and paved surfaces run-offs (oils, liquid fuels, bacteria, small particle suspensions), microbiological impurities (bacteria, viruses, fungi).

B. Classification based on harmfulness

• directly harmful,



• indirectly harmful (lead to a decrease of oxygen in water below the essential organisms’ requirement).

C. Classification based on contamination stability

• degradable—organic substances that undergo chemical transformations to form simple compounds,



• non-degradable—substances that do not yield to any chemical transformations and are not decomposable by microorganisms,



• stable—substances which only slightly undergo biological decomposition and stay in the habitat in an unchanged form for a long time.

D. Man-made

• urban and domestic—source: food serving facilities, hospitals, houses and apartments posing a hygienic and epidemiological threat,

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• rural—source: farms, pig fattening houses, animal farms, intensively fertilized fields,



• industrial—source: manufacture and processing of all branches of industry; this type of sewage is a major source of toxins,



• radioactive—source: scientific and health facilities, nuclear reactors; such types of wastes are especially dangerous to the habitat, therefore, they require special storage methods.

Pollutants–Municipal sewage Sewage is characterized by the following groups of organic and non-organic impurities:

• soluble substances,



• settling suspensions,



• liquid-suspended suspensions. Chemical impurities contained in sewage may be divided into:



• dissolved mineral substances (sulphates, chlorides, acids and neutral carbonates, calcium, magnesium, sodium, bases, nitrates, phosphates etc.),



• soluble gases (oxygen, hydrogen sulphide, carbon dioxide, nitrogen),



• soluble organic substances (proteins – about 40-60%, carbohydrates – about 25-50%, oils and fats – about 10%). One further classification of pollutants in sewage is as follows:



(a) physical impurities



(b) chemical impurities



(c) biological impurities

(a) physical pollutants of sewage are characterized by properties which can be detected by the senses (sight, smell). The properties of physical pollutants are: suspension, cloudiness, colour, smell, temperature. (b) organic pollutants are defined by three common parameters: BOD (biochemical oxygen demand), COD (chemical oxygen demand), TOC (total organic carbon).

• BOD – determines the amount of oxygen required by bacteria in order to biologically oxidize decomposable organic compounds in aerobic conditions in a temperature of 20°C. About 50% of pollutants are oxidized by microorganisms over a period of three days. Five days as the representative period is assumed to determine the characteristic of biochemical oxygen demand.



• COD – specifies the amount of oxygen required to oxidize organic compounds chemically.

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3.23

• TOC – specifies the amount of carbon contained in organic compounds.

(c) biological pollutants include microorganisms (viruses, bacteria, fungi), eggs of helminthes.

Biogenic pollutants: Dangers connected with them Biogenic pollutants are made up of mineral salts of elements which are essential for the development of living organisms. The basic ones are the compounds of phosphorus and nitrogen. After introduction into lakes and rivers, the above compounds increase their fertility causing eutrophication. Eutrophication is a term that describes a complex of unfavorable symptoms connected with over-fertilization. Urban sewage contains phosphates from human excrements, washing detergents and liquids, food wastes, food additives and other products. Another significant source of phosphate pollution of water is sewage from the agricultural industry. The presence of phosphorus in sewage introduced into water along with nitrates and nitric dioxides causes increased development of algae in both lotic and lentic waters. Increased eutrophication has been considered to be hazardous to water reservoirs as a consequence of uncontrollable growth of plant biomass.

Refraction pollutants Refraction pollutants are those which do not, or only to a minimal extent, undergo biological decomposition by microorganisms. Some of them demonstrate characteristics of dangerous poisons e.g. heavy metals, PAH (polycyclic aromatic hydrocarbons), PCB (polychlorinated biphenyls), dioxins, pesticides, nitrosamines. Elimination of contamination from industrial and municipal wastes, prior to their reintroduction to a receiving body of water, results from a need for rational management of water supply, environment protection and adequate sanitary conditions. Introduction of pollutants, depending on the watercourse, may decrease the water’s physical, chemical and sanitary conditions or even cause the disturbance of biological balance.

Main objectives of the wastewater treatment process The objectives of the wastewater treatment process are:

• lowering the content of organic carbon including compounds which are difficult to biodegrade as well as the toxic, mutagenic and carcinogenic ones,



• reduction of biogenic substances: mineral salts of nitrogen and phosphorus,



• elimination or inactivation of pathogenic microorganisms and parasites.

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Methods of the wastewater treatment Depending on the type of pollutants, there are different methods of purification used prior to reintroduction into a receiving body of water. The methods are classified as follows: • mechanical – in this method only non-soluble pollutants are removed by utilizing the following processes: gravitational and centrifugal sedimentation, flotation, source filtration, separation in hydrocyclones, which allow the removal of organic and mineral suspensions as well as floating bodies; • physical-chemical – utilizes the following operations and processes: coagulation, coprecipitation, sorption, ion exchange, electrolysis, reverse osmosis, ultrafiltration;

• chemical – utilizes neutralization, oxidation, reduction;



• biological – consists of sewage purification (elimination of organic pollutants as well as biogenic and some refraction compounds) during biochemical processes of mineralization conducted naturally by microorganisms in a water habitat (e.g. sprinkling of wastewater onto agricultural lands), or in special devices (on trickling filters or activated sludge).

Stages of sewage treatment A typical process of sewage treatment consists of four stages of purification:

• mechanical (stage I of purification),



• biological (stage II of purification),



• elimination of biogenic compounds (stage III of purification),



• water renovation (stage IV of purification).

Stage I of purification, primary treatment includes the so-called initial or mechanical purification. The goal of this stage is the removal of solid impurities. This stage is considered to be the preparation of sewage for further purification. By utilizing simple mechanical operations, the following impurities are removed during, the first stage:

• floating solid impurities,



• settling suspensions,



• oils and fats.

Stage II of purification, secondary treatment includes biological purification, which leads to the biodegradation of soluble organic impurities, colloidal systems and suspensions not removed during the first stage. The intensification of purification processes is obtained by utilizing trickling filters and activated sludge.

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Stage III of purification, tertiary treatment includes processes used to thoroughly clean sewage. The largest impurities removed during this process are the biogenic compounds (compounds of phosphorus and nitrogen). The nitrogenous compounds are removed during the process of biological nitrification and denitrification, whereas the compounds of phosphorus are eliminated by a process of chemical precipitation. The role of thorough cleaning of sewage in this stage is the prevention of water eutrophication. Stage IV of purification (water renovation) includes the processes of residual sewage removal, which are left over from the previous stages of purification. Water regeneration involves a set of methods which confer the properties of natural water onto the sewage so that it can be utilized in industrial facilities. Water regeneration allows the recycling of sewage, which is a significant element in water resource management, especially in regions low in water. There are several systems of water regeneration, from very simple ones, that use rapid filters or straining through microsieves, to very complex physical chemical processes: coagulation, membrane processes and disinfection, sedimentation, expelling of ammonium, recarbonization, absorption, ion exchange, and water demineralization.

3.1.9 Biological methods of wastewater treatment In biological methods of sewage treatment, bacteria which form zoogleal clusters in sewage play a crucial role. Methods of biological purification of sewage consist of inducing the enzymatic processes of saprophytic microorganisms that include partial oxidation of organic substances (sources of carbon) contained in sewage as well as their partial assimilation by microorganisms. As a result of these processes, an increase in cell mass of the active microorganisms occurs. Microorganisms flourish when the ratio of three basic elements C:N:P = 100:10:1. The biological processes of purification can be divided into natural and artificial, depending on where the processes take place—whether they occur in natural conditions or are intentionally triggered in specially designed artificial equipment. Biological purification can be conducted in oxygen-rich, oxygenpoor or oxygen-free conditions. It is a process of oxidation and mineralization of organic compounds from sewage using micro- and macroorganisms. During the process of biological purification the following phenomena take place:

• breakdown of organic substances down to CO2, H2O, NH3 (dependent on pH)



• nitrification (oxidation of NH3 by Nitrosomonas bacteria down to nitrites, and then by Nitrobacter bacteria down to nitrates),



• denitrification (transformation of nitrates to gaseous nitrogen N)

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Natural methods Natural methods of wastewater treatment include: purification in soil, field and forest irrigation (the method of irrigation and filtration fields) and soil filters.

Purification in soil Biological purification in a field soil consists in irrigation of a field with sewage. Biogenic substances contained in sewage lead to an average of 20% yield increase. A field used for agricultural purposes can receive an annual dose of 600 mm effluent per annum. After spreading, the sewage seeps into the soil and the contents of impurities are absorbed by the soil particles. Prior to introducing sewage onto the fields, sewage undergoes mechanical purification (screens, sand traps, primary settling tank) and is disinfected. Moreover the irrigated soil is checked for the content of metals. Field irrigation can be conducted only during the period of plant vegetation and the amount of the applied sewage has to be altered at different times. In the winter, sewage is purified on filtration fields. After some time, the absorbed organic compounds and microorganisms create a microscopic film around the particles of soil and the surface soil layer works like a biological filter. The final products of the mineralization process taking place in this layer, act as fertilizer for the soil. Only a limited amount of sewage can be purified by this method, otherwise the field becomes excessively loaded with sewage. In such situations, oxygen-free processes are triggered, that are accompanied by the formation of toxic substances and, odour release, causing the plant growth to stop. Due to sanitary reasons, prior to irrigation, sewage must be cleared of any helminth eggs. During the infiltration in soil, sewage gets purified and then carried over to a receiving body of water by a drainage system.

Soil filters Purification by soil filters consists of spreading waste upon the surface of soil that leads to its biological purification. Most often non-cultivable fields are utilized for such forms of purification. The lack of agricultural use allows utilization of greater amounts of sewage (annual dosage of sewage may go as high as 3000 mm/a). Loose and sandy soils with a grain diameter of 0.2-0.5 mm, with strata thickness between 1.5-2.0 m and with a low level of underground water are best suited for this purpose. The field is divided into drying beds (about 0.5 hectare area). Not cultivating the soil can result in greater amounts of sewage being purified. Prior to pouring out the sewage, it has to be mechanically cleaned, in order to eliminate the oily suspension that clogs up the source. The drying beds are flooded with wastes (thickness 5-10 cm) every 0.5-4 days. Purified waste is drained by a drainage system installed in the ground. After some time, the soil filters lose the ability to purify and have to be periodically excluded from operation in order to regenerate. Sewage may

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undergo purification in the winter when it is upon the filtrating fields. The number of active plots is then lowered in order to minimize losses, and instead, the depth of the flooding increases to 20-30 cm. The surface of the sewage covers with ice, under which the sewage is supplied and undergoes the process of purification while decreasing the quality of the outlet.

Sewage ponds Sewage ponds are earth reservoirs, in which the process of biological purification occurs naturally (utilizing microbes) thus; they are used in smaller towns, where the number of inhabitants does not exceed 20,000. Sewage ponds are either natural or artificial ground reservoirs, in which solar radiation reaches the bottom. Prior to introduction of sewage into the pond, sewage has to be preliminarily cleared of suspended matter. Sewage ponds, usually consist of a series of ponds: bacterial, algal and crustaceal. Oxidation of organic compounds by bacteria takes place in the bacterial pond, which leads to their mineralization, i.e. transformation into non-organic compounds known as biogenic salts. Sewage purified in that way is then directed to algal ponds, where algae flourish on it, assimilating the mineral compounds that evolved during the process of biodegradation. The final stage of such purification is conducted in the crustacean pond, where algae eating crustaceans flourish. Such systems of purification allow the elimination not only of organic substances but also of the excess biogenes, whose presence in the receiving body of water could cause eutrophication and consequently water blooming which lowers the oxygen content in water. This pond can be used for fish and duck breeding without a need for artificial feeding.

Hydrobotanic purification Hydrobotanic purification consists of utilization of self-purifying processes that take place in waterlogged ecosystems, thus it belongs to so-called wetland systems. Purification is a result of the co-operation of soil microorganisms and boggy plants. Microorganisms decompose the organic compounds contained in sewage, turning them into non-organic compounds, whereas plants absorb the produced mineral compounds creating a plant biomass. The adsorption of impurities by the particles of soil is improved, due to very small mineral particles (silt) present in the substrate. This type of treatment unit utilizes wetland vegetation (common-reed, reed-mace, basket willow, etc.) that has a high requirement for food, thus it absorbs large quantities of mineral salts. As a result, the vegetation desalts the sewage and does not lead to eutrophication of water reservoirs. There are three types of hydrobotanic purification plants: soil-plant filters—are the types of filters with horizontal (most common and the longest in use), vertical or combined flow; mainly sandy with rooted boggy vegetation (common-reed, reed-mace, schrubby willows).

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shallow reservoirs with rooted vegetation—these include water reservoirs or ducts of depths between 10-50 cm, they are occupied by boggy vegetation (common-reed, reed-mace, sedge). sealed reservoirs with floating vegetation—ponds of depth between 1-2 m, with sealed bottoms and side walls, with floating vegetation – in our climatic conditions it is duckweed – Lemna minor. Exploitation problems are connected with an even spread of duckweed throughout the surface of the pond and removal of the rapidly growing plants. In the winter, due to lack of vegetation, the purification plant plays the role of a normal pond.

3.1.10 Artificial methods of wastewater treatment Trickling filters: The treatment of sewage by trickling filters is conducted in reservoirs filled with loose, grainy and porous material. Sewage is sprayed upon the upper layer of the bed with sprinklers and then left to seep through its content. A mucous biological film forms upon the content of the bed. The film is composed of microorganisms such as: bacteria, protozoa and fungi. The role of the filter involves a constant supply of sewage and its flow through the trickling filter while maintaining contact with the biological film. During the flow, the sewage undergoes mineralization as a result of aerobic decomposition by microorganisms. The biological film is initially composed of zoogleal bacteria which produce mucous sheets. With time, the composition of species of the mucous membrane changes due to their succession. Besides

Trickling filter

The trickling filter process

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bacteria, the following appear: fungi, protozoa, annelida and fly larvae. Depending on the amount of treated sewage, the trickling filters may be subdivided into percolating and flushing filters. Depending on the amount of the organic load, the following types of biofilters are distinguished:

• Low–loaded – may be filled with natural or artificial material. The supplied organic material is less than 0.4kg BZT5/m3·d. In percolating filters, the film is more developed and the biological process of decomposition is almost complete. In the final phase of purification, intensive processes of nitrification occur, which lead to an increase of nitrates in a run-off to the secondary settling tank.



• Mid-loaded – are filled with natural-synthetic material and work with a load between 0.4-0.65 kg BZT5/m3·d. In order to ensure an adequate concentration of the supplied sewage, the recirculation of part of the purified sewage is utilized with this type of filters. The reduction of organic compounds upon these filters is adequate, and the processes of nitrification partially occur. The introduction of additional processes of purification is not necessary.



• High loaded (flushed) — are filled with natural-synthetic material, the filter is loaded with: 0.65-1.6kg BZT5/m3·d. In flushing filters, the intensity of sewage flow is greater, however, the biofilm is composed almost entirely of bacteria and does not develop as much as in the above stated case. Flowing sewage washes out used and dead biological material from the filter. The washed out material is transported in the form of flocy sediment. Only a partial mineralization of organic compounds occures on that type of filter and the nitrification process is inhibited. A low content of nitrates in effluent from filters testifies to partial mineralization of organic compounds. In complex systems, after these types of filters, re-purification is utilized, as the quality of the purified sewage does not usually meet the required standards.

Activated sludge The process of activated sludge relies on sewage purification by freely suspended matter. It consists of producing 50-100 mm flocs with highly developed surface areas. The floc is made up of brown or beige mineral nucleus, while on its surface, it contains heterotrophic bacteria within the mucous envelopes. The method of activated sludge requires delivery of oxygen into the substrate for bio-oxidation of organic pollutants, which should be > 0.5 mg/dm3 in order to ensure proper oxygen conditions for the bacteria.

Activated sludge characteristics Activated sludge is a type of flocculent suspended matter created during the aeration of sewage. Treating sewage with activated sludge consists of

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mineralization of organic compounds, conducted mainly by bacteria and following the same biochemical processes as observed in self-purification. However, the speed of the process is much greater. This results from the fact that the conditions of intensive aeration, triggered during sewage flow through aeration tanks, are conducive to the development of impurity-decomposing bacteria. Agglomeraters (flocs), which consist of heterotrophic bacteria coagulated with mucous, form during the process of aeration in aeration tanks (flocculation). The floccules absorb impurities contained in sewage, whereas microorganisms in floc decompose the absorbed substances. Activated sludge has a spongy, loose structure, made of small openings of various shapes. Undisturbed floccules easily settle and thus, allow the separation of the activated sludge from sewage. Biocenosis of activated sludge is, for the most part, composed of heterotrophic bacteria. In small percentages—and only under particular conditions and in some arrangements—it’s made up of chemolithotrophic bacteria, especially nitrifying bacteria. The most common species of activated sludge are: Zooglea ramigera , Pseudomonas fluorescens, Pseudomonas putida as well as bacteria of Achromobacter, Bacillus, Flavobacterium and Alcaligenes genera. The process of selection occurs naturally. The conditions in an aeration tank, especially the chemical composition, pH value and air conditions, are the determining factors for the diversity of the bacterial complex. In unfavourable conditions (overloading of aeration tanks with easily available substrates, high oxygen deficit), excess development of flocs occurs causing the so-called activesludge swelling. There are two distinguishable types of swelling: fibrous and non-fibrous swelling. Fibrous swelling is caused by excess filiform bacteria (Sphaerotilus natans, Beggiatoa alba or Thiothrix nivea) or fungi development. Non-fibrous swelling is caused by bacterial development, which produce excess amounts of mucous. Active sludge biocenosis is made up of not only bacteria but also protozoa, nematodes and rotifers. Even though these microorganisms do not play a major role, their presence is equally important. Protozoa feed upon bacterial cells forcing them to reproduce quickly, which essentially make them an important renewal and reactivating factor of the activated sludge. The most common protozoa are: Vorticella, Carchesium and Opercularia as well as Anthophysa, Oxytricha, Stylonychia and Lionotus. There is an inverse relationship between flagellates and ciliates within activated sludge. While a large number of flagellates indicate an overload of sludge, the presence of ciliates goes to show it is functioning properly. During the course of sewage purification with activated sludge, a characteristic succession of biocenosis is observed. Activated sludge process: Sewage is directed to aeration tanks filled with activated sludge (thick suspension of microorganisms) after its mechanical purification. The content of the aeration tank is constantly aerated in order

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to provide an adequate amount of oxygen, to keep the activated sludge in a suspended state and to ensure its constant mixing. The aeration tank is a device, in which the development of the activated sludge results from continuous cultivation. There is a state of equilibrium between the rate of sewage inflow, concentration of nutrients, bacterial reproductive rate, and the rate of the sewage outflow containing some activated sludge in it. During the time of contact of sewage with the activated sludge, the decomposition processes occurring simultaneously enable the development of activated sludge biomass. Separation of purified sewage is done in a secondary settlement tank. Both sedimentation and clarification of the purified sewage, which is then carried off to a receiving body of water, occurs in the device. Activated sludge may be used again for purification; it is then recycled into the aeration chamber. However, quite often, before reuse, the sludge is directed to a regenerative chamber, where it is aerated in order to bring back its particular physiological properties. When the sludge collected in a secondary settling tank is not recycled, then, as an excess sludge, it is removed and subjected to additional processing.

The activated sludge process

3.1.11 Methods of chemical wastewater treatment Purification of industrial sewage that contains mineral and organic compounds, and heavy metals, utilizes physical-chemical and chemical methods. They include the following processes: neutralization, coagulation, oxidation, reduction, sorption, flotation, membrane processes, extraction, electrolysis, distillation. Neutralization: It is a process of chemical neutralization of sewage in relation to the pH. Depending on the make-up of sewage and the type of the reacting substance used, neutralization may be accompanied by a chemical process of precipitation and coprecipitation. Neutralization may be conducted by mixing acidic sewage with bases. Hydroxides are substances most often

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used in the process of neutralization: NaOH in the form of 20-30% solution, Ca(OH)2 in the form of 5-15% milk of lime, Na2CO3 in solution form CaCO3, MgCO3, MgO, dolomite in the form of a grainy filter. Mineral acids are used for the neutralization of basic sewage: H2SO4, HCl, H3PO4 in the form of solutions as well as CO2 in the form of a clear gas. Coagulation is a process of binding colloidal particles and the suspension into clusters of particles called the agglomerates, which results in precipitation of the sediment in the form of coagulate. The factors which most often cause coagulation are: addition of an electrolyte solution to lower the electrolytic potential, addition of colloids of an opposite charge into the colloidal particles, creation of metal hydroxides that absorb ions, colloids and suspensions. Oxidation: An oxidation process is conducted in order to remove organic compounds, non-organic compounds and microorganisms from sewage. The reacting substances used in oxidation are: chlorine, chlorine-oxidizing compounds (NaOCl, Ca(OCl)2, chlorinated lime, chlorine dioxide, ozone. Reduction: The process of reduction used in sewage purification mainly concerns chromium. Chromium salts (VI) are toxic, carcinogenic, bacteriocidal and are irritants to skin. Its bacteriocidal properties slow down the process of water self-purification. Reduction of chrome from oxidation state of 6+ down to 3+ is conducted through reduction and precipitation of hydroxide, which belongs to a group of barely soluble compounds. Reduction is conducted either chemically or electrochemically. Sorption: Sorption consists of binding liquid soluble substances to the surface of solids. Depending on the characteristics of the process, it may be irreversible (chemiosorption), or reversible—adsorption. The characteristic of the process of sorption is determined by one of the components of force: • physical sorption — the result of van der Waals forces, • chemical sorption — the result of valence forces, • ion sorption — between groups of cations and anions in the structure of the substrate, • sieve sorption — at the molecular level according to the mechanism of a molecular sieve. Flotation: A process of structural separation consisting of raising the hydrophobic impurities into the foam along with the rising gas bubbles. As a result, the foam formed has a much higher concentration of pollutants than the rest of the sewage. Membrane processes: These processes consist of separation of particles by flowing through a porous layer (membrane). The following are the types of membrane processes: reversed osmosis, nanofiltration, ultrafiltration, electrodialysis. Extraction: This consists of transfer of components from one phase of the solution into the second liquid phase (dissolvent). Consequently, a solution of

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the component in the dissolvent is obtained. The required condition for the process is the presence of two liquid phases. Electrolysis: The process in which electrical energy invokes chemical changes of the electrolyte. As a result of the electrical field, the movement of ions toward the electrodes (upon which the process occurs) occurs: cathode

Me+ + e– → Me (reduction)

anode X– → X + e– → (oxidation) Distillation: Process that utilizes the difference between the composition of a liquid and vapour in the state of equilibrium.

3.1.12 Nucleic Acid - Based Techniques for Analyzing the Diversity, Structure, and Dynamics of Microbial Communities in Wastewater Treatment Biological wastewater treatment systems, like activated sludge basins, trickling filters, or anaerobic digesters, essentially can be interpreted as specialized aquatic ecosystems, where microorganisms are the main players. In order to fully characterize, understand, and, in the long run, control those microbial communities, knowledge of both their structure and function is necessary. Attention should, therefore, be given to identification, enumeration, and spatial distribution (structural parameters) as well as to in situ activities (functional parameter) of the community members. Furthermore, from an ecological and from an engineering point of view stability or dynamics of these microbial communities are important both for theory and practice. Until recently, identification of microorganisms required the isolation of pure cultures and the investigation of physiological and biochemical traits. Enumeration had to be done by plate counts or most probable number (MPN) techniques. However, since all cultivation-dependent techniques are not only time-consuming and laborintensive but select for certain organisms, they are inadequate for determining reliable cell numbers, and in many cases even for the identification of the main catalysts of a system. Questions like micro-scale distribution and in situ activity of microorganisms are almost impossible to address by classical methods. To circumvent these limitations, identification techniques based on nucleic acids have been developed and successfully applied to wastewater treatment systems during the last five years. This chapter intends to summarize the potential, current applications, and limitations of nucleic acid-based techniques for analyzing the microbial communities present in wastewater treatment systems.

Ribosomal Ribonucleic Acid (rRNA)-Based Methods Macromolecules like rRNA or proteins can be used as “molecular clocks” for evolutionary history. By comparative sequence analysis, reconstruction of

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evolution and classification of organisms based on their phylogeny is possible. For several reasons 16S, rRNA sequence comparison is currently considered the most powerful tool for the classification of microorganisms.

• Ribosomes and consequently rRNA molecules are present in all organisms. As an essential component of the protein synthesis apparatus, they have a homologous origin and show functional constancy. No lateral gene transfer has been shown for rRNA genes so far. Therefore, it is a valid assumption to reconstruct the phylogeny of the organisms based on these molecules.



• Some positions of the rRNA molecules are evolutionary more conserved than others. Consequently, sequence regions can be found that allow differentiation at any taxonomic level from species and genera up to kingdoms or domains.



• 16S rRNA sequences have been determined for many of the described bacterial species and deposited in public databases.



• The natural amplification of rRNA within microbial cells (usually more than 1,000, frequently several 10,000 copies) makes it easier and more sensitive to assay this molecule and gives, e.g., the opportunity for identification of single bacteria by fluorescent oligonucleotide hybridization.



• The presence and abundance of ribosomes and, consequently, rRNA in individual cells is connected to their viability and general metabolic activity, or at least their metabolic potential. Cells in a rapidly growing E. coli culture need and have more ribosomes than those in a slowly growing culture.

The applications of rRNA-based nucleic acid techniques to the analysis of wastewater treatment systems today range from a simple identification of isolates over the detection of bacterial diversity and population dynamics to attempts at fully and quantitatively describing the complex microbial communities.

Molecular Characterization of Isolates Despite the success of molecular methods in analyzing microbial communities, classical isolation of bacteria is still absolutely necessary for, e.g., investigations on the metabolic potential of the community members. However, nucleic acidbased techniques can support this classical approach by providing tools for screening, characterization, and identification of isolates. Compared with microbiological or biochemical methods, molecular methods are more rapid and more reliable since they are not affected by growth conditions and culture media.

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Restriction Fragment Length Polymorphism (RFLP) and Amplified Ribosomal DNA Restriction Analysis (ARDRA) A common methodology for a rapid molecular characterization of isolates is based on the generation of so-called “genetic fingerprints”. DNA of an organism is digested by rare cutting restriction enzymes, and the resulting fragments are separated by length using pulsed-field gel electrophoresis. Different species will have differing fragment lengths due to mutations of the restriction sites, insertions, or deletions, and these restriction fragment length polymorphisms (RFLPs) are used for comparison of the isolates. Since cutting the whole genome requires high quality DNA extraction prior to RFLP analysis and time-consuming, complicated separation of the rather large fragments by pulsed-field gel electrophoresis, most recent applications of RFLP are based only on part of the genome. For example, amplified ribosomal DNA restriction analysis (ARDRA) starts with the amplification of the genes encoding 16S rRNA by polymerase chain reaction (PCR), followed by restriction digestion and analysis of the fragment lengths by standard gel electrophoresis. The resulting patterns or fingerprints are compared to reference strains and can be used for cluster analysis of different isolates. In principle, a database of restriction patterns of reference organisms can be established to facilitate rapid identification of isolates by ARDRA. ARDRA allows the processing of numerous isolates in a very short time, and can yield valuable information on the similarity of isolates. Consequently, appropriate isolates can be selected for further physiologic and phylogenetic investigations. On the other hand, reliable identification using ARDRA is hampered by the enormous amount of reference patterns required. Furthermore, the information content in an ARDRA pattern is limited (frequently, only few bands) and this could cause false-positive identification.

Other Methods Besides ARDRA, the methods described in the following sections can also be applied. Denaturing gradient gel electrophoresis is ideal to screen a large set of isolates for redundancy. Hybridization with sets of rRNA–targeted probes also allows rapid screening of isolates for their phylogenetic affiliation. By this technique, e.g., the majority of isolates from a municipal activated sludge was characterized as members of the gamma subclass of Proteobacteria. In a second example, genus or species-specific probes were used for confirming the identification of Paracoccus sp., initially based on physiological tests. Finally, the full description of a new species, identified to be important in a given system, should always include the determination of its 16S rRNA sequence for valid phylogenetic classification and, if applicable, the DNA–DNA hybridization with related species or strains.

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Diversity and Dynamics of Microbial Communities Microbial diversity and population dynamics can be rapidly monitored using rRNA-based pattern or fingerprinting methods. These encompass extraction of total nucleic acids (DNA and/or RNA) from an environmental sample (like activated sludge or biofilms), amplification of part of the genes encoding 16S rRNA by PCR, and subsequent separation of the resulting gene fragments on a gel to form a pattern or fingerprint of the community.

Denaturing Gradient Gel Electrophoresis (DGGE) Method: By DGGE, DNA fragments of the same length but with different sequences can be separated. 16S rRNA gene fragments of a length of typically 200–500 bp are amplified by PCR with an additional 40 bp GC-rich sequence at the 5b end of one of the primers. When analyzed on a polyacrylamide gel containing an increasing gradient of DNA denaturants (a mixture of urea and formamide), for each of these DNA molecules (ds DNA), a transition from a double-stranded, helical to a partially single-stranded secondary structure will occur at a certain position in the gel. This will stop, or at least strongly slow down, the migration of the respective gene fragment. The GC-rich region

Flow chart of a community analysis by denaturing gradient gel electrophoresis (DGGE)

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acts as a “clamp” to prevent formation of single-stranded DNA (ssDNA). As sequence variations cause a difference in the melting behavior of ds DNA, sequence variants of particular fragments will stop at different positions in the denaturing gradient gel and hence can be separated effectively (Fig.1). The result of DGGE analysis of PCR products obtained from a microbial community is a band pattern, and the number of bands is a rough estimate for the microbial diversity of a given system. Applications: The patterns are frequently used for comparisons of different systems, e.g., aerobic and anaerobic biofilms or different activated sludge plants. DGGE is particularly useful to detect population changes addressing the question of stability and dynamics of microbial communities. The method can be modified by using rRNA instead of DNA as a template for 16S rDNA amplification. For that purpose, extracted rRNA is transcribed into ribosomal copy DNA (rcDNA) by the enzyme reverse transcriptase prior to the PCR amplification. While the rDNA-based DGGE pattern is solely determined by the presence of DNA and, therefore, in first approximation by the abundance of populations, the rRNA-based DGGE pattern should more strongly represent the metabolically active and, therefore, rRNA-rich parts of the community. A band at a given site of a DGGE gel has per se no biological meaning. Therefore, to learn more about the population represented by a certain band, this band needs to be further characterized, which can be achieved, in principle, by two techniques. The DGGE gel can be blotted to a nylon membrane and the pattern can be examined by hybridization analysis with taxon-specific probes. Alternatively, bands can be retrieved from the gel and subsequently sequenced; comparative sequence analysis then allows identification or at least affiliation with the closest relative. Limitations: Whereas DGGE analysis of rDNA PCR products is a powerful tool to analyze diversity and dynamics of microbial communities, it has severe limitations in the analysis of community structures, and is like any other method prone to specific biases: (1) The method involves extraction of nucleic acids and subsequent tPCR, which may both cause some bias: Not all cells lyse under the same conditions, and preferential amplification of certain templates can occur. Therefore, different intensities of DGGE bands must not be interpreted as quantitative measures of the abundance of species relative to each other. For qualitative statements on the development of a certain band (population) over time, i.e., to monitor its appearance or disappearance, identical treatment of all samples has to be ensured.

(2) Separation of DNA fragments with high resolution is restricted to a maximum size of about 500 bp. Consequently, the phylogenetic information that can be retrieved by sequencing is relatively little. In case of full identity with an rRNA sequence in a database, it might

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be sufficient for an identification, but in cases in which only distantly related sequences are available, classification becomes difficult if not impossible. Also characterization of bands by hybridization analysis with rRNA-targeted oligonucleotide probes is only possible if the probe target region is within the amplified fragment, which is only the case for a small fraction of the full set of available probes. (3) The main difficulty, however, is the “one band – one species” hypothesis. Especially in complex communities, bands might originate from two or more fragments that co-migrate on the denaturing gradient gel. Furthermore, single species might result in two or more DGGE bands due to inter-operon microheterogeneity.

Terminal Restriction Fragment Length Polymorphisms (T-RFLP) Recently, RFLP analysis has been modified for application to microbial communities: After extraction of total DNA from an environmental sample, 16S rDNA is amplified by PCR with one of the two primers being fluorescently labeled. The fluorescent PCR products are then digested with frequently cutting restriction enzymes, and analyzed by a standard high resolution gel electrophoresis in which the restriction fragments are separated solely by size. The abundance and length of only the fluorescent terminal fragments is determined in automated sequencing devices or by fluorimetry. This again yields a pattern or “community fingerprint”, like in the case of DGGE. The method may be suitable for analyzing diversity and dynamics of microbial communities as demonstrated for activated sludge. Besides similar advantages, it suffers even more from similar limitations as DGGE: The length of the terminal rDNA fragments ranges from 60–500 bp, too little information for valid phylogenetic affiliation or application of specific oligonucleotide probes. Different species may have in many cases the same terminal restriction fragment length, hence leading to an underestimation of the actual bacterial diversity. T-RFLP seems to be an easier, but also a less sensitive alternative to DGGE.

Community Structure The structure of a microbial community is mainly defined by two parameters: Identity and abundance of its members. A broader definition of structure would also include the spatial arrangement of species relative to each other, an information which might be especially important in stratified habitats like biofilms. Since both cultivation and fingerprint methods are not sufficient to address these questions, hybridization techniques applying rRNA-targeted oligonucleotide probes have been developed. Oligodeoxynucleotides are single-stranded pieces of DNA with a length of 15–25 nucleotides. When such oligonucleotides are labeled, e.g., with radioisotopes or fluorescent dye molecules, they become so-called probes that allow detection of complementary target sites by specific base pairing.

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For the reasons outlined before, target molecules are rRNAs, extracted and immobilized on a membrane (dot blot hybridization), or maintained in fixed cells (whole cell hybridization), respectively. In both cases, quantification is possible, either relative to total extracted rRNA, or relative to total cell counts.

3.2

AIR AS AN ENVIRONMENT OF MICROORGANISMS



• Air is an unfavorable environment for microorganisms, in which they cannot grow or divide. It is merely a place which they temporarily occupy and use for movement. • Therefore, there are no metabolic connections occurring between different microorganisms in air (such as in soil or water). As a result, they form only a random collection of microorganisms, not a microbiocenosis. • Microorganisms get into air as a consequence of wind movement, which sweeps them away from various habitats and surroundings (soil, water, waste, plant surfaces, animals, and others), or are introduced during the processes of sneezing, coughing, or sewage aeration. Why are the air conditions unfavourable for the microorganisms? There are 3 elementary limiting factors in the air: • a lack of adequate nutrients, • frequent deficit of water, threat of desiccation, • solar radiation. It is obvious that the first factor limits cell growth. As a matter of fact, air, and especially polluted air, contains some organic substances, but they are usually poorly decomposed and there is not enough to be utilized as food. Besides, there are other unfavorable factors contributing. Microorganisms contained in air are constantly subjected to drying, which definitely stops all processes. Some bacteria are especially sensitive to water deficits which cause bactericidal effects (e.g., gonococci or spirochete which die as soon as they enter the air). Many organisms, however, can successfully cope with water deficits and, although they cannot function properly, their dried up forms survive months and even years (endospores, fungi spores). Solar radiation is also damaging to microorganisms suspended in air as it causes mutation and desiccation (in water and soil, the solar radiation is usually very weak or simply does not exist)

3.2.1 Adaptation of microorganisms to the air environment What types of microorganisms occur in air? There are 3 main groups of microorganisms that occur in air: • viruses • bacteria • fungi

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Bacteria may exist as vegetative or resting forms, however, fungi occur in the form of spores or fragments of mycelium. Especially in the vegetative season, pollen of anemophilous plants (e.g., grasses and some trees) is abundant in the air. Besides the above, the following can be found in air as well: algae and protozoa cysts and small invertebrates such as worms in forms of eggs or cysts and mites. Besides living microorganisms, their fragments and products, which often exhibit toxic or allergic activities, may also occur in air. Which microorganisms are best adapted to a prolonged existence in air? The atmosphere can be occupied for the longest time by those forms which, due to their chemical composition or structure, are resistant to desiccation and solar radiation. They can be subdivided into the following groups:

• bacterial resting forms,



• bacterial vegetative forms which produce carotenoidal dyes or special protective layers (capsules, special structure of cell wall),



• spores of fungi,



• viruses with envelopes.

Resting forms of bacteria Endospores are the best known resting forms. These structures evolve within cells and are covered by a thick multi-layer casing. Consequently, endospores are unusually resistant to most unfavorable environmental conditions and are able to survive, virtually endlessly, in the conditions provided by the atmospheric air. They are only produced by some bacteria, mainly by Bacillus and Clostridium genera. Because each cell produces only one endospore, these spore forms cannot be used for reproduction. Another type of resting form is produced by very common soil bacteria, the actinomycetes. Their special vertical, filiform cells, of the so-called air mycelium, undergo fragmentation producing numerous ball-shaped formations. Due to the fact that their production is similar to the formation of fungal, they are also called conidia. Contrary to endospores, the conidia are used for reproduction. There are also other bacterial resting forms, among others, the cysts produced by azotobacters — soil bacteria, capable of molecular nitrogen assimilation. The production of carotenoidal dyes ensures cells with solar radiation protection. Carotenoids, due to the presence of numerous double bonds within a molecule (–C=C), serve a purpose as antioxidants, because, as strong reducing agents, they are oxidized by free radicals. Consequently, important biological macromolecules are being protected against oxidation (DNA, proteins, etc.). Bacteria devoid of these dyes quickly perish due to the photodynamic effect of photooxidation. That explains why the colonies of bacteria, which settle upon open agar plates, are often coloured (Fig. 2.2). The ability to produce

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carotenoids is possessed especially by cocci and rod-shaped actinomycetes. Rod-shaped actinomycetes, e.g. Mycobacterium tuberculosis, besides being resistant to light, also demonstrate significant resistance to drying due to a high content of lipids within their cell wall. High survival rates in air are also a characteristic for the bacteria which possess a capsule, e.g. Klebsiella genus, that cause respiratory system illnesses. Fungal spores: Spores are special reproductive cells used for asexual reproduction. Fungi produce spores in astronomical quantities, for example the giant puffball (Calvatia gigantea) produces 20,000,000,000 (20 billion!) spores, which get into the air and are dispersed over vast areas. A very common type of spores found in air is that of conidia. Conidia (gr. konia - dust) are a type of spore formed by asexual reproduction. They form in the end-sections of vertical hyphae called conidiophores and are dispersed by wind. The spores of common mould fungi such as Penicillium and Aspergillus are examples of the above. Spore plants such as ferns, horsetails and lycopods also produce spores. Plant pollen is also a kind of spores. Resistant viruses: Besides cells, the air is also occupied by viruses. Among those that demonstrate the highest resistance are those with enveloped nucleocapsids, such as influenza viruses. Among viruses without enveloped nucleocapsids, enteroviruses demonstrate a relatively high resistance. Of course, besides the previously mentioned resistant forms, the air is also occupied by more sensitive cells and viruses, but their survival is much shorter. It is believed, that among vegetative forms, gram-positive bacteria demonstrate greater resistance than gram-negative bacteria (especially for desiccation), mainly due to the thickness of their cell wall. Viruses are usually more resistant than bacteria.

Biological aerosols Microorganisms suspended in air as a colloidal system: Microorganisms in air occur in a form of colloidal system or the so-called bioaerosol. Every colloid is a system where, inside its dispersion medium, particles of dispersed phase occur whose size is halfway between molecules and particles visible with the naked eye. In the case of biological aerosols, it’s the air (or other gases) that has the function of the dispersion medium, whereas microorganisms are its dispersed phase. However, it is quite rare to have microbes independently occurring in air. Usually, they are bound with dust particles or liquid droplets (water, saliva, etc.), thus the particles of the bioaerosol often exceed microorganisms in size and may occur in two phases: • dust phase (e.g. bacterial dust) or • droplet phase (e.g. formed as the result of water-vapour condensation or during sneezing). The dust particles are usually larger than the droplets and they settle faster. The difference in their ability to penetrate the respiratory tract is dependent on

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the size of the particles; particles of the droplet phase can reach the alveoli, but dust particles are usually retained in the upper respiratory tract. The number of microorganisms associated with one dust particle is greater than in the droplet phase.

The size of bioaerosols The average size of bioaerosols ranges from about 0.02 ìm to 100 ìm. The sizes of certain particles may change under the influence of external factors (mainly humidity and temperature) or as a result of larger aggregates formed. By using size criterion, the biological aerosol can be subdivided into the following:

• fine particles (less than 1 µm), and



• coarse particles (more than 1 µm)

Fine particles are mainly viruses, endospores and cell fragments. They possess hygroscopic properties and make-up the so-called nucleus of condensation of water vapour. At high humidity, water collects around these particles creating a droplet phase. Then, the diameter of the particles increases. Coarse particles consist mainly of bacteria and fungi, usually associated with dust particles or with water droplets.

CHAPTER

4 4.1

Environmental Microbiology— Methods and Applications

MICROORGANISMS-METAL TRANSFORMATIONS

Microorganisms can mobilize metals and radionuclides through autotrophic and heterotrophic leaching, chelation by microbial metabolites and siderophores, and methylation, which can result in volatilization. Conversely, immobilization can result from sorption to cell components or exopolymers, transport into cells and intracellular sequestration or precipitation as insoluble organic and inorganic compounds, e.g. oxalates, sulphides or phosphates. In bioremediation, solubilization provides a route for removal from solid matrices such as soils, sediments, dumps and industrial waste. Alternatively, immobilization processes may enable metals to be transformed in situ into insoluble and chemically inert forms and are also particularly applicable to removing metals from mobile aqueous phases.

Simple model of microbial roles in the environmental mobility of metals. Metal entry into the environment [1] is shown as well as the importance of abiotic environmental components in affecting metal speciation and microbial populations [2] The major influence of microbes in effecting transformations between soluble and insoluble phases is emphasised [3,4]

Metal Mobilization Autotrophic (chemolithotrophic) leaching: Metals can be leached from solid matrices as a result of autotrophic metabolism. Most autotrophic leaching

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is carried out by chemolithotrophic, acidophilic bacteria which fix carbon dioxide and obtain energy from the oxidation of ferrous iron or reduced sulphur compounds. These metabolic processes yield Fe(III) or H2SO4 as the respective end products. The microorganisms involved in autotrotrophic leaching include sulphur-oxidizing bacteria, e.g. Thiobacillus thiooxidans, iron- and sulphur-oxidizing bacteria, e.g. Thiobacillus ferrooxidans and ironoxidizing bacteria, e.g. Leptospirillum ferrooxidans. As a result of sulphur and iron-oxidation by these bacteria, metal sulphides are solubilized and the pH of their immediate environment is decreased, which enhances the solubilization of other metal compounds. The autotrophic leaching of metal sulphides by Thiobacillus species and other acidophilic bacteria is well established for use in industrial scale biomining processes. In a bioremediation context, production of sulphuric acid by Thiobacillus species has been used to solubilize metals from sewage sludge, thus enabling separation from the sludge which can then be used as a fertiliser. Autotrophic leaching has been used to remediate other metal-contaminated solid materials including soil and red mud, the main waste product of Al extraction from bauxite. Heterotrophic (chemoorganotrophic) leaching: Heterotrophic metabolism can also lead to leaching as a result of the efflux of protons, organic acids and siderophores. Organic acids provide both protons and a metalchelating anion to complex the metal cation. Citrate and oxalate anions can form stable complexes with a large number of metals. Uranium forms very stable 1:1 and 1:2 uranium-citrate complexes with stability constants that are much higher than those of uranyl acetate, uranyl lactate, UEDTA and uranyl ascorbate complexes. Many metal citrates are highly mobile and not readily degraded and the presence of citric acid in soil may enhance contaminant metal solubility for a significant time. Oxalic acid can also act as a leaching agent for those metals that form soluble oxalate complexes, including Al and Fe. Many fungi are able to leach metals from industrial waste and byproducts, low-grade ores and metal-bearing minerals. Heterotrophic solubilization can have consequences for other remedial treatments for contaminated soils. Pyromorphite (Pb5(PO4) 3Cl) is a stable lead mineral and can form in urban and industrially-contaminated soils. Such insolubility reduces lead bioavailability and the formation of pyromorphite has been suggested as a remediation technique for lead-contaminated land, if necessary by means of phosphate addition. However, pyromorphite can be solubilized by phosphate-solubilizing fungi, e.g. Aspergillus niger, and plants grown with pyromorphite as a sole phosphorus source accumulate both P and Pb. During fungal transformation of pyromorphite, biogenic production of lead oxalate dihydrate was observed for the first time. This study emphasises the importance of considering microbial processes in developing remediation techniques for metal-contaminated soils. Another method for treatment of metal-contaminated sandy soil relied on siderophore-mediated metal solubilization by Alcaligenes eutrophus. Solubilized metals were adsorbed to

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the biomass and/or precipitated, with biomass separated from a soil slurry by a flocculation process. This resulted in a complete decrease in Cd, Zn and Pb bioavailability. Related to heterotrophic solubilization is fungal translocation of, e.g. Cs, Zn and Cd, which can lead to concentration in specific regions of the mycelium and/or in fruiting bodies. Whether the concentration factors observed in vitro can be reproduced in the field and, whether such amounts can contribute to soil bioremediation, remains uncertain. Reductive mobilization: Fe(III) and Mn(IV) oxides absorb metals strongly and this may hinder metal extraction from contaminated soils. Microbial reduction of Fe(III) and Mn(IV) may be one way for releasing such metals and this process may be enhanced with the addition of humic materials, or related compounds. Such compounds may also act as electron shuttles, for e.g., U(VI) and Cr(VI), converting them to less soluble forms, especially if located in tight pore spaces where microorganisms cannot enter. The solubility of certain radionuclides can also be increased by reduction and this may favour their removal from matrices such as soils. For example, iron-reducing bacterial strains solubilized 40% of the Pu present in contaminated soils within 6-7 days through reduction of Pu(IV) to the more soluble Pu(III) and both ironand sulphate-reducing bacteria were able to solubilize Ra from uranium mine tailings, although solubilization occurred largely by disruption of reducible host minerals. The mechanism of bacterial Hg2+ resistance is enzymic reduction of Hg2+ to non-toxic volatile Hg0 by mercuric reductase. Hg2+ may also arise from the action of organomercurial lyase on organomercurials. Since Hg0 is volatile, this could provide one means of mercury removal. Methylation of metalloids: Microbial methylation of metalloids to yield volatile derivatives, e.g. dimethylselenide or trimethylarsine, can be effected by a variety of bacteria, algae and fungi. Microbial methylation of selenium, resulting in volatilization, has also been used for in situ bioremediation of selenium-containing land and water at Kesterson Reservoir, California.

Metal Immobilization Biosorption: Biosorption is the uptake of organic and inorganic metal species, both soluble and insoluble, by physico-chemical mechanisms such as adsorption. In living cells, metabolic activity may also influence this process. Almost all biological macromolecules have some affinity for metal species with cell walls and associated materials being of the greatest significance. Biosorption can also provide nucleation sites for the formation of stable minerals as well as sorption to cellular surfaces cationic species can be accumulated within cells via transport systems of varying affinity and specificity. Once inside cells, metal species may be bound, precipitated, localised within intracellular structures or organelles, or translocated to specific structures depending on the element concerned and the organism. Freely-suspended and immobilized microbial biomass has received attention with immobilized systems possessing

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advantages which include higher mechanical strength and easier biomass/ liquid separation. Immobilized living biomass has mainly taken the form of bacterial biofilms on inert supports and is used in a variety of bioreactor configurations including rotating biological contactors, fixed bed reactors, trickle filters, fluidized beds and air-lift bioreactors. A range of specific and non-specific metal-binding compounds are also produced by microorganisms. Non-specific metal-binding compounds range from simple organic acids and alcohols to macromolecules such as polysaccharides, humic and fulvic acids. The metal-binding abilities of siderophores, metallothioneins, phytochelatins and other biomolecules also have potential for bioremediation. However, the earlier commercial promise and development of biosorption appears to have largely ceased and there is no adoption of biosorption as a commercially viable treatment method to date. The lack of commercial development is somewhat perplexing although the lack of specificity and lower robustness of biomassbased systems compared to ion exchange resins is often cited as a reason. Metal precipitation by metal-reducing bacteria: Where reduction of a metal to a lower redox state occurs, mobility and toxicity may be reduced, offering potential bioremediation applications. Such processes may also accompany other indirect reductive metal precipitation mechanisms, e.g. in sulphate-reducing bacterial systems where reduction of Cr(VI) can be a result of indirect reduction by Fe2+ and sulphide. A diverse range of metal reducing bacteria can use oxidized species of metallic elements, e.g. Fe(III), Cr(VI) or Mn(IV) as terminal electron acceptors. For example, a strain of Shewanella (Alteromonas) putrefaciens which reduces Fe(III) and Mn(IV) also reduces U(VI) to U(IV), forming a black precipitate of U(IV) carbonate. Bacterial uranium reduction has also been combined with chemical extraction to produce a potential process for soil bioremediation. Desulfovibrio desulphuricans can reduce Pd(II) to cell-bound Pd(0) with hydrogen-dependent reduction being O2-insensitive, providing a means of aerobic Pd recovery. Reduction of metalloid oxyanions: Se(VI) reduction to elemental insoluble Se(0) has been employed to remediate contaminated waters and soils. Some bacteria can use such reduction to support growth, making this a natural process for in situ applications. Though reduction of oxyanions of As and Se can occur by different mechanisms, the most environmentally significant process is dissimilatory reduction. Oxyanions of arsenic and selenium can be used in microbial anaerobic respiration as terminal electron acceptors, providing energy for growth and metabolism. Their reduction can be coupled to organic substrates, e.g. lactate, acetate and aromatics, with the bacteria found in a range of habitats and not confined to any specific genus. These organisms, and perhaps even the enzymes themselves, may have applications for bioremediation of selenium- and arsenic-contaminated environments.

Exposed reservoir sediments were flooded to create anoxic conditions, in which the natural bacterial population reduced and immobilized large

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quantities of the selenium that was present in the sediments. The incidental ability of a variety of microorganisms from all major groups to reduce Se(VI) and Te(VI) by additional, often uncharacterised, mechanisms offers additional scope for bioreactor-based approaches.

Metal precipitation by sulphate-reducing and other bacteria Sulphate-reducing bacteria (SRB) oxidize organic compounds or hydrogen coupled with the reduction of sulfate, producing sulphide. The solubility products of most heavy metal sulphides are very low, in the range of 4.65 × 10-14 (Mn) to 6.44 × 10-53 (Hg) so that even a moderate output of sulphide can remove metals to levels permitted in the environment with metal removal being directly related to sulphide production. Sulphate-reducing bacteria can also create extremely reducing conditions which can chemically reduce metals such as uranium(VI). In addition, sulphate reduction partially eliminates acidity from the system as a result of the shift in equilibrium when sulphate (dissociated) is converted to sulphide (largely protonated). This can result in the further precipitation of metals such as copper or aluminium as hydroxides as well as increasing the efficiency of sulphide precipitation. The sulphide produced from sulphate reduction plays a major role in metal sulphide immobilization in sediments but has also been applied to bioremediation of metals in water and leachates. Large-scale bioreactors have been developed using bacterial sulphate-reduction for treating metal-contaminated water. A process integrating bacterial sulphate-reduction with bioleaching by sulphuroxidizing bacteria has also been developed to remove contaminating toxic metals from soils. Sulphur- and iron-oxidizing bacteria liberated metals from soils in the form of an acid sulphate solution that enabled almost all the metals to be removed by bacterial sulphate reduction. SRB biofilm reactors may offer a means of process intensification and entrap or precipitate metals, e.g. Cu and Cd, at the biofilm surface. Regarding other organisms, the thiosulphate reductase gene from Salmonella typhimurium has been expressed in Escherichia coli. This resulted in sulphide production from inorganic thiosulphate which precipitated metals as metal-sulphide complexes (1). A Cd-resistant Klebsiella planticola also precipitated significant amounts of cadmium sulphide when grown in thiosulphate-containing medium. As an alternative to anaerobic sulphate reduction, a novel aerobic sulphate reduction pathway has been engineered for sulphide production. The assimilatory SO42--reduction pathway was redirected to cysteine production which was converted to S2- by cysteine desulphydrase, leading to CdS precipitation on bacterial cell surfaces. Another form of bacterial bioprecipitation is mediated by a phosphatase enzyme, which liberates inorganic phosphate from a supplied organic phosphate donor molecule, e.g. glycerol 2-phosphate. Metals/radionuclides are precipitated as phosphates on the biomass. Therefore, microorganisms play an important roles in the environmental fate of toxic metals and metalloids with physico-chemical and biological

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mechanisms effecting transformations between soluble and insoluble phases. Such mechanisms are important components of natural biogeochemical cycles with some processes being of potential application to the treatment of contaminated materials. Although the biotechnological potential of most of these processes has only been explored at the laboratory scale, some mechanisms, notably bioleaching, biosorption and precipitation, have been employed at a commercial scale. Of these, autotrophic leaching is an established major process in mineral extraction but has also been applied to the treatment of contaminated land. There have been several attempts to commercialise biosorption using microbial biomass but success has been limited, primarily due to competition with commercially-produced ion exchange media. As a process for immobilizing metals, precipitation of metals as sulphides has achieved large-scale application, and this holds out promise of further commercial development. Exploitation of other biological processes will undoubtedly depend on a number of scientific, economic and political factors, including the availability of a market.

4.2

MICROORGANISMS IN ENVIRONMENTAL MONITORING

In the past few decades, environmental pollution has become one of the world’s major concerns. A great number of toxic compounds, originating mostly from industrial and agricultural activities, are being released in to our environment continuously. In some cases, harmful chemicals induce strong acute toxic effects to exposed organisms when released to the environment, but frequently, the consequences are delayed due to the effects of bioaccumulation and biomagnification. Early detection of toxic chemical compounds in the environment, particularly in water, and their biological effects on organisms has therefore become increasingly important. The traditional approach to environmental pollution assessment is based on chemical analytical methods which only provide information about the absolute concentrations of known chemicals in the environmental sample without an adequate interpretation of its toxicity to biota in the context of bioavailability, which means it only provides information about their potential, not actual toxicity. Moreover, compounds that are toxic below the detection limit of chemical analytical method or new compounds that are not yet deposited in the databases, can not be detected this way. Another disadvantage of chemical methods is the lack of information about the combined toxicity of different compounds such as additive, synergistic or antagonistic effects. In order to get more relevant information about environmental pollution risk, it is therefore inevitable to supplement the chemical analytical data with the results of methods providing information on biological impacts. The negative biological effects of pollutants present in all kinds of environmental samples can be assessed using different living organisms or cells as ‘analytical devices’. The biological response following the exposure of

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living organisms or cells to environmental sample usually gives an information on toxicity, genotoxicity, estrogenicity, etc. of the whole mixture of chemical compounds present in that particular sample. Besides being sensitive only to the bioavailable fraction of pollutants, biotests also have the power to assess the integrated effect of interacting chemical compounds and to detect the compounds, which are toxic only due to bioactivation. According to the technical principle, methods of biological monitoring can be classified to

• bioassays,



• biosensors,



• immunoassays,



• estrogenicity tests and



• ecological methods,

but there also exist other types of classifications, for example, division to biomarkers, whole cell biotests and early warning biological systems. The first biotests for environmental monitoring were based on multicellular eucaryotic organisms, in particular, fish and mammals. As they were relatively expensive, time-consuming, difficult to standardize and ethically questionable, the need for alternative biological methods for environmental monitoring based on 3R strategy (Reduction, Replacement, Refinement) soon became evident. The development and standardisation of toxicity tests based on procarotic (bacteria) or eucarotic (protozoa, unicellular algae, yeasts) microorganisms, instead of higher organisms, has enabled fast and inexpensive screening of environmental samples for toxic and genotoxic effects. The first generation of biotests has been based on different naturally sensitive microbes, while the second generation includes genetically modified microorganisms to attain better sensitivity and/or specifity. The next step forward was combinig microbial cells or parts of the cells to physicochemical detection elements, forming new integrated devices, called biosensors.

Microbial toxicity and genotoxicity tests Bioassay or ecotoxicity assay is an experiment in which living test-species are exposed directly to an environmental sample (soil, sediment, surface water, ground water, waste water, etc.) or extract of an environmental sample to measure a potential biological effect due to the presence of potential contaminants. Microbial bioassays can roughly be divided to (general) toxicity assays and genotoxicity assays. The purpose of ecotoxicity bioassays is to assess the integral effect of an environmental sample on general physiological state of the test-species, while genotoxicity tests specifically show the effects resulting in changes of genetic material. Regarding the exposure time of the test-species to the investigated sample, bioassays can be subdivided into acute and chronic. The first group is formed of bioassays, where exposure time does not exceed 96 hours, while the chronic assay subgroup includes

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tests with longer exposure times. Common parameters calculated from acute bioassay data are EC50 (estimated toxicant concentration in a sample at which 50 per cent of the test organisms show an effect following a given exposure time) and LC50 (estimated toxicant concentration in a sample, at which 50 per cent of the test organisms die following a given exposure time), while the chronic test reference parameter is NOEC value (represents the highest toxicant concentration at which no significant effect can be detected when compared to the control sample).

Toxicity bioassays Several toxicity bioassays applying bacteria, microalgae, protozoa and yeast have already been developed. Many of them are also standardized and commercially available. Most common parameters measured by microbial toxicity assays are population growth, substrate consumption, respiration, ATP luminescence and bioluminescence inhibition. Vibrio fischeri bioluminescence inhibition assay has been most frequently used and is claimed to be the most sensitive across a wide range of chemicals compared to other bacterial assays (nitrification inhibition, respirometry, enzyme inhibition and ATP luminescence). Vibrio fischeri is a gram-negative marine bacterium possessing natural bioluminiscence properties. Light production in a culture of test species is directly proportional to the metabolic activity of the bacterial population, therefore, any inhibition of enzymatic activity causes a corresponding decrease in bioluminescence. The biochemical principle behind the bioluminescence reaction is the oxidation of reduced flavin mononucleotide (FMNH2) in to FMN and H2O upon reaction with molecular oxygen in the presence of aldehyde and luciferase enzyme. The surplus energy formed in this reaction is emitted as blue-green light of wavelength 490 nm, that can be measured by a luminometer. Vibrio fischeri bioluminescence inhibition assay is applicable for almost all kinds of environmental samples such as surface and groundwater, sediments, municipal and industrial waste effluents, etc. Other commonly used bacterial bioassays are based on assessing the inhibitory effect of a sample on b-galactosidase activity in E. coli. Commercially available variations of this assay use different chromogenic b-galactosidase substrates for colorimetric determination of enzymatic activity products. A similar principle is applied with Bacillus sp. dehydrogenase activity assay, where redox-potential induced changes in tetrazolium salt colour indicate inhibition of microbial respiration. Bioassays with unicellular algae have found wide application in environmental biomonitoring, too. A generally recognized method for testing the effects of pollutant chemicals, especially pesticides, is based on measuring the growth inhibition of green microalga Scenedesmus supspicatus following 72 hours exposure. Besides various modifications of this standardized test, several new algal bioassays based on different approaches have also been developed. An improvement on the standard microalgal growth inhibition

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test has been made by using flow cytometry. This is a rapid and sensitive method for quantitative measurement of light scattering and fluorescence of flowing cells, which enables lower cell density algal cultures to be used for the assay. Algae contain chlorophyll a, a pigment molecule which autofluoresces when excited by blue light, therefore, additional fluorophores for cell count experiments are not needed. By using biochemically specific fluorescent dyes, it is possible to get additional information about the physiological status of the cells and mechanisms of toxicant action. Staining of algal cells with fluorescein diacetate (FDA) enables measurement of algal esterase activity as an indicator of their physiological status. Healthy cells take up and hydrolise FDA-producing fluorescent fluorescein, therefore, decreased fluorescence at 530-560 nm indicates damage of algal cells, such as impaired enzyme activity or loss of cell membrane integrity. Short period incubation algal tests for toxicity estimation are based on ‘in vivo’ chlorophyll prompt (PF) and delayed fluorescence (DF) measurements, which indicate photosynthesis inhibition due to toxic chemicals. Many herbicides and some other chemical compounds like mercury and 3,5-dichlorophenol have already been detected in environmental water samples this way. An important difference between the 72-h algal growth inhibition test and shorter period tests is, that the first group of tests involve multiple cell generations, whereas the second group of tests only determine the effects of tested chemicals on one cell generation, which might result in lower sensitivity of short period tests to chemicals affecting multiple cell generations. Protozoa are eucaryotic microorganisms, known to be very sensitive to environmental changes, therefore, being an ideal early-warning indicators of aquatic ecosystem deterioration, as well as an important test-species for toxicological assays. Non-pathogenic, free-living ciliates Tetrahymena pyriformis and Tetrahymena termophila, being the first protozoa cultured axenically, are the organisms of choice for most toxicological studies. Common end points assessed in ecotoxicity protozoan assays are growth, viability/ mortality, grazing ability, ATP (adenosine-5’-triphosphate) content, ACP (acid phosphatase) activity and MMT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide) reduction capacity. Growth impairment bioassays are traditionally based on microscopic observations of morphological changes (cell shape and motility), what makes them simple and inexpensive techniques. Nevertheless, problems such as underestimating the true number of viable cells because of the assumption that all non-motile or shape-altered cells are dead, forced the development of an alternative method to standard direct counting. A novel method is based on using two fluorescent dyes: calcein/AM – non-fluorescent substance, which diffuses passively into cells where it is converted to green fluorescent calcein by intracellular esterases of viable cells and EthD-1, that enters only damaged

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(dead) cells and represent a source of red fluorescence when bound to DNA. Biotests based on detection of changes in metabolic state of protozoa often include colorimetric determination of acid phosphatase or dehydrogenase activity (MMT/tetrazolium reduction capacity) and determination of ATP content.

Genotoxicity assays Genotoxicity assays are used to measure the potential of environmental sample to induce changes in genetic material of test organisms. Types of DNA damage that may be assessed using these tests include mutagenicity, clastogenicity and aneuploidy. Mutagenicity results in changes of one or a small number of DNA base-pairs (point mutations), comprising substitutions, additions and deletions of base pairs. Clastogenicity involves structural changes in larger areas of chromosome, while changes in number of whole chromosomes lead to aneuploidy. Some widely used microbial genotoxicity assays are based on bacterium Salmonella typhimurium. The most widespread is the Ames test, which has also been established as a routine method of environmental water monitoring. It is based on a hystidine-dependent strain of S. typhimurium (TA98). Mutagenicity of the sample is determined by frequency of back mutations, which enable the growth of revertants on the medium without hystidine. Genotoxicity is detected by measuring the transcription of SOS-response genes, which code for enzymes involved in DNA repair. Fusion of SOS-response genes with â-galactosidase encoding reporter gene enables colorimetric detection of genotoxic compounds. The same principle is applied in SOS-chromotest, which applies Escherichia coli as test-species and is frequently used, too, because of its high sensitivity to certain groups of pollutants, such as chloride pesticides and chlorophenols. The genotoxicity bioassay developed by scientist Zimmermann and his group is based on recombinant Saccharomyces cerevisiae strains and enables not only detection of mutations, but also recombinations and loss of chromosomes. A novel commercially available yeast genotoxicity reporter assay has been developed recently. Green Screen assay (GSA) is sensitive to broad spectrum of mutagens and clastogenes. In this assay, the reporter system in yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to green fluorescent protein. Genotoxicity indicator assay, which has recently attracted much attention, is Comet assay (also called the Single-Cell Gel Electrophoresis Assay), which primarily measures single and double-DNA strand-breaks in single cells. Adapted protocols enable also the detection of oxidized bases and abasic sites. The protocol has originally been developed for detection of DNA damage in blood cells, but it has later been adapted for eukaryotic microorganisms, too. T. termophila have already been used for the purposes of environmental and drinking water genotoxicity monitoring by

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comet assay. Yeast cells have been applied equally successfully in monitoring of wastewater genotoxicity reduction by biological wastewater treatment plants.

Microbial biosensors A biosensor is defined as a self-contained, integrated device, consisting of a biological recognition element interfaced to a physical signal transducer, that together reversibly respond to a chemical species in a concentration-dependant manner. A wider definition also includes some other forms of biological sensors, including genetically engineered microorganisms, which respond in observable ways to target analyte or group of related analytes. A wide range of biological recognition elements have already been used in biosensors constructed for potential environmental applications. Whole microbial cells, cellular organelles and molecules such as enzymes, antibodies, different kinds of receptors or DNA are the most common bio-recognition elements of microbial origin. Regarding the type of transducer, biosensors could roughly be classified to electrochemical, optical, thermometric and piezoelectric. Microbial biosensors for environmental applications range in their development stages from proof of concept to full commercial availability. Regarding the target detection specificity, they may fall in one of two groups.

• Biosensors, which measure general biological effects/parameters or



• Biosensors for specific detection of target compounds.

The first group of biosensors is aimed to measure an integral toxicity, genotoxicity, estrogenicity or other general parameters of the sample, which affect living organisms. They essentially include whole microorganisms as biorecognition elements. The most often reported cell-based biosensors include genetically modified bacteria with artificially constructed fusions of particular regulatory system (native promoter) with reporter genes. The presence of an effector (non-specific stressor such as DNA damaging agents, heat shock, oxidative stress, toxic metals, organic environmental pollutants) results in transcription and translation of fused target genes, generating recombinant proteins which produce some measurable response. Frequently used reporter genes are lux (coding for luciferase) and gfp (coding for green fluorescence protein), expression of which correlates with luminescence– or fluorescence– based light emission. Colorimetric determination of target gene expression is possible by fusing it to reporter genes coding for â-galalactosidase (lacZ) or alkaline phosphatase (phoA). Recently, E.coli biosensor, capable of detecting both genotoxic and oxidative damage has been developed. This was achieved by introducing two plasmids: first one, with fusion of katG (gene encoding for an important antioxidative enzyme) promoter to the lux reporter genes, and another, with recA (gene encoding crucial enzyme for DNA repair) promoter with the gfp reporter gene. Besides genetically modified microorganisms (also named bioreporters), some

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other types of cellular biosensors have also been constructed. An example is an algal biosensor, based on amperometric monitoring of photosynthetic O2 evolution — the process affected by toxic compounds, which has been developed by coupling Clark electrode to cyanobacterium Spirulina subsalsa. Biosensors for specific determination of chemical compounds frequently contain molecules like enzymes, receptors and metal-binding proteins as recognition elements. A number of enzymes have been shown to be inhibited by toxic metals, pesticides and some other important contaminants, like endocrine-disrupting compounds. Limitations for the potential applications of many enzyme biosensors include limited sensitivity and selectivity, as well as interferences by environmental matrices. One recently introduced strategy to overcome the first two of these limitations uses inhibition ratio of two enzymes for the detection of specific compounds. Acetylcholinesterase and urease, co-entrapped in the sol–gel matrix with the sensing probe, FITCdextran, have succesfully been used for Cu, Cd and Hg detection, for example. Besides molecular biosensors, bioreporter cells may also be used for detection of specific target compounds. Recently, for example, a biosensor for nitrate monitoring has been constructed by transformation of E. coli with plasmid containing nitrate reductase operon fused to gfp reporter gene.

Immunoassays Immunochemical methods are based on specific and reversible binding of immunoglobulin molecules (antibodies) to their target antigenes. The most popular immunochemical technique in environmental analyses today is immunoassay, which has been shown to detect and quantify many compounds of environmental interest such as pesticides, industrial chemicals, and products of xenobiotic metabolism. Basic immunoassays are performed by detection of a specific marker molecule immobilized either to antibody (Ab) or the antigene (Ag). Marker molecules may be in the form of fluorescent or chemiluminescent compounds, radioisotopes or enzymes. Enzyme-based immunoassay offer many advantages over other immunotechniques, because of the great amount of product molecules, which results in signal amplification. The main enzymes used are horseradish peroxidase, alkaline phosphatase and â-galactosidase. A widely used immunoassay for environmental purposes is enzyme-linked immunosorbent assay (ELISA), which can be carried out according to different formats – direct competitive, indirect competitive or sandwich-type. Competitive assays are most common and can be performed in different ways. Analyte and the tracer (direct competitive ELISA) or analyte and the immobilised ligand (indirect ELISA) may compete for a limited number of binding sites. Sandwich-type ELISA is non-competitive assay, in which the analyte is recognised by two different antibodies-immobilized Ab and marker Ab. Flow-injection immunoassay (FIIA) is a technique, based on the introduction of the sample into carrier stream, which enters the reaction

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chamber where the immunoreaction takes place. FIIA has been successfully used for detection of different pollutants, e.g., triazines. At present, this method is integrated into different immunosensors. The role of microorganisms, related to immunoassays is mostly indirect, but still significant. They are used as artificial factories (expression systems) for recombinant antibody production. Since Escherichia coli provides the most popular expression system, much research has been done to maximise the expression levels of recombinant antibodies (rAbs) in this system. Main problems associated with prkaryotic expression systems are reducing environment inside microbial cell, that does not favour disulfide bond formation and leads to production of insoluble recombinant proteins in the form of inclusion bodies. The current approach to overcome this problem is to export the rAbs to the periplasm of E.coli. However, this strategy is still limited by the amount of proteins that can be exported. Eukryotic expression systems are also in use. They enable higher levels of Ab expression, whereas the functionality of Ab produced is highly dependent on individual single-chain antibody fragments. Different microscopic fungi have been used for recombinant antibody production, including yeast species Saccharomyces cerevisiae and Pichia pastoris. Besides being used as antibody-production systems, microorganisms may also represent a source of marker enzymes (alkaline phosphatase, â-galactosidase) used in certain type of immunoassays.

Endocrine disruptor (EDC) assays Endocrine disrupting compounds are a newly defined category of environmental contaminants, which interfere with the endocrine system function, which results in alternating the reproductive systems in wildlife and humans. Compounds, acting as agonists or antagonists of hormone (estrogene, androgene) receptors include a wide range of molecules, such as organochlorine pesticides, pthalates, alkylphenols, phyto-and mycoestrogenes, pharmaceutical estrogenes and many others. Estrogens are hormones, that play crucial functions in growth, differentiation and homeostasis of male and female reproductive organs. Besides, they also influence non-reproductive tissues, such as bone, liver and the cardiovascular system. When estrogenes enter the cells, they bind to specific receptors, forming homodimeric complexes. Ligand-receptor complexes induce the transcription of target genes by binding to specific regions on DNA, called ERES. Other mechanisms of action, which do not include hormone receptors, also exist. Faster responses to compounds with estrogenic activity, which take place in the cytoplasm or on membranes and involve different effector molecules are also of importance. Several bioassays have been developed to assess substances with estrogenic activity. Most of the ‘in vivo’ assays are based on a variety of end points and are therefore, time-consuming, expensive and require sacrification

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of numerous animals. Consequently, ‘in vitro’ assays applying microorganisms have been developed for the purpose of large-scale screening. Most of them are based on simple cell models, that express hormone receptor coding genes coupled to reporter genes, such as â-galactosidase or luciferase, when induced by estrogene-like compounds. The estrogene-induced signaling pathways are highly conserved in yeast and mammalian cells, which makes yeast cells a suitable system for modeling cellular response of mammalian cells when exposed to endocrine disrupters. Besides being less expensive and easier to culture, one important advantage of using yeasts instead of mammalian cells is their resistance to different contaminants, usually present in environmental samples. Numerous tests, using genetically modified yeasts for the detection of estrogenic and androgenic compounds, have been developed. They monitor either the transcriptional activation of the steroid receptor itself or its ligandinduced interaction with a transcriptional co-activator. Commercially available assay for estrogene screening (YES/Yeast Estrogen Screen by Glaxo) is based on genetically engineered Saccharomyces cerevisiae with human estrogene receptor (hER) fused to lacZ reporter gene.

Ecotoxicogenomic approaches in environmental monitoring

Rapid progress in the fields of genomics is lately beginning to provide tools that may assist our understanding of how chemicals can impact human and ecosystem health. A new scientific discipline, which integrates genomics (transcriptomics, proteomics and metabolomics) into ecotoxicology is named ecotoxicogenomics. It is defined as the study of the response of the genome to environmental toxicant exposure. Although the application of gene and protein expression analysis to ecotoxicology is still at an early stage, this holistic approach seems to have several potentials in different fields of ecological risk assessment. The most important advantage in using a gene expression profiling approach compared to most standardized methods used to asses the potential impact of chemicals on organisms, is the power of an insight into the precise mode of action (MOA) of toxicants. This may potentially be useful for prioritization of substances for extensive testing regarding their MOA and may therefore, allow optimization of resources and limit the use of animals for testing purposes. Moreover, the knowledge of precise toxicity pathways may reveal novel molecular biomarkers for early detection of environmental stress. Comparison of gene expression profiles of different test microorganisms and higher organisms may provide useful information about the possibility of extrapolation of the effects of toxic chemicals across species. Determining similarities and dissimilarities in toxicity mechanisms across species would give the answer where the extrapolation of chemical hazards from one species to another is technically valid. The knowledge about conservation of toxicity mechanisms in organisms will therefore enable to choose appropriate model

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organisms at lower levels of biological organization (e.g. microorganisms) for relevant monitoring of specific environmental toxicants. The use of microarray, proteomic and metabolomic techniques may also provide the possibility to predict toxic potential of unknown chemicals by comparing specific patterns of gene expression (fingerprints), reflecting mode of action of unknown chemical, with expression profiles of known Toxicants. To conclude, by providing both, mechanism of action and predictive tools, ecotoxicogenomic approach seems especially promising for studying the effect of pollutants at low, environmentally relevant concentrations, improvement of toxic mixture analysis and long-term exposure assessment of organisms. The use of biological methods in environmental monitoring is essential in order to complement chemical analysis with information about actual toxicity or genotoxicity of environmental samples. Microorganisms are widely applied test-species in different bioassays because of the ease and low costs of their culturing as well as the lack of ethical issues often accompanying the use of higher organisms. Combining biology to engineering skills has enabled the development of biosensors—new generation of analytical devices coupling biological recognition elements to physical signal transducers. Besides the direct application of whole microorganisms or their isolated parts for general toxicity assessment or detection of specific compounds, genetically modified microbes also represent an important source of recombinant antibody production, which makes them important also when talking about immunoassays. With the development of toxicogenomic approaches, the use of microorganisms for environmental monitoring purposes is expected to become even more extensive because of better knowledge about potential analogies in toxicity mechanisms between higher organisms and microbes.

4.3

APPLICATIONS OF THE POLYMERASE CHAIN REACTION IN ENVIRONMENTAL MICROBIOLOGY

The application of the polymerase chain reaction (PCR) to explore various areas of environmental microbiology has the potential to solve many difficult and unanswered questions about microbial activities in the environment at the physiological and molecular levels. This review describes the use of PCR for the detection of specific microbes in environmental samples and discusses how PCR may be used to answer future questions in molecular microbial ecology. The first two sections of the review will discuss preparation of nucleic acids from environmental microorganisms and PCR methodology specific to environmental microbiology. Subsequent sections present information on applying these methods to environmental problems such as: detection of genetically engineered microbes, detection of indigenous microorganisms in the environment and indicator microorganisms in water, detection of waterborne microbial pathogens and viable but nonculturable microorganisms, and environmental monitoring with multiplex PCR.

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Purification of nucleic acids from environmental microorganisms for PCR amplification Extraction and extensive purification of nucleic acids from environmental microorganisms is necessary for successful PCR amplification. Some of the general environmental contaminants that can inhibit the PCR reaction are the presence of humic materials, clay, and organics. For example, the addition of as little as 0.001 mg of montomorillite humic material in the PCR reaction inhibits the amplification process.

Purification of Nucleic Acids from Soil and Sediment Much effort has been devoted to developing convenient methods for removing humic materials from environmental samples to achieve successful PCR amplification of the target nucleic acids. Either direct lysis or isolation of bacterial cells followed by lysis can be used for extraction of DNA from microorganisms present in the environmental soil or sediment. ~61 Purification of the released DNA can be performed by applying a combination of the various standard purification methods such as phenol-chloroform extraction followed by ammonium acetate-ethanol precipitation, repeated polyvinylpolypyrrolidone (PVPP) treatment or dialysis, hydroxylapatite or affinity chromatography, and multiple CsC1- EtBr density gradient centrifugation. All of these methods produce positive PCR amplification from the environmental samples. Recently, Tsai and O1- son (7~ have described a direct extraction method using lysozyme followed by freeze-thaw disruption of the cells. The released DNAs were then purified by standard phenolchloroform extraction and chromatography. In another study, bacterial cells were differentially separated from soil colloids on the basis of their buoyant densities. In this method, a modified sucrose gradient centrifugation protocol is used to separate most of the soil colloids from the bacterial cells in the sample for PCR amplification of the target DNA. To isolate and purify RNA from environmental microorganisms, the samples can be treated with guanidinium hydrochloride, and phenolchloroform-isoamyl alcohol extraction followed by ethanol precipitation. Although significantly higher amounts of nucleic acids can be recovered by following the direct lysis method, the presence of eukaryotic DNA in the sample is a possibility.

Purification of Nucleic Acids from Microorganisms in Water Nucleic acids for PCR amplification from microorganisms present in the aquatic environment can be isolated and purified more easily than those from soil and sediment. A simple method for isolating nucleic acids from aquatic samples has been demonstrated by Sommersville and consists of collecting cells, followed by lysis and separation of plasmid DNA, chromosomal DNA, and RNA in a single filter cartridge. The dissolved and particulate DNA then can be conveniently purified for PCR analysis. In various other studies, the

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following procedures were found to be essential to yield sufficiently pure DNA for various molecular biological analyses and perhaps PCR analyses: CsCI-EtBr density gradient centrifugation/for all environmental DNAs, multiple PVPP treatment for rRNA studies, (and repeated phenol-chloroform extractions for total planktonic DNA t14,1s~ or cyanobacterial DNA. Microbial cells can be collected on a filter, lysed by repeated freeze-thaw methods, and used for PCR amplification without removing the filter from the reaction tube. Alternatively, filtered cells can be lysed directly on the filter by lysozyme, and the released DNA can be purified by phenol-choroform extraction followed by ethanol precipitation to yield adequately purified DNA for PCR amplification and analysis. Although the methods described above can remove the majority of the environmental contaminants and are useful for various molecular biological studies, there is no standard protocol for removing all possible inhibitors that can be applied for all types of environmental samples.

PCR Methodology for Environmental Applications Basically, PCR is the in vitro enzymatic amplification of a DNA fragment that is performed by using two flanking oligonucleotide primers at the two ends of the target DNA. This methodology has several advantages, as well as disadvantages, over conventional methodologies used in environmental microbiology. Some of the recent advancements in PCR technology that may be very useful in environmental applications and that we would like to discuss in some detail are: (1) “hot start,” (2) removal of PCR carryover contaminants, and (3) thermostable DNA polymerase from various thermotolerent microorganisms, some of them with additional activities such as reverse transcriptase.

Hot Start The specificity of oligonucleotide primers for the amplification of a specific target for detecting a specific microbial pathogen or a released genetically engineered microorganism (GEM) in the environment can be determined by testing varieties of microorganisms. However, because of the vast diversity of microorganisms in any environmental sample, it is possible that one may get non-specific PCR amplification. Thus, increased specificity is an important issue for environmental PCR. Methods such as increasing or decreasing MgC12 concentrations in the PCR reaction, high Tm value of the primers with an approximately 50% GC content, and a random base distribution minimum of 5-6 bases at the 3’ end of each of the primers can be used to remove non-specific amplified DNA. Also effective is the “hot start” method. This method is based on the fact that non-specific priming and subsequent production of unwanted amplified DNA bands generally result due to the retention of considerable enzymatic activity at temperatures below the optimum for DNA synthesis. Therefore, during the initial heating step of the PCR reaction, primers that anneal nonspecifically to a partially single-stranded template region can be

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Environmental Biotechnology

extended and stabilized before the reaction reaches the 72~ temperature for extension of specifically annealed primers. If the DNA polymerase is activated only after the reaction has reached high temperatures, nontarget amplification can be minimized by manual addition of an essential reagent, e.g., DNA polymerase, to the reaction tube at elevated temperatures. This approach improves specificity and minimizes the formation of “primer dimers.”

Removal of PCR Carryover Contamination Another potential problem is contamination of the PCR amplification reaction with products of a previous PCR reaction, i.e., product carryover, crosscontamination between samples, or contamination with exogenous nucleic acids from the laboratory environment, all of which can create false-positive results. Although some general precautions and good laboratory practice will reduce the possibility of such contamination problems, other simple and more effective preventive measures can be taken by exposing the reaction mixture to ultraviolet light, treating the reaction mixture with multiple restriction enzymes or DNase I, followed by inactivation of these enzymes, or photochemical modification of the contaminating DNA with psoralen or isopsoralen. Another effective approach is to make contaminating PCR products susceptible to degradation by substituting dUTP for dTTP in every PCR reaction and treating the subsequent PCR reaction mix with uracil DNA glycosylase, which will selectively eliminate dU-containing DNA by cleaving the uracil. This enzyme is active for both double- and single-stranded DNAs containing a basic polynucleotide (dUTP), but does not react on RNA template. Thus, when a PCR reaction is contaminated with RNA, this approach will not be useful and false amplification may result. Another problem with this approach is that the amplified DNA must be stored at high temperature until it is transferred to a freezer to prevent degradation of the newly amplified DNA product from any residual activity of the enzyme.

Thermostable DNA Polymerase Native Taq DNA polymerase and recombinant AmpliTaq DNA polymerase of Thermus aquaticus are commonly used for DNA amplification by PCR. Recently, several other thermostable DNA polymerases have been introduced and these may have potential advantages for environmental PCR technology. The Stoffel fragment, a modified version of the Taq DNA polymerase in which 289 amino acids are deleted from the amino-terminal end of the enzyme, is approximately two-fold more thermostable, exhibits optimal activity over a broader range of magnesium ion concentrations (2-10 mM), and lacks any intrinsic 5‘→3‘ exonuclease activity compared to the complete Taq DNA polymerase. As a result, when using the Stoffel fragment, the denaturation temperature can be raised several degreesf this is useful for templates that are

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G+C-rich or that contain complex secondary structures. Manv environmental microbes are identified by amplifying ribosomal RNA, which may have such complex secondary structure, as targets. In addition, when using the Stoffel fragment, the primer can be selected with higher annealing temperature for better specificity and the entire PCR reaction can be performed at an elevated temperature without losing much of the enzyme activity. Another thermostable DNA polymerase isolated from Thermus thermophilus (Tth) has significant reverse transcriptase activity, especially in the presence of MnC12 Tth polymerase can be useful for detecting gene expression and viable but nonculturable microorganisms in the environmental samples by using mRNA-cDNA-PCR amplification in a single reaction. Recently, several other thermostable DNA polymerases have been isolated and tested for their fidelity, stability at high temperatures, and exonuclease activities. The fidelity of the DNA polymerase isolated from Thennococcus litoralis (Vent DNA polymerase) has been studied and compared with other DNA polymerases such as Taq, Klenow, T4, and T7 DNA polyrnerases by Eckert and Kunkel. The advantage of using Vent DNA polymerase is that it has 3’~5’ exonuclease activity, which increases the fidelity of the reaction about six-fold as compared to the Taq DNA polymerase. It is noteworthy to mention that Vent DNA polymerase has a base substitution error rate of 1/31,000 in a reaction containing 1 mM dNTPs, which is similar to that observed for the Klenow DNA polymerase. Interestingly, T. litoralis grows at a temperature of 98~ in thermal vents on the ocean floor, and Vent DNA polyrnerase, isolated from this organism, retains its activity for over 2 hr at 100~ with a primer extension capacity of up to 13 kb. Recently, another therrnostable DNA polymerase has been isolated from a thermophilic, anaerobic, marine archaebacterium, Pyrococcus fitriosus. The pill DNA polymerase is a monomeric, 92-kD protein with both 5 ‘~ 3‘ and 3 ‘~ 5‘ exonuclease, activities has better thermostability at 95~ than some other thermostable DNA polymerases and has a 12-fold lower mutation frequency than Taq DNA polyrnerase. Eckert and Kunkel/have shown in a comparison of the fidelities of the various thermostable DNA polymerases that Tth and the Thermus flavis Replinase are lO-fold less accurate than the T4 or native T7 DNA polymerases. Although, the use of various newly introduced thermostable DNA polymerases with proofreading activities and lower misincorporation rates during synthesis may be an advantage to PCR users for direct cloning and sequencing of PCR products, PCR-based methods for mutagenesis, or detection of point mutations, the remaining concern is the degradation of primers in the PCR reaction mixtures by these enzymes. From a comparative study of the exonuclease activities and temperature profiles of Vent and pfu DNA polymerases, it has been claimed that pfu has a much lower primer degradation activity than the Vent DNA polymerase (E. Mathur, pers. comm.).

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Detection of Released GEMS in The Environment By PCR

There are many promising applications of GEMs in industry, agriculture, and medicine, but their use has been limited thus far because of a lack of sensitive methods for the monitoring and detection of GEMs after their release in the environment. Cultural methods and methods such as colony hybridization, which depend on the ability to recover and culture the organism from an environmental sample, lack sensitivity due to the limited efficiency of recovering bacteria from natural environments. Extraction of microbial DNA from the environmental sample, either directly or after recovery of microbial cells, followed by gene probe hybridization, though more sensitive than cultural methods, still lacks the level of sensitivity required to determine the ultimate fate of GEMs because of the limited relative numbers of target gene sequences, that may be present in the sample PCR amplification of the engineered genes from the released microorganisms to several million-fold, can potentially increase the sensitivity of detection of released GEMs in the environment. The PCR method was first applied to monitor GEMs by Steffan and Atlas. They detected by PCR amplification a portion of the 1.3-kb repeat sequence from Pseudomonas cepacia ACllO0, a herbicide (2,4,5-T)—degrading bacterium, after the organism was released in the soil. Their sensitivity of detection was 100 GEMs in 100 grams of sediment against a background of 1011 diverse non-target microorganisms, at least 103-fold higher than the sensitivity of nonamplified conventional dot-blot hybridization detection. In another example, a 0.3-kb unique DNA sequence from Pennisetum purpureum (napier grass) was cloned into pRCIO, a derivative of 2,4-dichlorophenoxyacetic acid-degrading plasmid, and transferred into Escherichia coll. This genetically altered microbe was then released into filter-sterilized lake and sewage-water samples at a concentration of 104 cells per milliliter. The microbe was detected by PCR at a sensitivity several-fold higher than the conventional plating technique, even after 10-14 days of incubation, using the unique cloned DNA sequence as a target. These studies show that the PCR method can be used for monitoring released GEMs in an environment consisting of a complex habitat of diverse microorganisms, in which it may be tedious and time-consuming to discriminate the GEMs from the indigenous microorganisms. In another study, a single copy of the transposon Tn5 was transferred into the genomic DNA of Rhizobium leguminosarum, which was released into the soil. These GEMs were detected by “double” PCR amplification using the transposon Tn5 as target to a sensitivity of 1-10 cfu/gram of soil. Although it is adequate to use Tn5, which contains an antibiotic resistance gene as a model target for PCR detection, this may not be an appropriate marker for releasing GEMs into the environment because of Tn5’s ability to be transferred into indigenous microorganisms, making them antibiotic resistant also. Detection of the indigenous Tn5 sequence by PCR may give false-positive results in such cases.

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Detection of Indigenous Microorganisms in the Environment by PCR Detection of Degrading Microorganisms: One of the potential ways to eliminate various pollutants and toxic wastes in the environment is efficient biodegradation and bioremediation by various indigenous microorganisms at the polluted sites. Sensitive detection of such degrading microorganisms in the polluted and toxic waste sites may be possible by using PCR. Although there might be variations in the genes of the same or different groups of microorganisms for the degradation of one or several types of pollutants, it may be possible to identify these microorganisms by detecting the conserved regions of these genes by PCR amplification. One such study used the nucleotide sequence information of a chlorocatechol dioxygenase-degrading gene from Alcaligenes eutrophus JMP134 (pJP4); oligonucleotide primers were designed for the detection of various chloro-aromatic-degrading bacteria by PCR amplification. PCR amplication using such oligonucleotide primers provides information quickly on the variations, similarities, and functional aspects of various pollutantde-grading genes present in closely or distantly related microorganisms in the environment. When such a polluted site is identified, it is important to investigate the possibility of the presence of various degrading microbes at that site. In many instances, conventional microbiological techniques do not detect all the pesticide-degrading indigenous microorganisms, as some of them may be in a viable but nonculturable condition. In another study, specific detection of a herbicide (2,4-dichlorophenoxyacetic acid)-degrading bacteria was achieved by PCR amplification of a region of/gene from pJP4 and its derivative plasmid pRO103 (I. Pepper, pers. comm.). In this study, by using direct PCR amplified DNA analysis, it was possible to detect approximately 3000 cfu or 15.6 pg of plasmid DNA.The sensitivity of such detection was onefold higher when DNA-DNA hybridization was performed with an oligonucleotide probe internal to the amplified DNA. Using such oligonucleotide primers and PCR amplification, it is possible to detect the specific microorganisms carrying the degrading gene from a complex-mixed microbial population in the environment.

Identification of Microorganisms in Biofilms Formation of biofilms on various surfaces by microorganisms in the environment can be beneficial or detrimental. For example, microbial aggregation or attachment is required for various water treatments; on the other hand, extensive corrosion and biodeterioration can be caused due to the formation of such microbial biofilms. Characterization and ecology of microbial populations in biofilms has been hindered because the available determinative techniques require culture of microorganisms in selective media. These methods eliminate many of the important microbes from the biofilms since they survive only in a mixed culture and live on the cometabolism

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(the gratuitous metabolic transformation of a substance by a microorganism growing on another substance). PCR amplification of specific targets makes it possible to identify a group of microbes in such a biofilm that may have been missed by the conventional techniques. To determine the feasibility of PCR, a sulfidogenic biofilm has been established in an anaerobic fixed-bed bioreactor. PCR amplification was performed for the detection of the population architecture of all the gramnegative sulfate-reducing bacteria using a region of the 16S ribosomal RNA conserved in the resident sulfate-reducing bacteria.

Detection of Indicator Microorganisms in Water The bacteriological safety of water supplies is tested by monitoring coliform bacteria whose presence in the water indicates potential human fecal contamination and the possibility of the presence of enteric pathogens. Coliform bacteria are traditionally detected by culturing on media such as Mac-Conkey, m-Endo, eosin methylene blue, or brilliant-green-lactose-bile media. The culture method for monitoring E. coli in environmental and potable waters has several problems associated with it. The conventional confirmative tests for the detection of E. coli, all of which require culturing of the organism, are time-consuming. Moreover, they do not detect viable but non-culturable bacteria, which may occur due to chlorine injury during the process of water purification and treatment. Also, the cells may die between the time of collection and the test. A colorimetric test, the Colilert test, for the detection of E. coli is based on the detection of β-D-glucuronidase enzyme produced by uidA gene. This method requires culturing of bacteria. In addition, this method fails to detect β-D-glucuronidase-negative E. coli. Bej and group have developed a PCR gene probe-based method for the detection of coliform bacteria. Amplification of a portion of the lacZ gene detects E. coli and other coliform bacteria, including Shigella spp. Amplification of part of the lamB gene detects E. coli, Salmonela, and Shigella spp. In another study, Bej and group developed a method for the detection of E. coli and Shigella spp. using four different regions of the uidA gene, which codes for the [β-glueruron dass glucuronidase enzyme, and part of the uidR gene, which is the regulatory region of the uidA gene, as targets. Besides being less time consuming and having higher specificity and sensitivity, the most important advantage of this method over conventional and other commercially available methods is that it can detect the uidA-negative E. coli that do not show a positive signal with the conventional tests because they lack the 13-glucuronidase enzyme. The sensitivity of the method is 1-10 fg of genomic DNA and 1-5 viable E. coli cells. Similarly, Cleuziat and Baudouy-Robert have used a large region of the uid gene of E. coli as a target for PCR amplification and gene probe detection of E. coli and Shigella spp. This PCR gene probe-based method has the specificity and sensitivity required for monitoring coliforms as indicator organisms in

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environmental and potable waters. A field evaluation of PCR detection of enteric pathogens and indicator microorganisms has been reported using uidA and lacZ as targets./Although these targets for PCR amplification detection show great promise, the targets for specific detection of E. coli, Shigella spp., and Salmonella spp. are yet to be described.

Detection of Water-borne Microbial Pathogens by PCR Apart from the detection and monitoring of indicator microorganisms for water quality assessment, it is also important to detect with high sensitivity and specificity various water-borne microbial pathogens.

PCR Detection of Legionella Legionella spp. is a water-borne microbial pathogen and can cause Legionnaires’ disease in humans via aerosol. Starnbach reported the detection of Legionella pneumophila by amplification of a fragment of DNA of unknown function from Legionella using PCR. Their sensitivity of detection was equivalent to 35 colony forming units detected by viable plating. Mahbubani et al. have developed a method based upon PCR and gene probes for detecting Legionella in environmental water sources. All species of Legionella, including all 15 serogroups of L. pneumophila, were detected by PCR amplification of a 104-bp DNA sequence that codes for a region of 5S rRNA followed by radiolabeled oligoprobe hybridization to an internal region of the amplified DNA. Strains of L. pneumophila (all serogroups) were specifically detected based upon amplification of a portion of the coding region of the macrophage infectivity potentiator (mip) gene. Pseudomonas spp. that exhibit antigenic cross-reactivity in serological detection methods did not produce positive signals in the PCR gene probe method using Southern blot analyses. Singlecell, single-gene Legionella detection was achieved with the PCR gene probe methods.

PCR Detection of Giardia Another microbial pathogen, Giardia lamblia, causes defined waterborne diarrhea in the United States and in many other parts of the world. Diagnosis of G. lamblia from environmental samples is performed by concentrating 100 gallons of water followed by microscopic examination using fluorescent dye. Using PCR amplification of different segments of the giardin gene of G. lamblia, it was possible to differentiate G. lamblia from G. muris. Also, a single Giardia cyst was detected by PCR amplification after separating the cyst by a micromanipulator. Although the specificity and sensitivity of the detection of Giardia shows great promise for rapid and reliable monitoring of this pathogen in water, application of this method for the detection of this pathogen in concentrates of 100 gallons of environmental water sample needs to be demonstrated.

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Multiplex PCR Amplification for Environmental Monitoring of Microorganisms It is possible that environmental samples and drinking water may contain more than one type of microbial pathogen in addition to the indicator microorganism. Use of multiplex PCR for amplification and detection of more than one target in a single PCR reaction can be useful for monitoring multiple microbial pathogens in a single environmental or water sample. This method was first described by Chamberlain for detecting human genes. A modification of this approach of simultaneous PCR amplification of multiple targets associated in different bacteria in the environmental samples has been demonstrated. Multiplex amplification of two different Legionella genes, one specific for Legionella pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered addition of two different sets of primers at two different concentrations. This method can detect genus Legionella and L. pneumophila should they be present in one sample. Using the same target genes, mip and 5S rRNA, a multiplex PCR assay for genus Legionella and L. pnemnophila was described. In this study, equal amplified products were achieved by adding equimolar quantities of each of the primers. This may be due to the fact that the two target sequences were closer in length than in the system, developed and described previously. In a field study of water quality monitoring, simultaneous PCR amplification was performed using lacZ and uidA as targets. In this study, it was posssible to detect in one sample total coliform bacteria by amplification of the lacZ gene, the indicator microorganism E. coli, and a pathogen Shigella spp. bv the amplification of the uidA gene. Also, in this study the lacZ PCR detection method gave results statistically equivalent to those of the conventional plate count and defined substrate methods accepted by the U.S. Environmental Protection Agency for water quality monitoring. The uidA PCR method was more sensitive than the 4-methylumbelliferyl-13-D-glucuronidebased defined substrate test for the specific detection of E. coli. In another study multiplex amplification of five different targets in a single PCR reaction has been achieved for the detection of non-pneumophila Legionella spp., L. pneumophila, total coliforms, E. coli and Shigella spp., and total eubacterial species. It may be desirable in future studies to group certain microbial pathogens and indicators in the environmental samples and design the primers for specific targets. For example, one can group all the environmental and water-borne respiratory pathogens and amplify all the specific target genes by PCR in a single reaction for their detection. When several GEMs are released together for the degradation of complex hazardous wastes and pollutants, they can be monitored together, possibly both qualitatively and quantitatively, in a single PCR reaction by amplifying a unique segment of the DNA of each of the GEMs, as well as by amplifying a common segment of all the GEMs that is not present in other eubacterial species.

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Detection of Viable But Non-culturable Microorganisms in the Environment There are several reports on the existence of many microorganisms, including human pathogens, in the environment in a viable but non-culturable, i.e., dormant, stage. These microbial pathogens are shown to be potentially infectious when suitable conditions prevail. One obvious difficulty in elucidating this potential hazard is the inability to detect these viable but nonculturable cells in the environment because routine microbiological methods will not allow them to grow (on agar media) or will not distinguish them from the dead cells (by microscopic technique). Recognizing that the terms “alive” and “viable” are subject to different definitions, the reasonably acceptable definition would be that the live cells are considered those capable of cell division, metabolism (respiration), or gene transcription (mRNA production). To detect those microbial cells that are in a viable but non-culturable state in the environment, it is desirable to target the mRNA rather than the DNA first for cDNA synthesis followed by PCR amplification. The potential problem of this approach is that most of the prokaryotic mRNAs have half-lives of only few minutes.

PCR Detection of L. pneumophila Mahbubani have shown that the mRNA of the mip gene of L. pneumophila can be stabilized simply by growing the cells for 10-15 min in the presence of chloramphenicol before harvesting. They have shown that the PCR amplification of the mip mRNA could be a potential means for the detection of metabolically active L. pneumophila cells. The use of chloram-phenicol for increasing the stability of bacterial mRNA is yet to be tested in other microorganisms. Another perplexing issue that may create additional problems in such an approach is the efficiency of gene expression of these dormant microbial pathogens. It is possible that the transcriptional or regulatory systems of the target genes in these microbial pathogens are inhibited by various environmental factors and inhibitors when they are present in the natural environment. Therefore, in this situation, the quantity of the target mRNA level may be so low that it may remain undetected even by a method as sophisticated as PCR. However, it has been shown that, targeting DNA for PCR amplification may be sufficient for the detection of culturable and nonculturable microbial pathogens. Both viable culturable and viable non-culturable cells of L. pneumophila, formed during exposure to hypochlorite, showed positive PCR amplification, whereas nonviable cells did not. Field verification of this approach for the detection of metabolically active (viable vs. dead) L. pneumophila from contaminated environmental samples is yet to be done.

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PCR Detection of Vibrio vulnificus Besides L. pneumophila, another important marine water-borne microbial pathogen, Vibrio vulnificus, which can cause fatal infections in humans who ingest contaminated raw oysters, has been found to enter in a viable but non-culturable state during the colder months and resuscitate from the non-culturable state when a suitable environment prevails. Using PCR amplification of the hemolysin gene, Scientists detected DNA from culturable and from non-culturable cells.

Difficulties in Detection of Non-culturable Cells by PCR Although the decreased sensitivity of detection of non-culturable cells by PCR is not well understood at this time, several possible explanations have been described. Among these possibilities, the important criteria that may be of concern in applying PCR methodology for the detection of viable but nonculturable microorganisms are: (1) less DNA content per cell, (2) difficulty in breaking open the cell because of the change in the cell wall that may occur due to carbon or nitrogen starvation or changes in the environmental conditions, and (3) modification of the target gene due to genetic rearrangement. However, Brauns did not attempt to use hemolysin mRNA as a target for PCR amplification from the non-culturable cells, a method that could have determined the exact nature of the ceils (i.e., alive or dead), and the gene expression of the target. A study by Mahbubani has shown that mRNA-PCR alone is not sufficient to distinguish live Giardia cysts from dead ones, since cysts killed by heat treatment or monochloramination also give positive mRNA-PCR amplification. Therefore, in this organism, if the giardin mRNA is used as a target for PCR amplification, it is necessary to include an mRNA induction step in the procedure to determine the viability of the cysts. Since in the viable but non-culturable stage there may be changes in the structure as well as in gene expression in many microorganisms, a modified version of the PCR method may need to be developed for the detection of such microbial pathogens in the environment.

Detection of Gene Expression in the Environment by PCR An important issue in environmental microbial molecular genetics is how various genes are regulated and expressed under various environmental conditions. One known fact is that some of the environmental microbial pathogens such as L. pneumophila and V. vulnificus alter their gene expression and remain in a dormant stage as non-culturable organisms in the environment. It has also been predicted that several biodegradative microorganisms may not express their degrading genes in the environment. As a result, one may not be sure whether the released GEMs or indigenous microorganisms are degrading the pollutants at a contaminated site. Using specific mRNA as a

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target for PCR amplification and developing a quantitative assay for such a method, it is possible to detect the level of mRNA production with high sensitivity in the environmental samples. A promising method for extraction of specific mRNA from soil seeded with naphthalene-degrading and mercury-resistant bacterial cells has been described. This method can be completed within a few hours; approximately 17 mg of total RNA per gram (wet weight) of soil containing 8.0 × 108 bacterial cells can be purified with a DNA-RNA hybridization detection sensitivity of 160 ng of specific target mRNA. Although, this method has potential for studying in situ gene expression, the humic acid compounds may precipitate with samples containing high-cation-exchange capacity, e.g., some sediments, which will greatly reduce the total RNA recovery efficiency and sensitivity of detection. Application of PCR for detecting specific mRNA extracted from various environmental samples by this method has yet to be evaluated. Although the application of PCR in the area of environmental microbiology has not progressed as much as applications in diagnostics, medicine, and molecular biology several studies have shown great promise in solving various difficult problems. One of the most important problems in environmental microbiology, is the detection and monitoring of released GEMs in the environment with high sensitivity. It has been shown that the application of the PCR method can detect 1-100 GEMs per gram of soil or sediment, which is a level of sensitivity several orders of magnitude higher than the conventional DNA-DNA hybridization method. One of the drawbacks of this approach is that PCR requires purified nucleic acid, which must be achieved from the environmental samples through several rigorous methodological steps. The application of PCR technology to monitoring pathogens and indicator microorganisms has reached a stage where it is safe to say that this method can be used, with greater specificity and sensitivity, as an alternative to the conventional methods. The most important criterion in applying PCR technology in this area of environmental microbiology is the removal of inhibitors and contaminants from the samples. Although several inhibitors from various environmental samples have been identified with possible removal procedures, unlimited numbers of such inhibitors may exist that have not yet been identified. For detection of microorganisms from soil and sediments, it has been found that the humic and fulvic acid compounds inhibit the polymerase activities and reduce the sensitivity of detection. Although, several procedures such as diluting the samples, ion-exchange chromatography, gel filtration chromatography, PVPP treatment, sucrose gradient purification, and so forth, have been used to remove humic and fulvic acids and other inhibitors from the samples, none of these methods seems to remove them totally. The application of PCR technology to various environmental samples for detection of pathogens and other microorganisms may be affected severely if a relatively universal method for removal of inhibitors from the environmental samples is not developed.

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Quantitation of microbial populations by conventional methods has several drawbacks. Although there are several reports on the quantitation of PCR-amplified products that can give information about the starting number of cells, this approach needs to be developed for the quantitation of a microbial population in a given environment. This will permit microbial succession, competition, and community structure in an ecosystem to be studied in the environment, including microbes living in many extreme environments. Another potential application of PCR methodology in the area of environmental microbiology is distinguishing the live cells from the dead ones in a given environmental sample. However, more research must be done before this approach can be applied to actual environmental samples. Alteration of gene expression in many microbial pathogens and other pollutant-degrading microbes due to various environmental conditions is a growing concern to human health, and detection of specific mRNA in the environment by PCR will provide information on the in situ activity of these microorganisms. PCR shows promise for cloning genes from environmentally important microorganisms, including those organisms that have not been cultured yet. In the near future, technological improvements and subsequent new development of the PCR method will solve many unanswered questions in the area of microbial ecology, microbial community structure, environmental health, and environmental analyses of molecular microbiology.

4.4 PESTICIDES AND OTHER POLLUTANTS DEGRADATION BY MICROORGANISMS AND GENETICALLY ENGINEERED MICROBES In the last few decades, highly toxic organic compounds have been synthesized and released into the environment for direct or indirect application over a long period of time. Pesticides, fuels, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), chlorophenols, and dyes are some of these types of compounds. The paramount pollution in our environment is a dire consequence of continually expanding population along with an exponential development in the industrial field. Biodegradation: According to the definition by the International Union of Pure and Applied Chemistry, the term biodegradation is “Breakdown of a substance catalyzed by enzymes in vitro or in vivo. This may be characterized for the purpose of hazard assessment such as:

• Primary. Alteration of the chemical structure of a substance resulting in loss of a specific property of that substance.



• Environmentally acceptable. Biodegradation to such an extent as to remove undesirable properties of the compound. This often corresponds to primary biodegradation but it depends on the circumstances under which the products are discharged into the environment.

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• Ultimate. Complete breakdown of a compound to either fully oxidized or reduced simple molecules (such as carbon dioxide/methane, nitrate/ ammonium and water). It should be noted that the biodegradation products can be more harmful than the substance degraded.”

Microbial degradation of chemical compounds such as pesticides and other pollutants in the environment is an important route for the removal of these compounds. Microbes are ubiquitous in nature and are being exposed to the continuous release of more and more recalcitrant xenobiotic compounds into the environment. No wonder, these microbes, inhabiting polluted environments, are armed with various resistance and catabolic potentials. The catalytic potential of microbes in nature is enormous and this is advantageous to mankind for a cleaner and healthier environment through bioremediation. In general, potential microbes with broad spectrum of activities from their native habitat have been screened, characterized, genetically modified and released back to their native habitat for better performance. By such studies, the core problem of pollution is tactfully attacked and benefits of decontamination add healthy atmosphere to mankind. The purified degrading enzymes, Nitrilase, Azoreductases and Oragnophosphate hydrolases could be effectively used in industry for the treatment of effluents. The systems developed are eco-friendly and economical and hence could effectively be integrated with physico-chemical methods for pollution control. The index of xenobiotic compounds released into the environment increases due to industrialization and combating pollution by the release of these compounds is essential for the sustenance of the future generation. In this context, microbes such as algae, fungi and bacteria, play an important role by giving us a helping hand in bioremediation of these xenobiotic compounds. Degradation of pesticides by different bacterial population proves to be the best example for citing the role of microbes in bioremediation of xenobiotic compounds. A large number of pesticides and insecticides like morpholine, methyl parathion, organophosphorous compounds and benzimidazoles are widely used to increase the agricultural output and has also contributed to the pollution load, as many of these man-made chemicals are non-biodegradable. The pollution control strategies involving physico-chemical methods many a time aggravate the problem, rather than eliminating it. Microbes play a very important role in the mineralization of pollutants either by natural selection or through recombinant DNA technology making bioremediation process an extension of normal microbial metabolism. Xenobiotic compounds are also widely employed in our day-to-day life. Microbes also mediate degradation of xenobiotic compounds like dyes and plastics. Understanding the molecular biology of the microorganisms, and the ability to genetically manipulate the microorganisms and infuse engineering principles into biology have led to novel strategies for combating environmental problems.

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Environmental Biotechnology

Construction of strains with broad spectrum of catabolic potential with heavy metal resistant traits makes them ideal for bioremediation of polluted environments in both aquatic and terrestrial ecosystems. The transfer of genetic traits from one organism to another paves way in creating Genetically Engineered Organisms (GEMs) for combating pollution in extreme environments making it a boon to mankind to cleanup the mess that has created in nature. Therefore, bioremediation protocols for treatment of industrial wastewaters like distillery effluent, textile mill effluent, tannery effluent and pharmaceutical effluent have been devised and managed by the author for commercial applications. Biodegradation of Wastewater and industrial effluents: Micro-organisms in sewage treatment plants remove the more common pollutants from waste water before it is discharged into rivers or the sea. Increasing industrial and agricultural pollution has led to a greater need for processes that remove specific pollutants such as nitrogen and phosphorus compounds, heavy metals and chlorinated compounds. New methods include aerobic, anaerobic and physico-chemical processes in fixed-bed filters and in bioreactors in which the materials and microbes are held in suspension. The costs of wastewater treatment can be reduced by the conversion of wastes into useful products.

• For example, heavy metals and sulphur compounds can be removed from waste streams of the galvanisation industry by the aid of sulphur metabolising bacteria and reused.



• Another example is the production of animal feed from the fungal biomass which remains after the production of penicillin. Most anaerobic waste watertreatment systems produce useful biogas.

Drinking and Process Water: Abundant supplies of water are vital for modern urban and industrial development. By the turn of this century, it is estimated that two-thirds of the world’s nations will be water stressed – using clean water faster than it is replenished in aquifers or rivers. A very important aspect of biotechnology is therefore its potential for the reclamation and purification of waste waters for re-use. Public concern has also increased over the quality of drinking water. Not only does water need to be recycled in the development of sustainable use of resources, overall quality must also be improved to satisfy consumers. In many agricultural regions of the world, animal wastes and excess fertilisers result in high levels of nitrates in drinking water. Biotechnology has provided successful methods by which these compounds can be removed from processed water before it is delivered to customers. Air and waste gases: Originally, industrial waste gas treatment systems were based on cheap compost-filled filters that removed odours. Such systems still exist. However, slow processing rates and the short life of such filters drove research into better methods such as bioscrubbers, in which the pollutants are washed out using a cell suspension and biotrickling filters, in which the

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pollutant is degraded by micro-organisms immobilised on an inert matrix and provided with an aqueous nutrient film trickling through the device. The selection of micro-organisms that are more efficient at metabolising pollutants has also led to better air and gas purifying biofilters.

• Examples are a bioscrubber based system for the simultaneous removal of nitrogen and sulphur oxides from the flue gas of blast furnaces which has been developed as an alternative to the classical limestone gypsum process, and the elimination of styrene from the waste gas of polystyrene-processing industries by a biofilter containing fungi.

Soil and land treatment: Both in situ (in its original place) and ex situ (somewhere else) methods are commercially exploited for the cleanup of soil and the associated groundwater. In situ treatments may include the introduction of micro-organisms (bioaugmentation), ventilation and/or adding nutrient solutions (biostimulation). Ex situ treatment involves removing the soil and groundwater and treating it above ground. The soil may be treated as compost, in soil banks, or in specialised slurry bioreactors. Groundwater is treated in bioreactors and either pumped back into the ground or drained into surface water. Bioremediation of land (biorestoration) is often cheaper than physical methods and its products are harmless if complete mineralisation takes place. Its action can however, be time-consuming, tying up capital and land. The in situ bioremediation of the ground under petrol stations has already become common practice but also for chlorinated solvents like tri- and tetrachloroethylene in situ bioremediation is possible. The applicability of in situ bioremediation is and probably will remain dependent on the physical parameters of the soil, mainly its transport properties. Bioremediation using plants is called phytoremediation. This technique is presently already used to remove metals from contaminated soils and groundwater and is being further explored for the remediation of other pollutants. The combined use of plants and bacteria may also be possible. Certain bacteria live closely associated with the roots of plants and depend on substances excreted by the roots. Such rhizobacteria, whose numbers are much higher than those of other soil bacteria, may be genetically modified to break down pollutants. Detection and monitoring of microorganisms used for bioremediation: When laboratory grown micro-organisms are inoculated into a bioremediation site (bioaugmentation), it often becomes necessary to monitor their presence and/or multiplication to check the progress of the process. This is especially true and even required when genetically modified micro-organisms are involved. The traditional technique to detect the presence of micro-organisms in soil is direct plating on selective media. This is greatly facilitated if the organism contains a marker which can be selected for. Newer techniques include the above mentioned immunological and light-based bioreporter techniques. The

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spatial distribution of specific microorganisms in a sample can be determined microscopically and non-invasively by using fluorescent in situ hybridisation (FISH) of micro-organisms. The most sensitive and specific technique is the direct isolation and amplification of DNA from soil, which is increasingly being used. Genetic Engineering: Recombinant DNA technology has had amazing repercussions in the last few years. Molecular biologists have mapped entire genomes, many new medicines have been developed and introduced and agriculturists are producing plants with novel types of disease resistance that could not be achieved through conventional breeding. Several of the previously mentioned examples like the amylose-free potato and the indigoproducing bacterium also involve the use of organisms genetically modified by recombinant DNA technology. Many enzymes are routinely produced by genetically modified organisms too.

• Given the overwhelming diversity of species, biomolecules and metabolic pathways on this planet, genetic engineering can, in principle, be a very powerful tool in creating environmentally friendlier alternatives for products and processes that presently pollute the environment or exhaust its non-renewable resources. Politics, economics and society will ultimately determine which scientific possibilities will become reality.



• Nowadays organisms can also be supplemented with additional genetic properties for the biodegradation of specific pollutants if naturally occurring organisms are not able to do that job properly or not quickly enough. By combining different metabolic abilities in the same microorganism, bottlenecks in environmental cleanup may be circumvented. Until now this has not been done on any significant scale. The main reason being the fact that, in most cases, naturally occurring organisms can be found or selected for, which are able to clean up a polluted site. Examples have been found where soil bacteria have developed new properties in response to the introduction of xenobiotics (that is, manmade chemicals, that are normally not found in nature).



• In some cases, they even appear to have acquired properties from other species. In the USA, some genetically modified bacteria have been approved for bioremediation purposes but large scale applications have not yet been reported. In Europe, only controlled field tests have been authorized. Because new organisms can be created by genetic engineering that may never be produced by spontaneous or selection driven evolution, concerns exist about the unpredictability of their possible interactions with the eco-system.



• Genetically modified organisms which are properly kept within the confines of their approved production facilities are much less a concern

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than genetically modified organisms which are meant to be released into the environment like disease-resistant plants or soil bacteria for bioremediation. The possible ecological effects of the latter are even more difficult to evaluate due to the fact that it is well known that soil bacteria frequently exchange genetic material (also between species). This together with the fact that we know little about the great majority of soil-inhabiting bacterial species, makes it almost impossible to predict the fate of every DNA copy of a newly introduced genetic property in a soil bacterium. If the extra DNA is derived from another soil bacterium, it may on the other hand be reasonable to argue that the genetically modified bacterium might also have evolved spontaneously some day due to the frequent exchange of genetic material in the soil. Use of genetic engineering and genetic manipulations for more efficient bioremediation: In recent years, efforts have been made to create genetically engineered microorganisms (GEMs) to enhance bioremediation. This is done to overcome some of the limitations and problems in bioremediation. These problems are:

• Sometimes the growth of microorganisms gets inhibited or reduced by the xenobiotics.



• No single naturally-occurring microorganisms has the capability of degrading all the xenobiotics present in the environmental pollution.



• The microbial degradation is a very slow process.



• Sometimes, certain xenobiotics get adsorbed on to the particulate matter of soil and thus, become unavailable for microbial degradation.

As the majority of genes responsible for the synthesis of enzymes with biodegradation capability are located on the plasmids, the genetic manipulations of plasmids can lead to the creation of new strains of bacteria with different degradative pathways. In 1970s, Chakrabarty and his team of coworkers reported the development of a new strain of bacterium Pseudomonas by manipulations of plasmid transfer which they named as “superbug”. This superbug had the capability of degrading a number of hydrocarbons of petroleum simultaneously such as camphor, octane, xylene, naphthalene, etc. In 1980, United States granted the patent to this superbug making it the first genetically engineered microorganism to be patented. In certain cases, the process of plasmid transfer was used. E.g., The bacterium containing CAM (camphor, degrading) plasmid was conjugated with another bacterium with OCT (octane, degrading) plasmid. Due to non-compatibility, these plasmids cannot coexist in the same bacterium. However, due to the presence of homologous regions of DNA, recombination occurs between these two plasmids which results in a single CAM-OCT plasmid giving the bacterium the capacity to degrade both camphor as well as octane.

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Environmental Biotechnology

DEGRADATION OF OIL BY MICROORGANISMS FOR THE PRODUCTION OF USEFUL PRODUCTS

Brief Introduction to Oil: Crude oil (or petroleum): a liquid mixture of a variety of hydrocarbon compounds derived from ancient algal and plant remains and found in reservoirs under the Earth’s surface. Nitrogen and sulfur-containing molecules (“resins”) are common constituents of some crude oils.

Crude oil components

• Volatile compounds—low molecular weight compounds, like methane (natural gas) or propane, that are normally gaseous or evaporate very quickly at room temperature.



• Saturated hydrocarbons—compounds with carbon and hydrogen atoms connected only by single bonds. Saturated hydrocarbons can be arranged in straight or branched chains of up to about 25 carbon atoms. Saturated hydrocarbons are readily biodegraded although degradability decreases with chain length.



• Aromatic compounds—compounds that contain rings of carbon atoms held together with double bonds between the carbon atoms. The smallest aromatic compounds in petroleum have six carbons in such a ring structure (e.g. benzene and toluene), but other compounds contain multiple rings. These are known as polycyclic aromatic hydrocarbons, often abbreviated ‘PAH’. Most aromatic molecules in petroleum have multiple attached hydrocarbon chains.

The smallest aromatic molecules (one- and two-rings) are both volatile and readily biodegraded, even with attached side-chains, but four-ring and larger aromatic compounds, are more resistant to biodegradation. They are, however, susceptible to photooxidation. Some larger PAHs are of concern because they are potentially carcinogenic; 16 different PAHs are designated as priority pollutants by the EPA. The percentage of PAHs in crude oil varies, but the ‘priority pollutants’ are present at low levels in crude oils; they are much more common as a byproduct of burning carbonaceous materials such as fuel, coal, wood, tobacco and other materials. Asphaltenes (used in making roads and roofing products) are examples of high molecular weight (heavy) PAHs that have additional chemical side chains attached to their aromatic rings. Asphaltenes are not soluble in water and most organic solvents. Oil-degrading bacteria: Bacteria usually are the dominant hydrocarbon degraders in aquatic systems such as oceans. They also posess diverse metabolic pahtways that are not present in fungi which allows them to utilize most recalcitrant petroleum hydrocarbons. Bacterial degradation of aromatic compounds can be divided into three steps:

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• modification and conversion of the many different compounds into a few central aromatic intermediates (ring-fission substrates); this step is referred as peripheral pathway and involves considerable modification of the ring and/or perhaps elimination of substituent groups;



• oxidative ring cleavage by dioxygenases, which are responsible for the oxygenolytic ring cleavage of dihdyroxylated aromatic compounds (catechol, protocatechuate, gentisate);



• further degradation of the non-cyclic, non-aromatic ring-fission products to intermediates of central metabolic pathways.

Long-chain hydrocarbons (C10-C18) can be used rapidly by many high G+C gram-positive bacteria. Only a few bacteria can oxidize C2-C8 hydrocarbons. Degradation of n-alkanes requires activation of the inert substrates by molecular oxygen with help of oxygenases by three possible ways that are associated with membranes:

• Monooxygenase attacks at the end producing alkan-1-ol:

R–CH3 + O2 + NAD(P)H + H+ → R–CH2OH _ NAD(P)+ + H2

Aerobic pathways for the degradation of alkanes by terminal and subterminal oxidation

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Environmental Biotechnology

• Dioxygenase attack produces the hydroperoxides, which are reduced to yield also alkan-1-ol:

R–CH3 + O2 → R–CH2OOH + NAD(P)H + H+ → R–CH2OH + NAD(P)+ + H2O

• Rarely, subterminal oxidation at C2 by monooxygenase yields secondary alcohols.

It is important to keep in mind that many strains within one species of bacteria usually exist. Usually, only some of strains are capable of hydrocarbon degradation and some of strains can cause opportunistic infections in humans and animals. Biotechnology and Control of Oil Spillage: Microorganisms can now be genetically engineered for use in oil recovery, pollution control, mineral leaching and recovery.

• In the petroleum industry, microorganisms can also be genetically engineered to produce chemicals useful for enhanced oil recovery .



• Cleaning up oil spills could, in the future, be left to geneticallyengineered bacteria. In the mining industries, micro-organisms with the property of enhanced leaching ability could be designed.



• Micro-organisms can bind metals to their surfaces and concentrate them internally. As a result of this, genetically improved strains can be used to recover valuable metals or remove polluting metals from dilute solution as in industrial waste.



• Research is already being carried out to improve the naturallyoccurring bacteria that can ‘eat oil’, for use following an oil spill.



• By applying bacteria to oilcovered beaches, the complex oil molecules would be broken down into harmless sugars. Many micro-organisms can degrade various kinds of environmental pollutants into relatively harmless materials before the death of the micro-organisms. This property could also be used in overcoming the environmental hazards of DDT, lead and other environmental pollutants like toxic wastes, globally.



• Strains of bacteria which can degrade fuel hydrocarbons have been designed and the use of genetically engineered micro-organisms to clean up oil spillages or treat sewages has been proposed and is undergoing production/manufacturing.

Oil Biodegradation in Marine Systems: Petroleum oil is toxic for most life forms and, episodic and chronic pollution of the environment by oil, causes major ecological perturbations. Marine environments are especially vulnerable since oil spills of coastal regions and the open sea are poorly containable and mitigation is difficult. In addition to pollution through human activities, millions of tons of petroleum enter the marine environment

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every year from natural seepages. Despite its toxicity, a considerable fraction of petroleum oil entering marine systems is eliminated by the hydrocarbondegrading activities of microbial communities, in particular, by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB). Alcanivorax borkumensis, a paradigm of HCB and probably the most important global oil degrader, was the first to be subjected to a functional genomic analysis. This analysis has yielded important new insights into its capacity for

(i) n-alkane degradation including metabolism, biosurfactant production and biofilm formation,

(ii) scavenging of nutrients and cofactors in the oligotrophic marine environment, as well as (iii) coping with various habitat-specific stresses. The understanding, thereby gained, constitutes a significant advance in efforts towards the design of new knowledge-based strategies for the mitigation of ecological damage caused by oil pollution of marine habitats. HCB also have potential biotechnological applications in the areas of bioplastics and biocatalysis.

4.6

DEGRADATION OF PLASTICS BY MICROORGANISMS FOR PRODUCTION OF USEFUL PRODUCTS

Microorganisms such as bacteria and fungi are involved in the degradation of both natural and synthetic plastics. The biodegradation of plastics proceeds actively under different soil conditions according to their properties, because the microorganisms responsible for the degradation differ from each other and they have their own optimal growth conditions in the soil. Polymers, especially plastics, are potential substrates for heterotrophic microorganisms. Biodegradation is governed by different factors that include: polymer characteristics, type of organism, and nature of pretreatment.

• The polymer characteristics such as its mobility, tacticity, crystallinity, molecular weight, the type of functional groups and substituents present in its structure, and plasticizers or additives added to the polymer all play an important role in its degradation. During degradation, the polymer is first converted to its monomers, then these monomers are mineralized.



• Most polymers are too large to pass through cellular membranes, so they must first be depolymerized to smaller monomers before they can be absorbed and biodegraded within microbial cells.



• The initial breakdown of a polymer can result from a variety of physical and biological forces. Physical forces, such as heating/cooling, freezing/ thawing, or wetting/drying, can cause mechanical damage such as the cracking of polymeric materials.

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• The growth of many fungi can also cause small-scale swelling and bursting, as the fungi penetrate the polymer solids. Synthetic polymers, such as poly (caprolactone), are also depolymerized by microbial enzymes, after which the monomers are absorbed into microbial cells and biodegraded. Abiotic hydrolysis is the most important reaction for initiating the environmental degradation of synthetic polymers like polycarboxylates, poly (ethylene terephthalate), polylactic acids and their copolymers, poly (α-glutamic acids), and polydimethylsiloxanes, or silicones.

Generally, an increase in molecular weight results in a decline of polymer degradability by microorganisms. In contrast, monomers, dimers, and oligomers of a polymer’s repeating units are much easily degraded and mineralized. High molecular weights result in a sharp decrease in solubility making them unfavorable for microbial attack because bacteria require the substrate to be assimilated through the cellular membrane and then further degraded by cellular enzymes. At least two categories of enzymes are actively involved in biological degradation of polymers: extracellular and intracellular depolymerases. During degradation, exoenzymes from microorganisms break down complex polymers yielding smaller molecules of short chains e.g., oligomers, dimers, and monomers, that are smaller enough to pass the semi-permeable outer bacterial membranes, and then to be utilized as carbon and energy sources. The process is called depolymerization. When the end products are CO2, H2O, or CH4, the degradation is called mineralization. It is important to note that biodeterioration and degradation of polymer substrate can rarely reach 100% and the reason is that a small portion of the polymer will be incorporated into microbial biomass, humus and other natural products. Dominant groups of microorganisms and the degradative pathways associated with polymer degradation are often determined by the environmental conditions. When O2 is available, aerobic microorganisms are mostly responsible for destruction of complex materials, with microbial biomass, CO2, and H2O as the final products. In contrast, under anoxic conditions, anaerobic consortia of microorganisms are responsible for polymer deterioration. The primary products will be microbial biomass, CO2, CH2 and H2O under methanogenic (anaerobic) conditions (e.g. landfills/ compost).

4.7

RECOVERY OF MINERALS BY MICROBES

The recovery of copper from the drainage water of mines was probably a widespread practice in the Mediterranean basin as early as 1000 B.C. Although such mining operations are difficult to document, it is known that

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the leaching of copper on a large scale was well established at the Rio Tinto mines in Spain by the 18th century. What none of the miners engaged in this traditional method of mineral extraction realized until about 25 years ago is, that bacteria take an active part in the leaching process. They help to convert the copper into a water-soluble form that can be carried off by the leach water Today, bacteria are being deliberately exploited to recover millions of pounds of copper from billions of tons of low-grade ore. Copper obtained in this way accounts for more than 10 per cent of the total U.S. production. In recent years, bacterial leaching has also been applied to the recovery of another non-ferrous metal: uranium.  Bioleaching: Bioleaching is leaching where the extraction of metal from solid minerals into a solution is facilitated by the metabolism of certain microbes—bioleaching microbes. Bioleaching is a process described as “the use of microorganisms to transform elements so that the elements can be extracted from a material when water is filtered through it”.

Schematic representation of the Bioleaching process

Bioleaching involves the use of micro-organisms to extract metals from low grade ores and has been performed successfully on earth to obtain gold, copper and uranium. • About 20% of the world’s copper is produced by bioleaching.

• Bioleaching of nickel, zinc and cobalt can be done with thermophyllic bacteria.

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Environmental Biotechnology

Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, Thiobacillus thiooxidans, Sulfolobus species and others have been used for bioleaching. Acidiphilium, Sulfobacillus, Ferroplasma, Sulfolobus, Metallosphaera, and Acidianus have also been used. These bacteria tolerate acids and metabolize sulfur. Weak solutions of acids are dripped through the ore and a bacterial liquor forms that is then electrolytically or chemically processed. Sometimes, this requires water and organic substrate like potato peels as well as solvents to extract the metals from the bacterial mass. Chaff from crops may be used for bioleaching rather than livestock feed. Precious water will be recycled. If bioleaching becomes a major industrial activity on the Moon we will be pressed to conserve our vital water and hydrogen resources for this instead of wasting them in the form of rocket fuel. Only ores containing sulfur can be bioleached because the bacteria feed on sulfur. Bioleaching does not require lots of energy but it is slow. High temperature roasting and smelting is not required, so there are decided benefits in addition to the fact that bioleaching can get metals from low-grade ores.

Factors Influencing Bioleaching

• Temperature (optimum between 30° & 50°



• pH (2.3-2.5)



• Iron supply



• Oxygen



• Availability of other nutrients required for growth

Recent progress in the genetic manipulation of microorganisms for industrial purposes promises to revitalize not only the bacterial leaching of metal-bearing ores but also the microbiological treatment of metalcontaminated wastewater. The enthusiasm of the microbiologists working on the development of the new “biomining” techniques is matched by a need in the minerals industry to find alternatives to conventional methods of mining, ore-processing and wastewater treatment. The need arises from recent trends in the industry: the continued depletion of high-grade mineral resources, the resulting tendency for mining to be extended deeper underground, the growing awareness of environmental problems associated with the smelting of sulfide minerals and the burning of sulfur-rich fossil fuels and the rising cost of the prodigious amounts of energy required in the conventional recovery methods. The current methods will surely prevail for many years to come, but biological processes are generally less energy-intensive and less polluting than most non-biological ones, and so the role of biological technology in mining, ore processing and wastewater treatment is likely to become increasingly important.  The bacteria involved in the leaching of metals from ores are among the most remarkable life forms known. The microorganisms are said to be

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chemolithotrophic (“rock-eating”); they obtain energy from the oxidation of inorganic substances. Many of them are also autotrophic, that is, they capture carbon for the synthesis of cellular components not from organic nutrients but from carbon dioxide in the atmosphere. The leaching bacteria live in environments that would be quite inhospitable to other organisms; for example, the concentration of sulfuric acid and of soluble metals is often very high. Some thermophilic, or heat-loving, species require temperatures above 50°C (122°F), and a few strains have been found at temperatures near the boiling point of water.  For many years, the only microorganism thought to be important in the leaching of metals from ores was the rod-shaped bacterium Thiobacillus ferrooxidans. This microorganism was discovered in the acidic water-draining coal mines; it was not until 1957 that a correlation was recognized between the presence of the bacterium and the dissolution of metals in copper-leaching operations. Since then, a great deal of information has been amassed on T. ferrooxidans and on its vital role in the leaching of metals.  T. ferrooxidans is acidophilic, or acid-loving; it tends to live in environments such as hot springs, volcanic fissures and sulfide ore deposits that have a high concentration of sulfuric acid. It is also moderately thermophilic, thriving in the temperature range between 20 and 35°C. The bacterium gets energy for growth from the oxidation of either iron or sulfur. The iron must be in the ferrous, or bivalent, form (Fe++), and it is converted by the action of the bacterium into the ferric, or trivalent, form (Fe++). Several forms of sulfur can be attacked. They include both soluble and insoluble sulfides (compounds containing the bivalent sulfur ion S- -), elemental sulfur and soluble compounds that incorporate either the thiosulfate ion (S2O3- -) or the tetrathionate ion (S4O6- -). In each case, the product of the transformation is a substance in which the sulfur atom has fewer valence electrons, culminating in the formation of the sulfate ion (SO4- -). T. ferrooxidans obtains carbon autotrophically from atmospheric carbon dioxide.  Although T. ferrooxidans is essential to the bacterial leaching of metals, it is by no means the only microorganism with an important role in the process. Among the other microorganisms taking part is T. thioxidans, a rod-shaped bacterium, not unlike T. ferrooxidans, that grows on elemental sulfur and some soluble sulfur compounds. Studies by Donovan P. Kelly and his associates at the University of Warwick have confirmed the importance of mixed cultures of bacteria in the extraction of metals from ores. T. ferrooxidans and T. thiooxidans combined, for example, are more effective in leaching certain ores than either organism is alone. Similarly, the combination of Leptospirillium ferrooxidans and T. organoparus can degrade pyrite (FeS2) and chalcopyrite (CuFeS2), a feat neither species can accomplish alone.  In acidic environments supporting leaching bacteria, one can often isolate a number of heterotrophic microorganisms: bacteria and fungi that scavenge the

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small amounts of organic matter present in these environments or that survive on the organic by-products of other organisms’ autotrophic metabolism. The role of the heterotrophic microorganisms in the leaching process is largely undetermined. Thiobacilli, that attack some sulfide minerals and certain soluble sulfur compounds under neutral conditions (that is, neither acidic nor alkaline), are often found in sulfide ore deposits and in other habitats where sulfur is available. Thiobacilli of this type may be responsible for the initial increase in acidity that establishes an environment conducive to the growth of the more acidophilic-leaching bacteria.  At temperatures between 60 and 75°C, and under neutral conditions the filamentous bacterium  Thermothrix thiopara  oxidizes sulfhydryl ions (HS-), sulfite ions (SO3-), thiosulfate ions and elemental sulfur to form sulfate ions. There is increasing evidence of the widespread existence of Thermothrix species and similar filamentous, sulfur-oxidizing bacteria, in thermal springs and near volcanic fissures. Few leaching sites have been tested for the presence of these bacteria. Their existence in sulfur-bearing springs, however, suggests they could colonize sulfidic ores and thereby, prepare such environments for the more acidophilic species.  The most robust of the leaching microorganisms are the extremely thermophilic and acidophilic species of the genus Sulfolobus. These bacteria flourish in acidic hot springs and volcanic fissures at temperatures that can exceed 60 degrees C. Some strains of Sulfolobus have been observed in springs at temperatures near the boiling point of water. The cell wall of the  Sulfolobus  bacteria has a different structure from that of most bacteria. Microorganisms of this type are thought to belong to the Archaebacteria, a group of unusual bacteria proposed as a separate kingdom of life forms. Sulfolobus acidocaldarius and S. brierleyi oxidize sulfur and iron for energy, relying on either carbon dioxide or simple organic compounds for carbon. Ordinarily, oxygen is required by Sulfolobus, — as in other aerobic organisms, the oxygen serves as the ultimate acceptor of the electrons removed in the process of chemical oxidation.  Sulfolobus  bacteria can also grow anaerobically, however. It has been demonstrated that molybdenum (Mo6+) and ferric iron can serve as electron acceptors in the absence of air. Minerals that resist most microorganisms, such as chalcopyrite and molybdenite (MoS2), are readily attacked by Sulfolobus, and the resulting soluble metals are not toxic to the organism. Molybdenum, which is extremely toxic even to the metal-tolerant thiobacilli, is readily endured by S. brierleyi in concentrations as high as 750 milligrams per liter. Sulfolobus has not been isolated from commercial leaching operations, but laboratory studies confirm the ability of the organism to proliferate in such environments. The potential of Sulfolobus species to leach metals from ores is only now being recognized: because of the extraordinary ability of these organisms to attack resistant mineral structures, however, they are certain to be among the leaching bacteria that will be successfully exploited in the future. 

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Indirect leaching, in contrast, does not proceed through a frontal attack, by the bacteria on the atomic structure of the mineral. Instead, the bacteria generate ferric iron by oxidizing soluble, ferrous iron; ferric iron in turn is a powerful oxidizing agent that reacts with other metals, transforming them into the soluble, oxidized form in a sulfuric acid solution. In this reaction, ferrous iron is again produced and is rapidly reoxidized by the bacteria. Indirect leaching is usually referred to as bacterially-assisted leaching. In an acidic solution without the bacteria, ferrous iron is stable, and leaching mediated by ferric iron would be slow. T ferrooxidans can accelerate such an oxidation reaction by a factor of more than a million.  Direct and indirect leaching by bacteria are difficult to differentiate quantitatively because most minerals include some iron. Even if leaching were to begin with the direct process exclusively, the iron would be released from the mineral and would establish an indirect leaching cycle. Direct leaching by thiobacilli has been demonstrated in the laboratory with iron-free synthetic metal sulfides.  In practice, the leaching of metals is far more complex than the above analysis might suggest; there are numerous processes in addition to direct enzymatic oxidation and bacterial generation of ferric iron. Some chemical reactions between ferric iron and metal-sulfide minerals result in the formation of secondary minerals and elemental sulfur which can “blind,” or inactivate the reactive surfaces. When sulfur is formed, T. thiooxidans plays an indispensable role in oxidizing the sulfur to sulfuric acid, thus exposing the metal for further leaching.  The control of acidity is of utmost importance in leaching, because an acidic environment must be maintained in order to keep ferric iron and other metals in solution. Acidity is controlled by the oxidation of iron, sulfur and metal sulfides, by the dissolution of carbonate ions and by the decomposition of ferric iron through reaction with water. The last reaction promotes leaching by generating hydrogen ions (which make the solution more acidic), but it may also be detrimental because precipitates of basic ferric sulfates may inactivate the surfaces of metal-sulfide minerals, and in some cases, may even prevent the flow of the leaching solution. The chemical and biological processes are part of a complex system whose functioning depends on elements of hydrology, geology, physics and engineering.  The dumps are not inoculated with the leaching bacteria. The organisms are ubiquitous, and when conditions in the rock pile become suitable for their growth, they proliferate. Rock samples collected near the top of a leach pile typically harbor more than a million bacteria of the species T. ferrooxidans per gram;  T thiooxidans bacteria are present in somewhat smaller numbers. The leach solution percolates through the leach dump, and the “pregnant,” or metal-laden, solution is collected in catch basins or reservoirs at the foot of the dump. The copper is removed from solution either by a cementation reation, in

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which ferrous iron replaces the copper in solution, or by solvent extraction, in which the copper is concentrated by transferring it from the aqueous leaching solution to an organic solution. The “barren,” or copper-free solution, is then recycled to the top of the dump.  In a large dump-leaching operation, the ore is processed for many years to recover as much of the copper as possible. Because of the construction methods employed and the volume of the solid material treated, dump leaching is a crude operation. The placement of the dump in a natural valley can impede the flow of air to the interior of the pile. The large size of some of the rocks limits contact among the metal-sulfide minerals, the oxidizing solution and the bacteria. During the dumps construction, large ore haulers compact the surface, creating impermeable zones in the pile. Gypsum (CaSO4), ferric hydroxide [Fe(OH)3] and basic ferric sulfate precipitates also decrease permeability, further reducing the contact of the solution with the sulfide rocks.  Although, studies of bacteria in the dump environment have barely begun, some factors that may adversely affect the populations are known. They include high metal concentrations, particularly of ions such as silver and mercury that are known to be toxic to the organisms, lack of air, and temperatures higher than those tolerated by the organisms. Because the limitations of dump leaching are more clearly defined today than they were in the past, considerable forethought now goes into the construction of the leaching piles. Special “finger” dumps allow greater air circulation, and haulage is controlled to minimize compaction. From a biological viewpoint, however, dump leaching remains an essentially uncontrolled process.  Extractive methods other than dump leaching offer somewhat more regulation of biological, chemical and engineering factors. Heap leaching, for example, is used to extract metals from sulfide and oxide minerals in ores of a somewhat higher grade than those subjected to dump leaching. In heap leaching, the rocks are often crushed to avoid the solution-contact problems encountered when leaching large boulders, and the heaps are built up on impermeable pads to prevent loss of the solution into the underlying soil. Aeration systems have been installed to increase the flow of air in the piles.  In-place leaching is a promising technique for the recovery of metals from low-grade ores in inaccessible sites. This technology, which has minimal impact on the environment, is currently employed to extract residual minerals from abandoned mine workings and to recover uranium from low-grade deposits. To leach metals from depleted mine workings, the leaching solution is applied directly to the walls and the roof of an intact stope (an underground excavation from which ore has been removed) or to the rubble of fractured workings. In-place leaching techniques have been successful in the recovery of both copper and uranium. 

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In the extraction of uranium, the bacteria do not directly attack the uranium mineral; instead, they generate ferric iron from pyrite and soluble ferrous iron. Ferric iron readily attacks minerals incorporating quadrivalent uranium (U4+), converting this ion into hexavalent uranium (U6+), which is soluble in dilute sulfuric acid.  The bacterially–assisted leaching of uranium could, in principle, be applied not only to the recovery of residual metals but also to the in-place leaching of low-grade uranium ore bodies. There are a number of low-grade deposits in the western and southwestern U.S., but they are low in pyritic material and include rocks that tend to neutralize acids. Such mineralogical conditions are not conducive to bacterial leaching, and an exclusively chemical method of leaching has been adopted instead. Wells are drilled into the isolated ore bodies at depths ranging from tens to hundreds of feet. A carbonate solution containing an oxidant is then injected into the mineralized zone, where the uranium is solubilized. The uranium-bearing solution is withdrawn from the formation through a precisely engineered pattern of recovery wells.  Although bacterial leaching is currently exploited only for the recovery of copper and uranium, the appetite of the leaching bacteria is fairly nonspecific. The organisms readily degrade other sulfide minerals, yielding zinc from sphalerite (ZnS) and lead from galena (PbS). The leaching bacteria can therefore be considered for the extraction of many other metals. The bacteria readily catalyze the dissolution of inorganic sulfur from coal, and recent advances indicate that organic sulfur may also be vulnerable to microbiological attack.  The microbiological processes currently exploited by the minerals industry are fairly simple in engineering design, and their effectiveness is sensitive to seasonal changes and sudden alterations in the chemistry of the system. Recent developments in the study of the uptake of metals by S. cerevisiae,  R. arrhizus and P. aeruginosa make it probable that these microorganisms can be utilized in precisely engineered processes for the recovery of metals from wastewater streams. The new tools of genetic engineering may well lead to the creation of modified organisms with, greater effectiveness in metal removal. It was noted above that only 44 per cent of the P. aeruginosa cells take part in the uranium-uptake process; Shumate and Strandberg speculate that if the factor or factors controlling the uptake can be identified, the bacteria may be genetically altered to increase the population that accumulates the metal.  The accumulation of metals by microorganisms, whether the process is intracellular uptake or surface accumulation, is fairly nonspecific: negatively charged groups of atoms on the surface of microorganisms attract any positively charged ions in the solution. Many organisms have cellular components that are highly metal-specific. One of the best-understood metal-binding agents is the protein metallothionein. Structural studies of metallothionein indicate there is a high concentration of sulfur-containing amino acid units, which, when they are brought into juxtaposition by the folding of the protein

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chain, form a sulfhydryl (HS-) chelation site. In the marine blue-green alga Synechococcus, a comparatively small cadmium-binding metallothionein can bind an average of 1.28 atoms of cadmium per molecule of protein. The identification of the gene or the genes that specify the structure of the metallothionein of this organism or any other may enable geneticists to isolate and clone the genes in selected microorganisms. Cells carrying the cloned genes could be directed to synthesize massive quantities of metallothionein with a specific metal-binding capacity. The small protein could be immobilized on an inert carrier and wastewater contaminated with metals could be passed over the fixed protein. Further studies of metallothioneins with the ability to bind specific metals may provide clues for the laboratory synthesis of simple compounds with an increased metal-binding capacity. Based on nature’s own workings, on controlled laboratory studies done by many workers and on current applications in the field, it is clear that microorganisms and their versatile activities will help man to lay claim to mineral wealth buried deep in the ground or available in amounts not economically feasible to recover at present. These small servants of man promise to help in cleaning the air and water while retrieving valuable metal resources.

4.8

BIOINDICATORS OF HAZARDOUS POLLUTANTS

The term ‘bioindicator’ is used for organisms or organism associations which respond to pollutant load with changes in vital functions, or which accumulate pollutants. Information about specific biological effects, supplements data on air pollutions generated by technical analysis methods. The most important reasons for using bioindicators are:

• the direct determination of biological effects,



• the determination of synergetic and antagonistic effects of multiple pollutants on an organism,



• the early recognition of pollutant damage to plants as well as toxic dangers to humans and



• relatively low cost compared to technical measuring methods.

The great potential of bioindicators for environmental monitoring is often confronted with difficult questions of methodology resulting from the use of “living measuring instruments”. The effects of environmental load cannot always be clearly differentiated from natural stress factors. Lack of practical experience with certain bioindicators sometimes makes clear interpretation of findings more difficult, especially, if no comparable pollutant measurements are available. Intensive research over the last decades has resulted in the availability of numerous bioindicators which satisfy the requirements of convenience, standardization, cost, and evaluative capability. Bioindicators are commonly grouped into accumulation indicators and response indicators. Accumulation

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indicators store pollutants without any evident changes in their metabolisms. Response indicators react with cell changes or visible symptoms of damage when taking up even small amounts of harmful substances. Biomonitoring is divided into passive and active:

• Passive biomonitoring is the use of organisms, organism associations, and parts of organisms which are a natural component of the ecosystem and appear there spontaneously.



• Active biomonitoring includes all methods which insert organisms under controlled conditions into the site to be monitored.

Aquifer (Underground Water) Indicators The organisms used as bioindicators must be characterized by much higher sensitivity than the best chemical indicators. Aquatic organisms accumulating pollutants allow us to detect them even when their water concentrations are too low to be detected. An example may be determination of radioisotope activity in plankton, which is several times higher than in water. Sometimes, the level of toxic substances in the abiotic part of a given area is low and does not suggest any threat to the environment, even in the case of further pollutant leakage. Analysis based on bioindicators, may at the same time, show that the concentration of toxic substances in living organisms is so high that its further increase may result in irreversible damage to particular populations or the whole organic world in the biotope examined. To make global analyses uniform, international organizations have established a set of principles to be followed during toxicity determination, and compiled a list of indicatory organisms. Bioindicators should be selected according to the following criteria:

• sedentary life,



• abundance, wide distribution,



• simple procedure of identification and sampling,



• high tolerance for the pollutants analyzed,



• population stability,



• high accumulating capacity.

The water purity state should be determined using organisms sensitive to pollution, characterized by a narrow range of tolerance. The following tests and bioindicators can be applied to analysis of water and sewage toxicity:

• test based on Chlorella vulgaris – a unicellular green alga, widespread in fresh waters. Diluted sewage solutions are introduced into laboratory algal cultures, then absorbance is measured with a spectrophotometer in the visible range;



• test based on Daphnia magna Straus – a crustacean living in fresh waters. Young organisms are placed in crystallizers with sewage solutions of different concentrations. The count of bioindicators showing the test

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effect (organism immobilization) is determined after 24 and 48 hours. These data allow to determine sample toxicity;

• test Spirotox, based on the protozoan Spirotostomum ambiguum, present in clean rivers and lakes. Ciliates are placed in the sample and observed under slight magnification. The cells of these very sensitive organisms undergo dissolution (lysis) when affected by toxicants. Sample toxicity is determined by its dilution, causing lysis of 50% of the population;



• test Microtox, which consists in measurement of the natural luminescence of bacteria Vibrio fischeri, suspended in the solution of the sample, analyzed. Toxic chemical compounds inhibit the activity of bacterial enzymes, which reduces the intensity of luminescence. The measurement is performed by the spectrophotometric method.

One of the criteria of water cleanliness is the fecal pollution index, referred to as the coli index, showing the degree of pollution with intestinal pathogenic bacteria. Also the so-called saprobiotic index is applied to evaluate running water purity. Water quality is determined on the basis of the count of indicatory organisms in a given site, and the catalogue value of the saprobiotic index. The suitability of particular animal species as bioindicators depends on their specific requirements towards the environment. Following table presents selected indicatory organisms typical of different water purity classes. Occurrence of selected bioindicators depending on water purity class Oligosaprobiotic zone

b-mesosaprobiotic zone

Diatoms Ceratoneis arcus Meridion cerculare Chrysophyte Hydrulus foetidus Red algae Betrachospermum vagum Zoobenthos Perla sp. Caddis-flies Molanna angustata

Snails Planorbis comeus Viparus viparus Lymne stagualis Common mayflies Ephemera vulgata Diatoms Melosira gramirata Melostra variens Blue-green algae Microcistis aeruginoza Bivalves Pisidhon amnicum

a-mesosaprobiotic zone Fungi Leptomitus lapteus Single diatoms Navicula viridula Zoobenthos Asselus aquaticus Erpobdella actoculata Bivalves Sphertum corneian

Polysaprobiotic zone Bacteria Spherotilus nataus Zoogla ramigera Bacterhim cynusii Thiothrix nivea Beggioata Zoobenthos Dipteran’s larvae Chironomus phomosus Eristalomya

The oligosaprobiotic zone is characterized by the presence of all systematic groups, corresponds to the first water purity class and is suitable for Salmonidae breeding. The most common bioindicators here are dipteran’s larvae, hemipterans and caddis-flies. The β-mesosaprobiotic zone (second water purity class) is suitable for breeding fish other than the family Salmonidae. The most popular bioindicators here are snails and diatoms. In the α-mesosaprobiotic zone (third water purity class) bioindicators are first of all fungi, whereas in the polysaprobiotic zone (fourth water purity class) – bacteria.

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Today the indices of water cleanliness are also determined on the basis of the species composition and count of different organisms (e.g. plankton, periphytons, benthos), as well as analysis of matter production and destruction processes. In clean waters, a state of equilibrium is maintained between these processes. An increase in organic matter supply results in the domination of destruction over production and macroconsumption. The only consumers left in the ecosystem are destructors. The presence or absence of certain indicatory species (algae, insects, crustaceans, fish) may provide detailed information on the purity or pollution state of aquatic ecosystems.

Use of Whole Organism for Detection of Pesticides and Heavy Metals A wide range of biological methods are already in use to detect pollution incidents and for the continuous monitoring of pollutants such as pesticides and heavy metals. Long established measures include:

• counting the number of plants,



• animal and microbial species,



• counting the numbers of individuals in those species or analysing the levels of oxygen, methane or other compounds in water.

More recently, biological detection methods, using biosensors and immunoassays, have been developed and are now being commercialised. Most biosensors are a combination of biological and electronic devices—often built onto a microchip. The biological component might be simply an enzyme or antibody, or even a colony of bacteria, a membrane, neural receptor, or an entire organism. Immobilised on a substrate, their properties change in response to some environmental effect in a way that is electronically or optically detectable. It is then possible to make quantitative measurements of pollutants with extreme precision or to very high sensitivities. The sensors can be designed to be very selective, or sensitive to a broad range of compounds.

• For example, a wide range of herbicides can be detected in river water using algal-based biosensors; the stresses inflicted on the organisms being measured as changes in the optical properties of the plant’s chlorophyll.

Microbial biosensors are micro-organisms which produce a reaction upon contact with the substance to be sensed. Usually they produce light but cease to do so upon contact with substances which are toxic to them. Both naturallyoccurring light-emitting microorganisms as well as specially developed ones are used. Positively acting bacterial biosensors have been constructed which start emitting light upon contact (and subsequent reaction) with a specific pollutant. In the USA, such a light emitting bacterium has been approved for the detection of polyhalogenated aromatic hydrocarbons in field tests. Immunoassays use labelled antibodies (complex proteins produced in biological response to specific agents) and enzymes to measure pollutant

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levels. If a pollutant is present, the antibody attaches itself to it; the label making it detectable either through colour change, fluorescence or radioactivity. Immunoassays of various types have been developed for the continuous, automated and inexpensive monitoring of pesticides such as dieldrin and parathion. The nature of these techniques, the results of which can be as simple as a colour change, make them particularly suitable for highly sensitive field testing where the time and large equipment needed for more traditional testing is impractical. Their use is however limited to pollutants which can trigger biological antibodies. If the pollutants are too reactive, they will either destroy the antibody or suppress its activity and so also the effectiveness of the test. Detection and monitoring of microorganisms used for bioremediation: When laboratory grown micro-organisms are inoculated into a bioremediation site (bioaugmentation), it often becomes necessary to monitor their presence and/or multiplication to check the progress of the process. This is especially true and even required when genetically modified microorganisms are involved. The traditional technique to detect the presence of micro-organisms in soil is direct plating on selective media. This is greatly facilitated if the organism contains a marker which can be selected for. Newer techniques include the above mentioned immunological and light-based bioreporter techniques. The spatial distribution of specific microorganisms in a sample can be determined microscopically and non-invasively by using fluorescent in situ hybridisation (FISH) of micro-organisms. The most sensitive and specific technique is the direct isolation and amplification of DNA from soil, which is increasingly being used. Detection and monitoring of ecological effects: Bioremediation is aimed at improving the quality of the environment by removing pollutants. However, the disappearance of the original pollutant is not the only criterion by which the success of a bioremediation operation is determined. (Even more) toxic metabolites may be produced from the pollutant or the biodegrading bacterium may cause diseases or produce substances that are harmful to useful microorganisms, plants, animals or humans. All these negative effects, are of course, excluded as much as possible in advance by getting as familiar as possible with the organism through extensive literature searches and microcosm studies in which the bioremediation process is simulated in the laboratory. To avoid unexpected effects, especially after the release of new member of the eco-system like a genetically modified organism, the monitoring of the ecological effects of a bioremediation operation may be required. The problem with monitoring ecological effects is, what to monitor. Numerous ecological effects are possible but not all of them may be relevant or permanent or even the result of the bioremediation operation. The parameters to be monitored are usually determined case-by-case. Monitoring techniques may include all of those mentioned in the two previous subsections on detection and monitoring.

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Biogeochemical Methods Biogeochemistry integrates several disciplines, including biology, geology, geography, and chemistry. Biogeochemical studies can be diverse and may include topics such as nutrient cycling, biotic and abiotic weathering processes, carbon cycling, microbiological-macrobiological interactions, among others.

• The world is complex and Earth systems do not work independently of one another; that is, the biotic world is inherently tied to the abiotic world. Therefore, a more wholistic understanding of the Earth system can be achieved by integrating biological, geological, and geographic studies, among others.

Ion Chromatography Ion chromatography is used for water chemistry analysis. Ion chromatographs are able to measure concentrations of major anions, such as fluoride, chloride, nitrate, nitrite, and sulfate, as well as major cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium in the parts-per-billion (ppb) range. Concentrations of organic acids can also be measured through ion chromatography. How Does Ion Chromatography Work? Ion chromatography, a form of liquid chromatography, measures concentrations of ionic species by separating them based on their interaction with a resin. Ionic species separate differently, depending on species type and size. Sample solutions pass through a pressurized chromatographic column where ions are absorbed by column constituents. As an ion extraction liquid, known as eluent, runs through the column, the absorbed ions begin separating from the column. The retention time of different species determines the ionic concentrations in the sample. Applications: Some typical applications of ion chromatography include:

• Drinking water analysis for pollution and other constituents.



• Determination of water chemistries in aquatic ecosystems.



• Determination of sugar and salt content in foods.



• Isolation of select proteins.

How to – Sample Collection, Preparation and Concerns Liquid Samples: Liquid samples should be filtered prior to evaluation with an ion chromatograph to remove sediment and other particulate matter as well as to limit the potential for microbial alteration before the sample is run. Aqueous samples should be collected using a sterile syringe or bottle rinsed three times with sample water and then filtered through 0.45 um (or smaller) filters. The collection vial should, likewise, be rinsed three times with filtrate before being filled brimfull of sample filtrate. Samples should be stored cold until they can be processed. The minimum sample required for analysis is approximately 5 mL, with no maximum limits.

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Solid samples and Organic Liquids Solid samples can be extracted with water or acid (cations) to remove ions from the sample surface. Liquid samples must also be filtered and stored cold until analysis can be performed. The minimum sample required for a solid sample is approximately 2-3 cm2 for solids, with no maximum limits.

Measuring Primary Production Using 14C Radiolabeling Primary Productivity: Primary productivity is the process by which organisms make their own food from inorganic sources. The majority of primary producers are terrestrial plants and microbial life, such as algae. These organisms are known as autotrophs, since they can use inorganic substrates and solar energy to carry out metabolic processes and build cellular material. Primary productivity due to photosynthesis is commonly measured by quantifying oxygen production or CO2 assimilation. How Does This Method Work?

The theory behind using 14C to measure productivity involves using a labeled tracer to quantify assimilated carbon. The 14C method estimates the uptake and assimilation of dissolved inorganic carbon (DIC) by planktonic algae in the water column. The method is based on the assumption that biological uptake of 14C-labelled DIC is proportional to the biological uptake of the more commonly found 12C-DIC. In order to determine uptake, one must know the concentration of DIC naturally occurring in the sample water, the amount of 14C-DIC added, and the amount of 14C retained in particulate matter (14C-POC) at the end of the incubation experiment. A 5% metabolic discrimination factor may be applied to the data as well, since organisms preferentially take up lighter isotopes. Carbon uptake can be measured by the following equation: C uptake = (naturally occurring DIC × 14C-POC × 1.05)/( 14C-DIC added) Applications: Aquatic primary productivity generally governs the biological activity of a lake. Since primary productivity makes up the bottommost trophic level, it provides essential nutrients and energy to higher trophic levels and higher organisms. For instance, primary productivity in a lake may supply oxygen to aerobic organisms such as insects and fish, and so, times of stressed productivity may result in a decline in the fish population. Low primary productivity may thus hinder other lake biota by limiting available nutrients. Conversely, extremely high primary productivity may result in algal blooms, which may eventually lead to mass kills at other trophic levels due to nutrient depletion or by high turbidity from massive concentrations of plankton. So, relatively high rates of primary productivity may support the most diverse and largest amount of biomass, but productivity can also become too high for a system to handle.

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How To- Sample Collection, Protocol, Analysis, and Considerations Protocols and Analyses: There are several protocols for measuring rates of primary productivity via 14C. The basic idea remains the same: before dawn, water samples are collected in a series of bottles—generally, two clear and one amber/dark-colored bottle through which light is not able to penetrate. The water samples are inoculated with 14C, capped, and placed in the environment they were collected from, for a day (this time may vary). Following the incubation, the samples are collected under low light conditions and filtered through a 0.2 um filter. The filter is placed into a liquid scintillation vial, acidified to purge excess 14C, and kept cold until it can be analyzed in a scintillation counter. The following resources provide step-by-step protocols for measuring primary productivity using 14C. • Limnological Methods for the McMurdo Long Term Ecological Research Program, • Primary Productivity.

Considerations The 14C method for measuring productivity has several important considerations, including:

• This method assumes that 14C will be taken up proportionally to the more naturally prevalent 12C species.



• Bottles should be inoculated and collected in low-light conditions (predawn or post-sunset) in order to ensure that samples collected from subsurface waters will not receive sunlight they would not naturally receive.



• Samples should be filtered as soon as possible after collection. Excess (non-incorporated) 14C can be purged from the filter, using a small amount of 1M HCl (this will drive off excess 14C as CO2.



• When running a scintillation count, dissolving the filter in the scintillation cocktail is not necessarily bad, since it ensures that all 14C will be in solution, and thus, be counted.

Results Analysis: Rates of primary productivity will vary by environment. Factors influencing rates of primary productivity include:

• Availability of nutrients,



• Availability of Photosynthetically Active Radiation (PAR), or available light. This can be influenced by lake depth, turbidity/suspended sediment load, and shading by macrophytes, or plants,



• Biota dynamics in the system (e.g. population composition, number of primary producing organisms, competition, population establishment time vs. disturbance).

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Rates of primary productivity can range from 0-9000 kcal/m2/yr, with desert regions having lower rates and estuaries having higher rates. More constrained rates of primary productivity for specific environments may be researched by looking at primary literature for a specific site.

Measuring Dissolved and Particulate Organic Carbon (DOC and POC) Dissolved organic carbon (DOC) is defined as the organic matter that is able to pass through a filter (filters generally range in size between 0.7 and 0.22 um). Conversely, particulate organic carbon (POC) is that carbon, that is too large and is filtered out of a sample. If you have ever seen a body of water that appears straw, tea, or brownish in color, it likely has a high organic carbon load. This color comes from the leaching of humic substances from plant and soil organic matter. This organic matter contributes acids to the stream, resulting in the yellow-brown coloration as well as weathering of the soils. Organic carbon can be allochthonous, or sourced from outside the system (e.g. by atmospheric deposition, or transported long distances via stream flow) or it can be autochthonous, or sourced from the immediate surroundings of the system (e.g. plant and microbial matter and sediments/soils within the catchment). High amounts of organic matter are common in low-oxygen areas, such as bogs and wetlands. • Dissolved and particulate organic carbon are important components in the carbon cycle and serve as a primary food sources for aquatic food webs. In addition, DOC alters aquatic ecosystem chemistries by contributing to acidification in low-alkalinity, weakly buffered, freshwater systems. Furthermore, DOC forms complexes with trace metals, creating water-soluble complexes which can be transported and taken up by organisms. Finally, organic carbon, as well as other dissolved and particulate matter, can affect light penetration in aquatic ecosystems, which is important for the ecosystem’s phototrophs that need light to subsist. • Dissolved organic carbon can be measured via several different techniques. High temperature combustion and UV/persulfate oxidation methods are discussed in detail below, but both methods share the same sample preparation protocol: • The sample is collected in a glass container that has been baked in the laboratory at 550°C for 2-4 hours (the baking process removes any residual carbon, in or on the collection vessel, that may cause contamination). • The sample is then filtered with a glass filtration device. Commonly used filters include glass fiber filters (GF/F), silver membrane filters, or a nitrocellulose/polypro filters and range between 0.7-0.25 mm in pore size. The nitrocellulose/polypro filters are the least expensive of these filters, but may leach DOC, so they should be cleaned by passing deionized water through them, before collection.

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• Once collected, samples should be stored cold (e.g. in the refrigerator or on ice) until they can be processed. They should be processes as soon as possible to prevent post-filtering sample alteration.



• Measuring DOC By High Temperature Combustion: The high temperature combustion method for measuring DOC involves conversion of inorganic carbon to dissolved CO2, and purging this from the sample. The remaining (organic) carbon is then oxidized at a high temperature to CO2 which can be detected by the instrument’s nondispersive infrared (NDIR) sensor and directly correlated to total organic carbon (TOC) content.



• Measuring DOC By UV/Persulfate Oxidation: This method combines the sample with an acid, lowering the sample pH to 2.0. This process converts inorganic carbon to dissolved CO2, which is then purged from the sample. A persulfate reagent is then added to the sample and the remaining carbon is oxidized by UV radiation to form CO2, which can be detected by the NDIR sensor and directly correlated to total organic carbon (TOC) content.

Particulate organic carbon is measured by determining mass lost, upon combustion of a sample. In aqueous samples, this can be done by measuring the dry mass of a filter that had a known amount of water passed through it before and after it is subjected to combustion via heating the filter to 550° C. This method requires that the filter is purged of extraneous POC before filtration (by combusting it at 550°C for 2 hours), and that the filter and sample are dry (this can be done by putting them in a warm oven) at their precombustion weight measurement. The method also requires that the sample has a measurable amount of organic carbon present. POC in soil samples can also be measured by mass loss by measuring the dry weight of a given volume of sample before and after combustion. These methods assume that the mass loss is attributable solely to carbon, rather than any other sample component. In addition to measuring DOC concentrations in a sample, DOC can be characterized to determine its reactivity (including quality and composition), source, and potential importance in its ecosystem. Characterization via absorbance and fluorescence are discussed below. Absorbance: Terrestrial and microbial-derived humic substances differ in their carbon to nitrogen ratio (C:N ratio).

• Terrestrially-derived humic acids have high C:N ratios because they are derived from lignin, and lignin does not contain nitrogen. These humics contain large amounts of carbon in the form of aromatic carbons and phenols.



• Microbially-derived humic substances have high N contents, relative to terrestrial sources, along with a low aromatic carbon and phenolic content.

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Utilizing these differences, UV absorbance can provide an estimate of the aromaticity of the DOC in a sample, and thereby, determine its source. Fluorescence: DOC can be characterized by differences in fluorescence spectra also, which is associated with different sources of organic carbon in aquatic systems. This method involves excitation of a sample and looking at its 3-d excitation-emission matrix for fulvic acids. Basically, a sample is excited and its corresponding emission intensity is used to determine its source. The emission intensity ratio is generally higher for microbially-derived fulvic acids than it is for terrestrially-derived acids. Results Analysis: As stated above, DOC is an important component in an ecosystem. It provides a primary food source for aquatic food webs, suggesting that high DOC is beneficial to an ecosystem. However, DOC can also contribute to the acidity of a water body and can increase light attenuation, thus, detrimentally affecting phototrophic organisms in an aquatic environment. Therefore, as with most things, moderation is key for DOC content. Depending on factors such as buffering capacity, or the ability of an aquatic system to stabilize its acidity/alkalinity, biomass composition and amount, and water depth, DOC necessary to support an ecosystem varies by area. Typical DOC values for various environments are commonly reported in scientific literature such as peer-reviewed journal articles and textbooks.

CHAPTER

5

Beneficial and Effective Microorganisms for a Sustainable Agriculture and Environment

The uniqueness of microorganisms and their often unpredictable nature and biosynthetic capabilities, given a specific set of environmental and cultural conditions, has made them likely candidates for solving particularly difficult problems in the life sciences and other fields, as well. The various ways in which microorganisms have been used over the past 50 years to advance medical technology, human and animal health, food processing, food safety and quality, genetic engineering, environmental protection, agricultural biotechnology, and more effective treatment of agricultural and municipal wastes provide a very impressive record of achievement. Many of these technological advances would not have been possible using straightforward chemical and physical engineering methods, or if they were, they would not have been practically or economically feasible. Nevertheless, while microbial technologies have been applied to various agricultural and environmental problems with considerable success in recent years, they have not been widely accepted by the scientific community because it is often difficult to consistently reproduce their beneficial effects. Microorganisms are effective only when they are presented with suitable and optimum conditions for metabolizing their substrates including available water, oxygen (depending on whether the micro-organisms are obligate aerobes or facultative anaerobes), pH and temperature of their environment. Meanwhile, the various types of microbial cultures and inoculants available in the market today have increased rapidly because of these new technologies. Significant achievements are being made in systems where technical guidance is coordinated with the marketing of microbial products. Since microorganisms are useful in eliminating problems associated with the use of chemical fertilizers and pesticides, they are now widely applied in nature farming and organic agriculture Environmental pollution, caused by excessive soil erosion and the associated transport of sediments, chemical fertilizers and pesticides to surface waters and groundwater, and improper

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Environmental Biotechnology

treatment of human and animal wastes has caused serious environmental and social problems throughout the world. Often, engineers have attempted to solve these problems using established chemical and physical methods. However, they have usually found that such problems cannot be solved without using microbial methods and technologies in coordination with agricultural production. For many years, soil microbiologists and microbial ecologists have tended to differentiate soil microorganisms as beneficial or harmful according to their functions and how they affect soil quality, plant growth and yield, and plant health. As shown in following Table 1, beneficial microorganisms are those that can fix atmospheric nitrogen, decompose organic wastes and residues, detoxify pesticides, suppress plant diseases and soil-borne pathogens, enhance nutrient cycling, and produce bioactive compounds such as vitamins, hormones and enzymes that stimulate plant growth. Harmful microorganisms are those that can induce plant diseases, stimulate soil-borne pathogens, immobilize nutrients, and produce toxic and putrescent substances that adversely affect plant growth and health. Some common functions of beneficial and harmful soil micro-organisms as they affect soil quality, crop production, and plant health Functions of Beneficial Microorganisms • Fixation of atmospheric nitrogen

Functions of Harmful Microorganisms • Induction of plant diseases

• Decomposition of organic wastes • Stimulation of soil-borne and residues pathogens • Suppression of soil-borne pathogens

• Immobilization of plant nutrients

• Recycling and increased availability of plant nutrients

• Inhibition of seed germination

• Degradation of toxicants including pesticides

• Inhibition of plant growth and development

• Production of antibiotics and other bioactive compounds

• Production of phytotoxic substances

• Production of simple organic molecules for plant uptake • Complexation of heavy metals to limit plant uptake • Solubilization of insoluble nutrient sources • Production of polysaccharides to improve soil aggregation

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A more specific classification of beneficial microorganisms has been suggested by Higa (1991; 1994; 1995) which he refers to as “Effective Microorganisms” or EM. This report presents some new perspectives on the role and application of beneficial microorganisms, including EM, as microbial inoculants for shifting the soil microbiological equilibrium in ways that can improve soil quality, enhance crop production and protection, conserve natural resources, and ultimately create a more sustainable agriculture and environment. The report also discusses strategies on how beneficial microorganisms, including EM, can be more effective after inoculation into soils.

5.1 THE CONCEPT OF EFFECTIVE MICROORGANISMS: THEIR ROLE AND APPLICATION The concept of Effective Microorganisms (EM) was developed by Professor Teruo Higa, University of the Ryukyus, Okinawa, Japan. EM consists of mixed cultures of beneficial and naturally-occurring microorganisms that can be applied as inoculants to increase the microbial diversity of soils and plants. Research has shown that the inoculation of EM cultures to the soil/plant ecosystem can improve soil quality, soil health, and the growth, yield, and quality of crops. EM contains selected species of microorganisms including predominant populations of lactic acid bacteria and yeasts, and smaller numbers of photosynthetic bacteria, actinomycetes and other types of organisms. All of these are mutually compatible with one another and can coexist in liquid culture. EM is not a substitute for other management practices. It is, however, an added dimension for optimizing our best soil and crop management practices such as crop rotations, use of organic amendments, conservation tillage, crop residue recycling, and biocontrol of pests. If used properly, EM can significantly enhance the beneficial effects of these practices. Throughout the discussion which follows, we will use the term “beneficial microorganisms” in a general way to designate a large group of often unknown or ill-defined microorganisms that interact favorably in soils and with plants to render beneficial effects which are sometimes difficult to predict. We use the term “Effective Microorganisms” or EM to denote specific mixed cultures of known, beneficial microorganisms that are being used effectively as microbial inoculants.

5.2 UTILIZATION OF BENEFICIAL MICROORGANISMS IN AGRICULTURE What Constitutes an Ideal Agricultural System: Conceptual design is important in developing new technologies for utilizing beneficial and Effective

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Environmental Biotechnology

Microorganisms for a more sustainable agriculture and environment. The basis of a conceptual design is simply to first conceive an ideal or model and then to devise a strategy and method for achieving the reality. However, it is necessary to carefully coordinate the materials, the environment, and the technologies constituting the method. Moreover, one should adopt a philosophical attitude in applying microbial technologies to agricultural production and conservation systems. There are many opinions on what an ideal agricultural system is. Many would agree that such an idealized system should produce food on a longterm sustainable basis. Many would also insist that it should maintain and improve human health, be economically and spiritually beneficial to both producers and consumers, actively preserve and protect the environment, be self-contained and regenerative, and produce enough food for an increasing world population. Efficient Utilization and Recycling of Energy: Agricultural production begins with the process of photosynthesis by green plants, which requires solar energy, water, and carbon dioxide. It occurs through the plant’s ability to utilize solar energy in “fixing” atmospheric carbon dioxide into carbohydrates. The energy obtained is used for further biosynthesis in the plant including essential amino acids and proteins. The materials used for agricultural production are abundantly available with little initial cost. However, when it is observed as an economic activity, the fixation of carbon dioxide by photosynthesis has an extremely low efficiency mainly because of the low utilization rate of solar energy by green plants. Therefore, an integrated approach is needed to increase the level of solar energy utilization by plants so that greater amounts of atmospheric carbon dioxide can be converted into useful substrates. Although the potential utilization rate of solar energy by plants has been estimated theoretically at between 10 and 20%, the actual utilization rate is usually less than 1%. Even some C 4 plants, such as sugarcane, with very high photosynthetic efficiencies, will seldom exceed a utilization rate of 6 or 7% during the maximum growth period. The utilization rate is normally less than 3% even for optimum crop yields. Past studies have shown that photosynthetic efficiency of the chloroplasts of host crop plants cannot be increased much further; this means that their biomass production has reached a maximum leve1. Therefore, the best opportunity for increasing biomass production is to somehow utilize the visible light, which chloroplasts cannot presently use, and the infrared radiation; together, these comprise about 80% of the total solar energy. Also, we must explore ways of recycling organic energy contained in plant and animal residues through direct utilization of organic molecules by plants. Thus, it is difficult to exceed the existing limits of crop production unless the efficiency of utilizing solar energy is increased, and the energy contained

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in existing organic molecules (amino acids, peptides and carbohydrates) is utilized either directly or indirectly by the plant. This approach could help to solve the problems of environmental pollution and degradation caused by the misuse and excessive application of chemical fertilizers and pesticides to soils. Therefore, new technologies that can enhance the economic-viability of farming systems with little or no use of chemical fertilizers and pesticides are urgently needed and should be a high priority of agricultural research, both now, and in the immediate future. Preservation of Natural Resources and the Environment: The excessive erosion of topsoil from farmland caused by intensive tillage and row-crop production has caused extensive soil degradation and also contributed to the pollution of both surface waters and groundwater. Organic wastes from animal production, agricultural and marine processing industries, and municipal wastes (e.g., sewage and garbage), have become major sources of environmental pollution in both developed and developing countries. Furthermore, the production of methane from paddy fields and ruminant animals and, of carbon dioxide from the burning of fossil fuels, land clearing and organic matter decomposition have been linked to global warming as “green house gases”. Chemical-based, conventional systems of agricultural production have created many sources of pollution that, either directly or indirectly, can contribute to degradation of the environment and destruction of our natural resource base. This situation would change significantly if these pollutants could be utilized in agricultural production as sources of energy. Therefore, it is necessary that future agricultural technologies be compatible with the global ecosystem and with solutions to such problems in areas different from those of conventional agricultural technologies. An area that appears to hold the greatest promise for technological advances in crop production, crop protection, and natural resource conservation is that of beneficial and Effective Microorganisms applied as soil, plant and environmental inoculants.

5.3 BENEFICIAL AND EFFECTIVE MICROORGANISMS FOR A SUSTAINABLE AGRICULTURE Integration of Essential Components for Optimum Crop and Livestock Production: Agriculture, in a broad sense, is not an enterprise which leaves everything to nature without intervention. Rather, it is a human activity in which the farmer attempts to integrate certain agro-ecological factors and production inputs for optimum crop and livestock production. Thus, it is reasonable to assume that farmers should be interested in ways and means of controlling beneficial soil microorganisms as an important component of the agricultural environment. Nevertheless, this idea has often been rejected by naturalists and proponents of nature farming and organic agriculture. They

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Environmental Biotechnology

argue that beneficial soil microorganisms will increase naturally when organic amendments are applied to soils as carbon, energy and nutrient sources. This indeed may be true where an abundance of organic materials are readily available for recycling which often occurs in small-scale farming. However, in most cases, soil microorganisms, beneficial or harmful, have often been controlled advantageously when crops in various agro ecological zones are grown and cultivated in proper sequence (i.e., crop rotations) and without the use of pesticides. This explains why scientists have long been interested in the use of beneficial microorganisms as soil and plant inoculants to shift the microbiological equilibrium in ways that would enhance soil quality and the yield and quality of crops. Most would agree that a basic rule of agriculture is to ensure that specific crops are grown according to their agro climatic and agro ecological requirements. However, in many cases the agricultural economy is based on market forces that demand a stable supply of food, and land to its full productive potential throughout the year. The purpose of crop breeding is to improve crop production, crop protection, and crop quality. Improved crop cultivars along with improved cultural and management practices have made it possible to grow a wide variety of agricultural and horticultural crops in areas where it once would not have been culturally or economically feasible. The cultivation of these crops in such diverse environments has contributed significantly to a stable food supply in many countries. However, it is somewhat ironic that new crop cultures are almost never selected with consideration of their nutritional quality or bioavailability after ingestion. To enhance the concept of controlling and utilizing beneficial microorganisms for crop production and protection, one must harmoniously integrate the essential components for plant growth and yield including light (intensity, photoperiod, and quality), carbon dioxide, water, nutrients (organic-inorganic), soil type, and the soil microflora. Because of these vital interrelationships, it is possible to envision a new technology and a more energy-efficient system of biological production.

5.4

BENEFICIAL MICROORGANISMS FOR SOIL QUALITY AND A MORE SUSTAINABLE AGRICULTURE:

As will be discussed later, crop growth and development are closely related to the nature of the soil microflora, especially those in close proximity to plant roots, i.e., the rhizosphere. Thus, it will be difficult to overcome the limitations of conventional agricultural technologies without controlling soil microorganisms. This particular tenet is further reinforced because the evolution of most forms of life on earth and their environments are sustained by microorganisms. Most biological activities are influenced by the state of these invisible, minuscule units of life. Therefore, to significantly increase

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food production, it is essential to develop crop cultivars with improved genetic capabilities (i.e., greater yield potential, disease resistance, and nutritional quality) and with a higher level of environmental competitiveness, particularly under stress conditions (i.e., low rainfall, high temperatures, nutrient deficiencies, and aggressive weed growth). Low agricultural production efficiency is closely related to a poor coordination of energy conversion which, in turn, is influenced by crop physiological factors, the environment, and other biological factors including soil microorganisms. The soil and rhizosphere microflora can accelerate the growth of plants and enhance their resistance to disease and harmful insects by producing bioactive substances. These microorganisms maintain the growth environment of plants, and may have primary effects on both soil quality and crop quality. A wide range of results are possible depending on their predominance and activities at anyone time. Nevertheless, there is a growing consensus that it is possible to attain maximum economic crop yields of high quality, at higher net returns, without the application of chemical fertilizers and pesticides. Until recently, this was not thought to be a very likely possibility using conventional agricultural methods. However, it is important to recognize that the best soil and crop management practices to achieve a more sustainable agriculture will also enhance the growth, numbers and activities of beneficial soil microorganisms that, in turn, can improve the growth, yield and quality of crops. In essence, soil quality is the very foundation of a more sustainable agriculture.

5.5

CONTROLLING THE SOIL MICROFLORA: PRINCIPLES AND STRATEGIES

Principles of Natural Ecosystems and the Application of Beneficial and Effective Microorganisms: The misuse and excessive use of chemical fertilizers and pesticides have often adversely affected the environment and created many problems associated with, a) food safety and quality and b) human and animal health. Consequently, there has been a growing interest in nature farming and organic agriculture by consumers and environmentalists as possible alternatives to chemical-based, conventional agriculture. Agricultural systems which conform to the principles of natural ecosystems are now receiving a great deal of attention in both developed and developing countries. A number of books and journals have recently been published which deal with many aspects of natural farming systems. New concepts such as alternative agriculture, sustainable agriculture, soil quality, integrated pest management, integrated nutrient management and even beneficial microorganisms, are being explored by the agricultural research establishment. Although, these concepts and associated methodologies hold

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Environmental Biotechnology

considerable promise, they also have limitations. For example, the main limitation in using microbial inoculants is the problem of reproducibility and lack of consistent results. Unfortunately, certain microbial cultures have been promoted by their suppliers as being effective for controlling a wide range of soil-borne plant diseases, when in fact, they were effective only on specific pathogens under very specific conditions. Some suppliers have suggested that their particular microbial inoculant is akin to a pesticide that would suppress the general soil microbial population while increasing the population of a specific beneficial microorganism. Nevertheless, most of the claims for these single-culture microbial inoculants are greatly exaggerated and have not proven to be effective under field conditions. One might speculate that if all of the microbial cultures and inoculants that are available as marketed products were applied at the same time, some degree of success might be achieved because of the increased diversity of the soil microflora and stability that is associated with mixed cultures. While this, of course, is a hypothetical example, the fact remains that there is a greater likelihood of controlling the soil microflora by introducing mixed cultures of compatible microorganisms, rather than single, pure cultures. Even so, the use of mixed cultures in this approach has been criticized because it is difficult to demonstrate conclusively which microorganisms are responsible for the observed effects, how the introduced microorganisms interact with the indigenous species, and how these new associations affect the soil plant environment. Thus, the use of mixed cultures of beneficial microorganisms as soil inoculants to enhance the growth, health, yield, and quality of crops has not gained widespread acceptance by the agricultural research establishment because conclusive scientific proof is often lacking. The use of mixed cultures of beneficial microorganisms as soil inoculants is based on the principles of natural ecosystems which are sustained by their constituents; that is, by the quality and quantity of their inhabitants and specific ecological parameters, i.e., the greater the diversity and number of the inhabitants, the higher the order of their interaction and the more stable the ecosystem. The mixed culture approach is simply an effort to apply these principles to natural systems such as agricultural soils, and to shift the microbiological equilibrium in favor of increased plant growth, production and protection. It is important to recognize that soils can vary tremendously as to their types and numbers of microorganisms. These can be both beneficial and harmful to plants, and often, the predominance of either one depends on the cultural and management practices that are applied. It should also be emphasized that most fertile and productive soils have a high content of organic matter and, generally, have large populations of highly diverse microorganisms (i.e., both species and genetic diversity). Such soils will also usually have a wide ratio of beneficial to harmful microorganisms.

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5.6 CONTROLLING THE SOIL MICROFLORA FOR OPTIMUM CROP PRODUCTION AND PROTECTION The idea of controlling and manipulating the soil microflora through the use of inoculants, organic amendments, and cultural and management practices to create a more favorable soil microbiological environment for optimum crop production and protection is not new. For almost a century, microbiologists have known that organic wastes and residues, including animal manures, crop residues, green manures, municipal wastes (both raw and composted), contain their own indigenous populations of microorganisms, often with broad physiological capabilities. It is also known that when such organic wastes and residues are applied to soils, many of these introduced microorganisms can function as biocontrol agents by controlling or suppressing soil-borne plant pathogens through their competitive and antagonistic activities. While this has been the theoretical basis for controlling the soil microflora, in actual practice, the results have been unpredictable and inconsistent, and the role of specific microorganisms has not been well-defined. For many years, microbiologists have tried to culture beneficial microorganisms for use as soil inoculants to overcome the harmful effects of phyto-pathogenic organisms, including bacteria, fungi, and nematodes. Such attempts have usually involved single applications of pure cultures of microorganisms which have been largely unsuccessful for several reasons. First, it is necessary to thoroughly understand the growth and survival characteristics of each particular beneficial microorganism, including their nutritional and environmental requirements. Second, we must understand their ecological relationships and interactions with other microorganisms, including their ability to coexist in mixed cultures, both before and after application to soils. There are other problems and constraints that have been major obstacles to controlling the microflora of agricultural soils. First and foremost is the large number of types of microorganisms that are present at any one time, their wide range of physiological capabilities, and the dramatic fluctuations in their populations that can result from man’s cultural and management practices applied to a particular farming system. The diversity of the total soil microflora depends on the nature of the soil environment and those factors which affect the growth and activity of each individual organism including temperature, light, aeration, nutrients, organic matter, pH and water. While there are many microorganisms that respond favorably to these factors, or a combination thereof, there are some that do not. Microbiologists have actually studied relatively few of the microorganisms that exist in most agricultural soils, mainly because we don’t know how to culture them; i.e., we know very little about their growth, nutritional, and ecological requirements.

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Environmental Biotechnology

The diversity and population factors associated with the soil microflora have discouraged scientists from conducting research to develop control strategies. Many believe that, even when beneficial microorganisms are cultured and inoculated into soils, their number is relatively small compared with the indigenous soil inhabitants, and they would likely be rapidly overwhelmed by the established soil microflora. Consequently, many would argue that even if the application of beneficial microorganisms is successful under limited conditions (e.g., in the laboratory), it would be virtually impossible to achieve the same success under actual field conditions. Such thinking still exists today, and serves as a principal constraint to the concept of controlling the soil microflora. It is noteworthy that most of the microorganisms encountered in any particular soil are harmless to plants with only a relatively few that function as plant pathogens or potential pathogens. Harmful microorganisms become dominant if conditions develop that are favorable to their growth, activity and reproduction. Under such conditions, soilborne pathogens (e.g., fungal pathogens) can rapidly increase their populations with devastating effects on the crop. If these conditions change, the pathogen population declines just as rapidly to its original state. Conventional farming systems that tend toward the consecutive planting of the same crop (i.e., monoculture) necessitate the heavy use of chemical fertilizers and pesticides. This, in turn, generally increases the probability that harmful, disease-producing, plant pathogenic microorganisms will become more dominant in agricultural soils. Chemical-based conventional farming methods are not unlike symptomatic therapy. Examples of this are, applying fertilizers when crops show symptoms of nutrient-deficiencies, and applying pesticides whenever crops are attacked by insects and diseases. In efforts to control the soil microflora some scientists feel that the introduction of beneficial microorganisms should follow a symptomatic approach. However, we do not agree. The actual soil conditions that prevail at any point in time may be most unfavorable to the growth and establishment of laboratory-cultured, beneficial microorganisms. To facilitate their establishment, it may require that the farmer make certain changes in his cultural and management practices to induce conditions that will: (a) allow the growth and survival of the inoculated microorganisms and (b) suppress the growth and activity of the indigenous plant pathogenic microorganisms. An example of the importance of controlling the soil microflora and how certain cultural and management practices can facilitate such control is useful here. Vegetable cultivars are often selected on their ability to grow and produce over a wide range of temperatures. Under cool, temperate conditions there are generally few pest and disease problems. However, with the onset of hot weather, there is a concomitant increase in the incidence of diseases and insects making it rather difficult to obtain acceptable yields without applying

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pesticides. With higher temperatures, the total soil microbial population increases as do certain plant pathogens such as Fusarium, which is one of the main putrefactive, fungal pathogens in soil. The incidence and destructive activity of this pathogen can be greatly minimized by adopting reduced tillage methods and by shading techniques to keep the soil cool during hot weather. Another approach is to inoculate the soil with beneficial, antagonistic, antibiotic-producing microorganisms such as actinomycetes and certain fungi.

5.7

APPLICATION OF BENEFICIAL AND EFFECTIVE MICROORGANISMS

A New Dimension for a Sustainable Agriculture and Environment: Many microbiologists believe that the total number of soil microorganisms can be increased by applying organic amendments to the soil. This is generally true because most soil microorganisms are heterotrophic, i.e., they require complex organic molecules of carbon and nitrogen for metabolism and biosynthesis. Whether the regular addition of organic wastes and residues will greatly increase the number of beneficial soil microorganisms in a short period of time, is questionable. However, we do know that heavy applications of organic materials, such as seaweed, fish meal, and chitin from crushed crab shells, not only helps to balance the micronutrient content of a soil but also increases the population of beneficial antibiotic-producing actinomycetes. This can transform the soil into a disease-suppressive state within a relatively short period. The probability that a particular beneficial microorganism will become predominant, even with organic farming or nature farming methods, will depend on the ecosystem and environmental conditions. It can take several hundred years for various species of higher and lower plants to interact and develop into a definable and stable ecosystem. Even if the population of a specific microorganism is increased through cultural and management practices, whether it will be beneficial to plants is another question. Thus, the likelihood of a beneficial, plant-associated microorganism becoming predominant under conservation-based farming systems is virtually impossible to predict. Moreover, it is very unlikely that the population of useful anaerobic microorganisms, which usually comprise only a small part of the soil microflora, would increase significantly even under natural farming conditions. This information then emphasizes the need to develop methods for isolating and selecting different microorganisms for their beneficial effects on soils and plants. The ultimate goal is to select microorganisms that are physiologically and ecologically compatible with one another and that can be introduced as mixed cultures into soil where their beneficial effects can be realized. Principles and Fundamental Considerations: Microorganisms are utilized in agriculture for various purposes; as important components of

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Environmental Biotechnology

organic amendments and composts, as legume inoculants for biological nitrogen fixation, as a means of suppressing insects and plant diseases to improve crop quality and yield, and for reduction of labor. All of these are closely related to one another. An important consideration in the application of beneficial microorganisms to soils is the enhancement of their synergistic effects. This is difficult to accomplish if these microorganisms are applied to achieve symptomatic therapy, as in the case of chemical fertilizers and pesticides. If cultures of beneficial microorganisms are to be effective after inoculation into soil, it is important that their initial populations be at a certain critical threshold level. This helps to ensure that the amount of bioactive substances produced by them will be sufficient to achieve the desired positive effects on crop production and/or crop protection. If these conditions are not met, the introduced microorganisms, no matter how useful they are, will have little, if any, effect. At present, there are no chemical tests that can predict the probability of a particular soil inoculated microorganism to achieve a desired result. The most reliable approach is to inoculate the beneficial microorganism into soil as part of a mixed culture, and at a sufficiently high inoculum density to maximize the probability of its adaptation to environmental and ecological conditions. The application of beneficial microorganisms to soil can help to define the structure and establishment of natural ecosystems. The greater the diversity of the cultivated plants that are grown and the more chemically complex the biomass, the greater the diversity of the soil microflora as to their types, numbers and activities. The application of a wide range of different organic amendments to soils can also help to ensure a greater microbial diversity. For example, combinations of various crop residues, animal manures, green manures, and municipal wastes applied periodically to soil will provide a higher level of microbial diversity than when only one of these materials is applied. The reason for this is that each of these organic materials has its own unique indigenous microflora which can greatly affect the resident soil microflora after they are applied, at least for a limited period.

5.8

CLASSIFICATION OF SOILS BASED ON THEIR MICROBIOLOGICAL PROPERTIES

Most soils are classified on the basis of their chemical and physical properties; little has been done to classify soils according to their microbiological properties. The reason for this is that a soil’s chemical and physical properties are more readily defined and measured than their microbiological properties. Improved soil quality is usually characterized by increased infiltration, aeration, aggregation and organic matter content and by decreased bulk density, compaction, erosion and crusting. While these are important indicators of potential soil productivity, we must give more attention to soil biological

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properties because of their important relationship (though poorly understood) to crop production, plant and animal health, environmental quality, and food safety and quality. Research is needed to identify and quantify reliable and predictable biological/ecological indicators of soil quality. Possible indicators might include total species diversity or genetic diversity of beneficial soil microorganisms as well as insects and animals. The basic concept here is not to classify soils for the study of microorganisms but for farmers to be able to control the soil microflora so that biologicallymediated processes can improve the growth, yield, and quality of crops as well as the tilt, fertility, and productivity of soils. The ultimate objective is to reduce the need for chemical fertilizers and pesticides.

5.8.1 Functions of Microorganisms: Putrefaction, Fermentation, and Synthesis Soil microorganisms can be classified into decomposer and synthetic microorganisms. The decomposer microorganisms are subdivided into groups that perform oxidative and fermentative decomposition. The fermentative group is further divided into useful fermentation (simply called fermentation) and harmful fermentation (called putrefaction). The synthetic microorganisms can be subdivided into groups having the physiological abilities to fix atmospheric nitrogen into amino acids and/or carbon dioxide into simple organic molecules through photosynthesis. Fermentation is an anaerobic process by which facultative microorganisms (e.g., yeasts) transform complex organic molecules (e.g., carbohydrates) into simple organic compounds that often can be absorbed directly by plants. Fermentation yields a relatively small amount of energy compared with aerobic decomposition of the same substrate by the same group of microorganisms. Aerobic decomposition results in complete oxidation of a substrate and the release of large amounts of energy, gas, and heat with carbon dioxide and water as the end products. Putrefaction is the process by which facultative heterotrophic microorganisms decompose proteins anaerobically, yielding malodorous and incompletely oxidized metabolites (e.g., ammonia, mercaptans and indole) that are often toxic to plants and animals. The term “synthesis”, as used here, refers to the biosynthetic capacity of certain microorganisms to derive metabolic energy by “fixing” atmospheric nitrogen and/or carbon dioxide. In this context, we refer to these as “synthetic” microorganisms, and if they should become a predominant part of the soil microflora, then the soil would be termed a “synthetic” soil. Nitrogen-fixing microorganisms are highly diverse, ranging from “free-living”, autotrophic bacteria of the genus Azotobacter to symbiotic, heterotrophic bacteria of the genus Rhizobium, and blue-green algae (now, mainly classified as blue-green bacteria), all of which, function aerobically. Photosynthetic microorganisms fix atmospheric carbon dioxide in a manner similar to that of green plants.

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Environmental Biotechnology

They are also highly diverse, ranging from blue-green algae and green algae, that perform complete photosynthesis aerobically to photosynthetic bacteria, which perform incomplete photosynthesis anaerobically.

5.8.2 Relationships Between Putrefaction, Fermentation, and Synthesis The processes of putrefaction, fermentation, and synthesis proceed simultaneously according to the appropriate types and numbers of microorganisms that are present in the soil. The impact on soil quality attributes and related soil properties is determined by the dominant process. The production of organic substances by microorganisms results from the intake of positive ions, while decomposition serves to release these positive ions. Hydrogen ions play a pivotal role in these processes. A problem occurs when hydrogen ions do not recombine with oxygen to form water, but are utilized to produce methane, hydrogen sulfide, ammonia, mercaptans and other highly reduced putrefactive substances, most of which are toxic to plants and produce malodors. If a soil is able to absorb the excess hydrogen ions during periods of soil anaerobiosis and if synthetic microorganisms such as photosynthetic bacteria are present, they will utilize these putrefactive substances and produce useful substrates from them which helps to maintain a healthy and productive soil. The photosynthetic bacteria, which perform incomplete photosynthesis anaerobically, are highly desirable, beneficial soil microorganisms because they are able to detoxify soils by transforming reduced, putrefactive substances such as hydrogen sulfide into useful substrates. This helps to ensure efficient utilization of organic matter and to improve soil fertility. Photosynthesis involves the photo-catalyzed splitting of water which yields molecular oxygen as a by-product. Thus, these microorganisms help to provide a vital source of oxygen to plant roots. Reduced compounds, such as methane and hydrogen sulfide are often produced, when organic materials are decomposed under anaerobic conditions. These compounds are toxic and can greatly suppress the activities of nitrogen-fixing microorganisms. However, if synthetic microorganisms, such as photosynthetic bacteria that utilize reduced substances, are present in the soil, oxygen deficiencies are not likely to occur. Thus, nitro nitrogen-fixing microorganisms, coexisting in the soil with photosynthetic bacteria, can function effectively in fixing atmospheric nitrogen even under anaerobic conditions. Photosynthetic bacteria not only perform photosynthesis but can also fix nitrogen. Moreover, it has been shown that, when they coexist in soil with species of Azotobacter, their ability to fix nitrogen is enhanced. This then is an example of a synthetic soil. It also suggests that by recognizing the role, function, and mutual compatibility of these two bacteria and utilizing them effectively to their full potential, soils can be induced to a greater synthetic

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capacity. Perhaps, the most effective synthetic soil system results from the enhancement of zymogenic and synthetic microorganisms; this allows fermentation to become dominant over putrefaction and useful synthetic processes to proceed.

5.9

CLASSIFICATION OF SOILS BASED ON THE FUNCTIONS OF MICROORGANISMS

As discussed earlier, soils can be characterized according to their indigenous microflora which perform putrefactive, fermentative, synthetic and zymogenic reactions and processes. In most soils, these functions are going on simultaneously, with the rate and extent of each, determined by the types and numbers of associated microorganisms, that are actively involved at any time. A simple diagram, showing a classification of soils based on the activities and functions of their predominant microorganisms, is presented in Figure 5.1. Disease-Inducing Soils. In this type of soil, plant pathogenic microorganisms such as Fusarium fungi can comprise 5 to 20 per cent of the total microflora. If fresh organic matter with a high nitrogen content is applied to such a soil, incompletely oxidized products can arise that are malodorous and toxic to growing plants. Such soils tend to cause frequent infestations of disease-causing organisms, and harmful insects. Thus, the application of fresh organic matter to these soils is often harmful to crops. Probably more than 90 per cent of the agricultural land devoted to crop production worldwide can be classified as having disease-inducing soils. Such soils generally have poor physical properties, and large amounts of energy are lost as “greenhouse gases,” particularly in the case of rice fields. Plant nutrients are also subject to immobilization into unavailable forms. Disease-Suppressive Soils. The microflora of disease-suppressive soils is usually dominated by antagonistic microorganisms that produce copious amounts of antibiotics. These include fungi of the genera Penicillium, Trichoderma, and Aspergillus, and actinomycetes of the genus Streptomyces. The antibiotics they produce can have biostatic and biocidal effects on soil-borne plant pathogens, including Fusarium which would have an incidence in these soils of less than 5 per cent. Crops planted in these soils are rarely affected by diseases or insect pests. Even if fresh organic matter with a high nitrogen content is applied, the production of putrescent substances is very low and the soil has a pleasant earthy odor after the organic matter is decomposed. These soils generally have excellent physical properties; for example, they readily form water-stable aggregates and they are well-aerated, and have a high permeability to both air and water. Crop yields in the disease-suppressive soils are often slightly lower than those in synthetic soils. Highly acceptable crop yields are obtained whenever a soil has a predominance of both diseasesuppressive and synthetic microorganisms.

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Environmental Biotechnology

Zymogenic Soils. These soils are dominated by a microflora that can perform useful kinds of fermentations, i.e., the breakdown of complex organic molecules into simple organic substances and inorganic materials. The organisms can be either obligate or facultative anaerobes. Such fermentationproducing microorganisms often comprise the microflora of various organic materials, i.e., crop residues, animal manures, green manures and municipal wastes including composts. After these amendments are applied to the soil, their numbers and fermentative activities can increase dramatically and overwhelm the indigenous soil microflora for an indefinite period. While these microorganisms remain predominant, the soil can be classified as a zymogenic soil which is generally characterized by: a) pleasant, fermentative odors, especially, after tillage, b) favorable soil physica1 properties (e.g., increased aggregate stability, permeability, aeration and decreased resistance to tillage), c) large amounts of inorganic nutrients, amino acids, carbohydrates, vitamins and other bioactive substances which can directly or indirectly enhance the growth, yield and quality of crops, d) low occupancy of Fusarium fungi which is usually less than 5 per cent, and e) low production of greenhouse gases (e.g., methane, ammonia, and carbon dioxide) from croplands, even where flooded rice is grown. Synthetic Soils. These soils contain significant populations of microorganisms which are able to fix atmospheric nitrogen and carbon dioxide into complex molecules such as amino acids, proteins and carbohydrates. Such microorganisms include photosynthetic bacteria which perform incomplete photosynthesis anaerobically, certain Phycomycetes (fungi, that resemble algae), and both green algae and blue-green algae which function aerobically. All of these are photosynthetic organisms that fix atmospheric nitrogen. If the water content of these soils is stable, their fertility can be largely maintained by regular additions of only small amounts of organic materials. These soils have a low Fusarium occupancy, and they are often of the disease-suppressive type. The production of gases from fields where synthetic soils are present is minimal, even for flooded rice. This is a somewhat simplistic classification of soils based on the functions of their predominant types of microorganisms, and whether they are potentially beneficial or harmful to the growth and yield of crops. While these different types of soils are described here in a rather idealized manner, the fact is that, in nature, they are not always clearly defined because they often tend to have some of the same characteristics. Nevertheless, research has shown that a disease-inducing soil can be transformed into disease-suppressive, zymogenic and synthetic soils by inoculating the problem soil with mixed cultures of Effective Microorganisms (EM). Thus, it is somewhat obvious that the most desirable agricultural soil for optimum growth, production, protection, and quality of crops would be the composite soil indicated in Figure 2, i.e., a soil that is highly zymogenic and synthetic, and has an established disease-suppressive capacity. This, then is the principal reason for seeking ways and means of controlling the microflora of agricultural soils.

Beneficial and Effective Microorganisms for a Sustainable Agriculture

5.17

Classification of soils based on the activities and functions of their predominant microorganisms Disease-inducing soils can be transformed into diesease-suppressing, zymogenic and synthetic soils that are more conductive growth and health of plants by introducing beneficial microorganisms as microbial inoculate and following best management practices. The ideal soil for a more sustainable agriculture is a composite of the other three soil types and contains associative groups of beneficial microorganisms to enhance the optimum growth, yield and quality of crops.

Controlling the soil microflora to enhance the predominance of beneficial and Effective Microorganisms can help to improve and maintain the soil chemical and physical properties. The proper and regular addition of organic amendments are often an important part of any strategy to exercise such control. Previous efforts to significantly change the indigenous microflora of a soil by introducing single cultures of extrinsic microorganisms have largely been unsuccessful. Even when a beneficial microorganism is isolated from a soil, cultured in the laboratory, and reinoculated into the same soil at a very high population, it is immediately subject to competitive and antagonistic effects from the indigenous soil microflora, and its numbers, soon decline. Thus, the probability of shifting the “microbiological equilibrium” of a soil and controlling it to favor the growth, yield and health of crops is much greater if mixed cultures of beneficial and Effective Microorganisms are introduced that are physiologically and ecologically compatible with one another. When these mixed cultures become established, their individual beneficial effects are often magnified in a synergistic manner. Actually, a disease-suppressive microflora can be developed rather easily by selecting and culturing certain types of gram-positive bacteria that produce antibiotics and have a wide range of specific functions and capabilities; these organisms include facultative anaerobes, obligate aerobes, acidophilic and alkalophilic microbes. These microorganisms can be grown to high populations in a medium consisting of

5.18

Environmental Biotechnology

rice bran, oil cake and fish meal and then applied to soil along with well-cured compost that also has a large stable population of beneficial microorganisms, especially facultative anaerobic bacteria. A soil can be readily transformed into a zymogenic/synthetic soil with disease-suppressive potential if mixed cultures of Effective Microorganisms with the ability to transmit these properties are applied to that soil. The desired effects from applying cultured beneficial and Effective Microorganisms to soils can be somewhat variable, at least, initially. In some soils, a single application (i.e., inoculation) may be enough to produce the expected results, while for other soils, even repeated applications may appear to be ineffective. The reason for this is that, in some soils, it takes longer for the introduced microorganisms to adapt to a new set of ecological and environmental conditions and to become well-established as a stable, effective and predominant part of the indigenous soil microflora. The important consideration here is the careful selection of a mixed culture of compatible, Effective Microorganisms, properly cultured, and provided with acceptable organic substrates. Assuming that repeated applications are made at regular intervals during the first cropping season, there is a very high probability that the desired results will be achieved. There are no meaningful or reliable tests for monitoring the establishment of mixed cultures of beneficial and Effective Microorganisms after application to a soil. The desired effects appear only after they are established and become dominant, and remain stable and active in the soil. The inoculum densities of the mixed cultures and the frequency of application serve only as guidelines to enhance the probability of early establishment. Repeated applications, especially during the first cropping season, can markedly facilitate early establishment of the introduced Effective Microorganisms. Once the “new” microflora is established and stabilized, the desired effects will continue indefinitely and no further applications are necessary unless organic amendments cease to be applied, or the soil is subjected to severe drought or flooding. Finally, it is far more likely that the microflora of a soil can be controlled through the application of mixed cultures of selected beneficial and Effective Microorganisms than by the use of single or pure cultures. If the microorganisms comprising the mixed culture can coexist and are physiologically compatible and mutually complementary, and if the initial inoculum density is sufficiently high, there is a high probability that these microorganisms will become established in the soil and will be effective as an associative group, whereby such positive interactions would continue. If so, then it is also highly probable that they will exercise considerable control over the indigenous soil microflora which, in due course, would likely be transformed into or replaced by a “new” soil microflora.

CHAPTER

6

Phytoremediation

Phytoremediation combines the Greek word “phyton” (plant), with the Latin word “remediare” (to remedy) to describe a system whereby certain plants, working together with soil organisms, can transform contaminants into harmless and often, valuable forms. This practice is increasingly used to remediate sites contaminated with heavy metals and toxic organic compounds. Phytoremediation can be defined as “the efficient use of plants to remove, detoxify or immobilize environmental contaminants in a growth matrix (soil, water or sediments) through the natural, biological, chemical or physical activities and processes of the plants”. Plants are unique organisms equipped with remarkable metabolic and absorption capabilities, as well as transport systems that can take up nutrients or contaminants selectively from the growth matrix, soil or water. Phytoremediation involves growing plants in a contaminated matrix, for a required growth period, to remove contaminants from the matrix, or facilitate immobilization (binding/containment) or degradation (detoxification) of the pollutants. The plants can be subsequently harvested, processed and disposed. Plants evolved a great diversity of genetic adaptations to handle the accumulated pollutants that occur in the environment. Growing and, in some cases, harvesting plants on a contaminated site as a remediation method is a passive technique that can be used to clean up sites with shallow, low to moderate levels of contamination. Phytoremediation can be used to clean up metals, pesticides, solvents, explosives, crude oil, polyaromatic hydrocarbons, and landfill leachates. It can also be used for river basin management through the hydraulic control of contaminants. Phytoremediation has been studied extensively in research and small-scale demonstrations, but full-scale applications are currently limited to a small number of projects. Further research and development will lead to wider acceptance and use of phytoremediation.

6.2

6.1

Environmental Biotechnology

PRINCIPAL MECHANISM OF PHYTOREMEDIATION

There are several ways in which plants are used to clean up, or remediate, contaminated sites. To remove pollutants from soil, sediment and/or water, plants can break down, or degrade, organic pollutants or contain and stabilize metal contaminants by acting as filters or traps. The uptake of contaminants in plants occurs primarily through the root system, in which the principal mechanisms for preventing contaminant toxicity are found. The root system provides an enormous surface area that absorbs and accumulates the water and nutrients essential for growth, as well as other non-essential contaminants. Researchers are finding that the use of trees (rather than smaller plants) is effective in treating deeper contamination because tree roots penetrate more deeply into the ground. In addition, deep-lying contaminated ground water can be treated by pumping the water out of the ground and using plants to treat the contamination. Plant roots also cause changes at the soil-root interface as they release inorganic and organic compounds (root exudates) in the rhizosphere. These root exudates affect the number and activity of the microorganisms, the aggregation and stability of the soil particles around the root, and the availability of the contaminants. Root exudates, by themselves, can increase (mobilize) or decrease (immobilize) directly or indirectly the availability of the contaminants in the root zone (rhizosphere) of the plant through changes in soil characteristics, release of organic substances, changes in chemical composition, and/or increase in plant-assisted microbial activity. Phytoremediation is an alternative or complimentary technology that can be used along with or, in some cases, in place of mechanical conventional cleanup technologies that often require high capital inputs and are labour and energy-intensive. Phytoremediation is an in situ, remediation technology that utilizes the inherent abilities of living plants. It is also an ecologically friendly, solar energy-driven clean-up technology, based on the concept of—using nature to cleanse nature.

6.2

PHYTOREMEDIATION PROCESSES

Depending on the underlying processes, applicability, and type of contaminant, phytoremediation can be broadly categorised as: Phytoremediation includes the following processes and mechanisms of contaminant removal S.No.

Process

Mechanism

Contaminant

1

Rhizofiltration

Rhizosphere accumulation

Organics/ Inorganics

2

Phytostabilization

Complexation

Inorganics

3

Phytoextraction

Hyper-accumulation

Inorganics

6.3

Phytoremediation

4

Phytovolatilization

Volatilization by leaves Organics/ Inorganics

5

Phytotransformation

Degradation in plants

Organics

6.2.1  Rhizofiltration Rhizofiltration is similar in concept to Phytoextraction but is concerned with the remediation of contaminated groundwater rather than the remediation of polluted soils. The contaminants are either adsorbed onto the root surface or are absorbed by the plant roots. Plants used for rhizofiltration are not planted directly in situ, but are acclimated to the pollutant first. Plants are hydroponically grown in clean water rather than soil, until a large root system has developed. Once a large root system is in place, the water supply is substituted for a polluted water supply to acclimatize the plant. After the plants become acclimatized, they are planted in the polluted area, where the roots uptake the polluted water and the contaminants along with it. As the roots become saturated, they are harvested and disposed of safely. Repeated treatments of the site can reduce pollution to suitable levels as was exemplified in Chernobyl where sunflowers were grown in radioactively contaminated pools.

6.2.2  Phytostabilization Phytostabilization is the use of certain plants to immobilise soil and water contaminants. Contaminant are absorbed and accumulated by roots, adsorbed onto the roots, or precipitated in the rhizosphere. This reduces or even prevents the mobility of the contaminants preventing migration into the groundwater or air, and also reduces the bioavailability of the contaminant, thus preventing spread through the food chain. This technique can also be used to re-establish a plant community on sites that have been denuded due to the high levels of metal contamination. Once a community of tolerant species has been established the potential for wind erosion (and thus, spread of the pollutant) is reduced and leaching of the soil contaminants is also reduced.

6.2.3  Phytoextraction Phytoextraction (or Phytoaccumulation) uses plants or algae to remove contaminants from soils, sediments or water into harvestable plant biomass (organisms that take larger-than-normal amounts of contaminants from the soil are called hyperaccumulators). Phytoextraction has been growing rapidly in popularity worldwide for the last twenty years or so. In general, this process has been tried more often for extracting heavy metals than for organics. At the time of disposal, contaminants are typically concentrated in the much smaller volume of the plant matter than in the initially contaminated soil or sediment. ‘Mining with plants’, or phytomining, is also being experimented with. The main advantage of phytoextraction is environmental friendliness. Traditional

6.4

Environmental Biotechnology

methods that are used for cleaning up heavy metal-contaminated soil disrupt soil structure and reduce soil productivity, whereas phytoextraction can clean up the soil without causing any kind of harm to soil quality. Another benefit of phytoextraction is that it is less expensive than any other clean-up process. Disadvantages of this process is that this process is controlled by plants, it takes more time than anthropogenic soil clean-up methods. There are two versions of phytoextraction: • natural hyper-accumulation, where plants naturally take up the contaminants in soil unassisted, and • induced or assisted hyper-accumulation, in which a conditioning fluid containing a chelator or another agent is added to soil to increase metal solubility or mobilization so that the plants can absorb them more easily. In many cases, natural hyperaccumulators are metallophyte plants that can tolerate and incorporate high levels of toxic metals.

Enzymes in plant roots break (degrade) organic contaminant. The fragments are incorporate into new plant material.

6.2.4  Phytovolatilisation Phytovolatilization is the process where plants uptake contaminants which are water soluble and release them into the atmosphere as they transpire the water. The contaminant may become modified along the way, as the water travels along the plant’s vascular system from the roots to the leaves, whereby the contaminants evaporate or volatilize into the air surrounding the plant. There are varying degrees of success with plants as phytovolatilizers, with one study showing poplar trees to volatilize up to 90% of the TCE they absorb.

6.2.5  Phytotransformation or Phytostimulation or Rhizodegradation Rhizodegradation (also called enhanced rhizosphere biodegradation, phytostimulation, and plant-assisted bioremediation) is the breakdown of organic contaminants in the soil by soil-dwelling microbes which is enhanced by the rhizosphere’s presence. Certain soil-dwelling microbes digest organic pollutants such as fuels and solvents, producing harmless products through

Phytoremediation

6.5

a process known as Bioremediation. Plant root exudates such as sugars, alcohols, and organic acids act as carbohydrate sources for the soil microflora and enhance microbial growth and activity. Some of these compound may also act as chemotactic signals for certain microbes. The plant roots also loosen the soil and transport water to the rhizosphere, thus, additionally enhancing microbial activity.

Enzymes in plant roots break down (degrade) organic contaminants. The fragments are incorporated into new plant material.

6.3

PHYTOREMEDIAITON OF ORGANIC POLLUTANTS

To avoid the toxicity associated with hazardous chemicals that are present in the environment, researchers have developed strategies that employ plants to degrade, remove or stabilize a range of different compounds from polluted soils. These environmental pollutants may include metals such as lead, zinc, cadmium, selenium, chromium, cobalt, copper, nickel and mercury; inorganic compounds such as arsenic, sodium, nitrate, ammonia and phosphate; radioactive compounds like uranium, cesium and strontium; or organic compounds, including chlorinated solvents like trichloroethylene (TCE), explosives such as trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5hexahydrotriazine (RDX), petroleum hydrocarbons such as benzene, toluene and xylene (BTX), polycyclic aromatic hydrocarbons (PAHs), and pesticides such as atrazine and bentazon. While some organic compounds can be metabolized (remediated) by soil bacteria in the absence of plants, this process is often slow and inefficient. This notwithstanding, the field of bacterial bioremediation has been expanding. Contaminant-degrading bacteria have been isolated from a wide range of impacted soils. In addition, it has been suggested that these contaminantdegrading bacteria may be found in virtually all soils. Following isolation and characterization of contaminant-degrading bacteria, attempts have been made to inoculate contaminated field soils with the isolates; however, as indicated above, this strategy has generally proven to be unsuccessful. This lack of success may be attributed to: (i) the inability of introduced bacterial isolates to compete with existing microflora and microfauna in the soil;

6.6

Environmental Biotechnology

(ii) the inability of the bacteria to reach sub-surface contaminants; (iii) the lack of sufficient nutrients in contaminated soils to support bacterial growth; (iv) the low bioavailability of many contaminants; (v) the preferential utilization by the degradative bacteria of carbon compounds other than the contaminant of interest; and (vi) the presence of other toxicants within the soil that may inhibit bacterial growth. However, in the area around plant roots (the rhizosphere), some organic soil contaminants can be completely degraded and mineralized by plant enzymes through the process of phytodegradation. This process occurs because many plants produce, and secrete to the environment, enzymes that can degrade a wide range of organic compounds. Phytoremediation of organic compounds may occur by phytostabilization (stabilizing pollutants in the soil to make them less bioavailable and therefore less hazardous); phytostimulation (the stimulation of microbial biodegradation in the rhizosphere—sometimes called rhizodegradation); or by phytotransformation—the absorption and degradation of organic contaminants by the plant. The biodegradation of recalcitrant organic compounds in the soil is often enhanced around the roots of plants. This is a direct consequence of the high level of nutrients (including sugars, amino acids and organic acids) that most plants release (exude) into the soil as root exudates, nutrients that typically support a bacterial concentration in the rhizosphere that is often 100- to 1000-fold greater than the bacterial concentration in the bulk soil. Some rhizosphere bacteria are directly involved in the degradation of the organic soil contaminants, while others (plant growth-promoting bacteria) can positively affect plant growth and health, enhancing root development or increasing plant tolerance to various environmental stresses. As a direct consequence of their interaction with plant growth-promoting bacteria, plants grow larger and healthier, and are better able to phytoremediate a range of organic soil contaminants. Unfortunately, inorganic environmental pollutants cannot readily be degraded. They must either be stabilized in the soil to make them less bioavailable and thereby reduce their spread in the environment; extracted, transported, accumulated and concentrated from the soil into plant roots and/ or shoots (phytoextraction); removed from liquid effluents via the use of plant roots (rhizofiltration); or transformed into volatile forms (phytovolatilization). Following phytoextraction, plants may be harvested, dried and converted to ash to recover the concentrated metal. A serious impediment to more effective phytoextraction of metals is the tight binding of metals to soil particles so that often only a small fraction of the metal that is present in the soil can be mobilized and taken up by plant roots.

Phytoremediation

6.7

As a result of the testing of numerous plants, several, that are naturally able to accumulate large amounts of metal per unit of plant biomass, have been identified and are being studied for possible use in the phytoremediation of metallic contaminants. These plants are called hyperaccumulators and are often found growing in soils with elevated metal concentrations. A practical limitation of using hyperaccumulators is that many of the plants that are most effective at removing metals from the soil, such as Thlaspi caerulescens (Alpine pennycress) and Alyssum bertolonii, are small, containing only a low level of biomass, and they are slow-growing, thus reducing their potential for metal phytoextraction from soil (on a large scale) in the field. Moreover, the growth of metal-resistant, metal-accumulating plants, that are capable of hyperaccumulating metals, can be severely inhibited when the concentration of available metal in the contaminated soil is very high. This results in a decrease in plant biomass and, thereby, in the efficiency of phytoremediation. To be effective for the remediation of metal polluted soils, plants must be tolerant to one or more metals, highly competitive, fast-growing, and produce a high aboveground biomass. Because of their high biomass and extensive root system, some species of trees (e.g. poplar) have been considered to be attractive for phytoremediation; however, metal accumulation by trees is generally low. Finally, a convergence of phytoremediation and bacterial bioremediation strategies has led to a more successful approach to remediation of contaminants, particularly, organic compounds. Bacteriaassisted phytoremediation, both with bacteria already present in the soil and with bacteria deliberately introduced by seed inoculation, has been investigated in a number of laboratory, greenhouse and field studies. In this regard, phytoremediation is most effective when the introduced bacteria can both degrade the soil contaminant(s) and promote the growth of plants. Given the above mentioned considerations, it is currently possible to develop phytoremediation strategies to clean up a large number of the sites contaminated with organic compounds (this process may require several field seasons depending on the particular plant, soil, bacteria, contaminants and climate involved). On the other hand, phytoremediation is not yet a practical approach for the removal of inorganic compounds from contaminated soil environments.

6.4

PLANTS’ RESPONSE TO HEAVY METALS

Heavy metals are elements having atomic weight between 63.54 and 200.59, and a specific gravity greater than 6. Trace amount of some heavy metals are required by living organisms, however, any excess amount of these metals can be detrimental to the organisms. Non-essential heavy metals include arsenic, antimony, cadmium, chromium, mercury, lead, etc; these metals are of particular concern to surface water and soil pollution. Heavy metals exist in colloidal, ionic, particulate and dissolved phase. Metals also have a high

6.8

Environmental Biotechnology

affinity for humic acids, organic clays, and oxides coated with organic matter. The soluble forms are generally ions or unionized organometallic chelates or complexes. The solubility of metals in soil and groundwater is predominantly controlled by pH amount of metal, cation exchange capacity, organic carbon content, the oxidation state of the mineral components, and the redox potential of the system. In general, soil pH seems to have the greatest effect of any single factor on the solubility or retention of metals in soils with a greater retention and lower solubility of metal cations occurring at high soil pH . Under the neutral to basic conditions, typical of most soils, cationic metals are strongly adsorbed on the clay fractions and can be adsorbed by hydrous oxides of iron, aluminium, or manganese present in soil minerals. Elevated salt concentration creates increased competition between cations and metals for binding sites. Also competitive adsorption between various metals has been observed in experiments involving various solids with oxide surfaces; in several experiments, Cd adsorption was decreased by the addition of Pb or Cu. Plants have three basic strategies for growth on metal-contaminated soil.

6.4.1  Metal excluders They prevent metal from entering their aerial parts or maintain low and constant metal concentration over a broad range of metal concentration in soil, they mainly restrict metal in their roots. The plant may alter its membrane permeability, change metal-binding capacity of cell walls or exude more chelating substances.

6.4.2  Metal indicators Species which actively accumulate metal in their aerial tissues and generally reflect metal level in the soil. They tolerate the existing concentration level of metals by producing intracellular metal binding compounds (chelators), or alter metal compartmentalisation pattern by storing metals in non-sensitive parts.

6.4.3  Metal accumulator plant species They can concentrate metal in their aerial parts, to levels, far exceeding than soil. Hyperaccumulators are plants that can absorb high levels of contaminants concentrated either in their roots, shoots and/or leaves. Baker and Brooks have defined metal hyperaccumulator as plants that contain more than, or up to, 0.1% i.e. more than (1000 mg/g) of copper, cadmium, chromium, lead, nickel cobalt or 1% (>10,000 mg/g ) of zinc or manganese in the dry matter. For cadmium and other rare metals, it is > 0.01% by dry weight. Researchers have identified hyperaccumulator species by collecting plants from the areas where soil contains greater than usual amount of metals as in case of polluted areas or, geographically rich in a particular element. Approximately, 400 hyper accumulator species from 22 families

Phytoremediation

6.9

have been identified. The Brassicaceae family contains a large number of hyperaccumulating species with widest range of metals, these include 87 species from 11 genera.

Conceptual response strategies of metal concentrations in plant tops in relation to increasing total metal concentrations in the soil.

6.5

HYDRAULIC CONTROL OF POLLUTANTS

Hydraulic control is the term given to the use of plants to control the migration of subsurface water through the rapid uptake of large volumes of water by the plants. The plants are effectively acting as natural hydraulic pumps which when a dense root network has been established near the water table can transpire up to 300 gallons of water per day. This fact has been utilized to decrease the migration of contaminants from surface water into the groundwater (below the water table) and drinking water supplies. There are two such uses for plants:

6.5.1  Riparian corridors Riparian corridors and buffer strips are the applications of many aspects of phytoremediation along the banks of a river or the edges of groundwater plumes. Phytodegradation, phytovolatilization, and rhizodegradation are used to control the spread of contaminants and to remediate polluted sites. Riparian strips refer to these uses along the banks of rivers and streams, whereas buffer strips are the use of such applications along the perimeter of landfills.

6.5.2  Vegetative cover Vegetative cover is the name given to the use of plants as a cover or cap growing over landfill sites. The standard caps for such sites are usually plastic or clay. Plants used in this manner are not only more aesthically pleasing they may also help to control erosion, leaching of contaminants, and may also help to degrade the underlying landfill.

6.10

6.6

Environmental Biotechnology

ADVANTAGES AND DISADVANTAGES OF PHYTOREMEDIATION

As with most new technologies phytoremediation has many pros and cons. When compared to other more traditional methods of environmental remediation, it becomes clearer what the detailed advantages and disadvantages actually are. Advantages of phytoremediation compared to classical remediation

• It is more economically viable, using the same tools and supplies as agriculture.



• It is less disruptive to the environment and does not involve waiting for new plant communities to recolonize the site.



• Disposal sites are not needed.



• It is more likely to be accepted by the public as, it is more aesthetically pleasing then traditional methods.



• It avoids excavation and transport of polluted media, thus reducing the risk of spreading the contamination.



• It has the potential to treat sites polluted with more than one type of pollutant. Disadvantages of phytoremediation compared to classical remediation:



• It is dependent on the growing conditions required by the plant (i.e., climate, geology, altitude, temperature).



• Large scale operations require access to agricultural equipment and knowledge.



• Success is dependant on the tolerance of the plant to the pollutant.



• Contaminants collected in senescing tissues may be released back into the environment in autumn.



• Contaminants may be collected in woody tissues used as fuel.



• Time taken to remediate sites far exceeds that of other technologies.



• Contaminant solubility may be increased leading to greater environmental damage and the possibility of leaching.

The low cost of phytoremediation (up to 1000 times cheaper than excavation and reburial) is the main advantage of phytoremediation; however many of the pros and cons of phytoremediation applications depend greatly on the location of the polluted site, the contaminants in question, and the application of phytoremediation.

6.7

PHYTOREMEDIATION & BIOTECHNOLOGY

The first goal in phytoremediation is to find a plant species which is resistant to or, tolerates a particular contaminant with a view to maximizing it’s potential for phytoremediation. Resistant plants are usually located growing on soils

Phytoremediation

6.11

with underlying metal ores or, on the boundary of polluted sites. Once a tolerant plant species has been selected, traditional breeding methods are used to optimize the tolerance of a species to a particular contaminant. Agricultural methods such as the application of fertilizers, chelators, and pH adjusters can be utilized to further improve the potential for phytoremediation. Genetic modification offers a new hope for phytoremediation as GM approaches can be used to overexpress the enzymes involved in the existing plant metabolic pathways or to introduce new pathways into plants. Richard Meagher and colleagues introduced a new pathway into Arabidopsis to detoxify methylmercury, a common form of environmental pollutant, to elemental mercury, which can be volatilized by the plant.

• The genes originated in gram-negative bacteria.



• MerB encodes a protein, organomercurial lyase converts methylmercury to ionic mercury.



• MerA encodes mercuric reductase, which reduces ionic mercury to the elemental form.



• Arabidopsis plants were transformed with either MerA or MerB coupled with a constitutive 35S promoter.



• The MerA plants were more tolerant to ionic mercury, volatilized elemental mercury, and were unaffected in their tolerance of methylmercury.



• The MerB Plants were significantly more tolerant to methylmercury and other organomercurials and could also convert methylmercury to ionic mercury, which is approximately 100 times less toxic to plants.



• MerA MerB double transgenics were produced in an F2 generation. These plants not only showed a greater resistance to organic mercury when compared to the MerA, MerB, and wildtype plants but also were capable of volatilizing mercury when supplied with methylmercury.



• The same MerA/MerB inserts have been used in other plant species including tobacco (Nicotiana tabacum), yellow poplar (Liriodendron tulipifera).



• Wetland species (bulrush and cat-tail) and water tolerant trees (willow and poplar) have also been targeted for transformation.

6.7.1 Risk Assessment The use of phytoremediation in the field is subject to many environmental concerns, especially, in the light of the recent public hysteria about the release of GM crops into the environment. Even if non GM strains of plants are used, there are still many concerns:

• It is unknown what ecological effects hyperaccumulator plants may have if ingested by animals.

6.12

Environmental Biotechnology



• Fallout from senescing tissues in autumn may also re-enter the food chain.



• Do volatilized contaminants remain at ‘safe’ levels in the atmosphere?



• Exposure of the ecosystem to contaminants is prolonged as phytoremediation is a relatively slow process.



• However there are other issues that affect the risk assessment for the use of transgenic organisms as phytoremediators. Not only do such organisms have the same risks as wild type remediates, but they also have the same risks as releasing any GM organism into the field have:



• The potential genetic pollution of native species.



• Potential for the gene to recombine with other genes possibly leading to the hyperaccumulation of non-contaminant compounds.



• Reporter/marker genes may also escape into the environment.



• The GM plants may revert to a wild type genotype.

6.7.2 Future of Phytoremediation One of the key aspects to the acceptance of phytoextraction pertains to the measurement of its performance, ultimate utilization of by-products and its overall economic viability. To date, commercial phytoextraction has been constrained by the expectation that site remediation should be achieved in a time comparable to other clean-up technologies. So far, most of the phytoremediation experiments have taken place in the lab scale, where plants grown in hydroponic setting are fed heavy metal diets. While these results are promising, scientists are ready to admit that solution culture is quite different from that of soil. In real soil, many metals are tied up in insoluble forms, and they are less available and that is the biggest problem. The future of phytoremediation is still in research and development phase, and there are many technical barriers which need to be addressed. Both agronomic management practices and plant genetic abilities need to be optimized to develop commercially useful practices. Many hyperaccumulator plants remain to be discovered, and there is a need to know more about their physiology. Optimization of the process, proper understanding of plant heavy metal uptake and proper disposal of biomass produced is still needed. Phytoremediation is a fast developing field; since last ten years; lot of field application were initiated all over the world—it includes Phytoremediation of Organic, Inorganic and Radionuclides. This sustainable and inexpensive process is fast emerging as a viable alternative to conventional remediation methods, and will be most suitable for a developing country like India. Most of the studies have been done in developed countries and knowledge of suitable plants is particularly limited in India. In India, commercial application of Phytoremediation of soil Heavy metal or Organic compounds is in its earliest phase. Fast growing plants with high biomass and good metal uptake ability

Phytoremediation

6.13

are needed. In most of the contaminated sites, hardy and tolerant weed species exist and phytoremediation through these and other non-edible species can restrict the contaminant from being introduced into the food web. However, several methods of plant disposal have been described, but data regarding these methods are scarce. Composting and compaction can be treated as pre-treatment steps for volume reduction, but care should be taken to collect leachate resulting from compaction. Between the two methods that significantly reduce the contaminated biomass, incineration seems to be least time-consuming and environmentally sound than direct burning or ashing.

CHAPTER

7

Solid Waste Disposal and Management

Waste is unwanted or undesired material left over after the completion of a process. In other words, it can also be stated that any substance or object, which the holder discards or intend to discard, is also waste. Waste is a continually growing problem at global and regional as well as at local levels. A waste product is regarded as a pollutant when it damages the environment. Often wastes and pollutants are intricately linked. In simple words, pollutants are generally wastes but all wastes are not pollutants. Wastes may be:

• Biological,



• Chemical, or



• Physical in nature, and may originate from the following activities:

— Manufacturing — Agriculture and dairy — Energy production — Transport and — House building and house keeping. Basically, the waste can be categorised into (according to their Properties):

• Biodegradable Waste and



• Non-Biodegradable Waste.



— Biodegradable waste is a type of waste, typically originating from Plant or Animal sources, which may be broken down by other living organisms. With the proper treatment, Biodegradable waste can be used for composting, animal feed, or converted into energy. Biodegradable waste accounts for approximately 60% of municipal waste; the most common types of biodegradable waste are food waste, garden waste, paper and cardboard waste, and biodegradable plastics.

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— Waste that cannot be broken down by other living organisms may be called non-biodegradable. Examples are plastics, metal and glass. Some dangerous chemicals and toxins are also non-biodegradable, as are plastic grocery bags, Styrofoam (polystyrene), and other similar materials, but will eventually break down over time.

Waste is broadly segregated into solid, liquid and gaseous waste materials. Solid wastes arise from human and animal activities that are normally discarded as useless or unwanted. In other words, solid wastes may be defined as the organic and inorganic waste materials produced by various activities of the society and which have lost their value to the first user. As the result of rapid increase in production and consumption, urban society rejects and generates solid material regularly which leads to considerable increase in the volume of waste generated from several sources such as, domestic wastes, commercial wastes, institutional wastes and industrial wastes of most diverse categories.

7.1

TYPES OF SOLID WASTE

Solid waste can be classified into different types depending on their source:

• Municipal solid waste (Household waste), 



• Industrial solid waste (Hazardous waste) and 



• Biomedical waste (Hospital waste as infectious waste).

7.1.1 Municipal solid waste The term Municipal Solid Waste (MSW) is normally assumed to include all of the waste generated in a community, with the exception of waste generated by municipal services, treatment plants, and industrial and agricultural processes. In the urban context the term municipal solid wastes is of special importance. The term refers to all wastes collected and controlled by the municipality and comprises of most diverse categories of wastes. It comprises of wastes from several different sources such as, domestic wastes, commercial wastes, institutional wastes and building materials wastes. Following are the different types of wastes.

• Biodegradable waste: food and kitchen waste, green waste, paper (can also be recycled).



• Recyclable material: paper, glass, bottles, cans, metals, certain plastics, etc.



• Inert waste: construction and demolition waste, dirt, rocks, debris.



• Composite wastes: waste clothing, Tetra Packs, waste plastics such as toys.



• Domestic hazardous waste (also called “household hazardous waste”) and toxic waste: medication, e-waste, paints, chemicals, light bulbs,

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fluorescent tubes, spray cans, fertilizer and pesticide containers, batteries, shoe polish. In 1947, cities and towns in India generated an estimated 6 million tonnes of solid waste in 2011; it was about 90 million tonnes. More than 25% of the municipal solid waste is not collected at all; 70% of the Indian cities lack adequate capacity to transport it and there are no sanitary landfills to dispose of the waste. Over the last few years, the consumer market has grown rapidly leading to products being packed in cans, aluminium foils, plastics, and other such non-biodegradable items that cause incalculable harm to the environment. The type of litter we generate and the approximate time it takes to degenerate Type of litter

Approximate time it takes to degenerate the litter

Organic waste such as vegetable and fruit a week or two. peels, leftover foodstuff, etc. Paper

10–30 days

Cotton cloth

2–5 months

Wood

10–15 years

Woolen items

1 year

Tin, aluminium, and other metal items such as 100–500 years cans Plastic bags

one million years

Glass bottles

Undetermined

7.1.2 Industrial solid waste Industrial waste is defined as waste generated by manufacturing or industrial processes. The types of industrial waste generated include cafeteria garbage, dirt and gravel, masonry and concrete, scrap metals, trash, oil, solvents, chemicals, weed grass and trees, wood and scrap lumber, and similar wastes. Industrial solid waste—which may be solid, liquid or gases held in containers—is divided into hazardous and non-hazardous waste.

• Hazardous waste may result from manufacturing or other industrial processes. Certain commercial products such as cleansing fluids, paints or pesticides discarded by commercial establishments or individuals, can also be defined as hazardous waste.



• Non-hazardous industrial wastes are those that do not meet the EPA’s definition of hazardous waste—and are not municipal waste.

Industrial waste has been a problem since the industrial revolution. Industrial waste may be toxic, ignitable, corrosive or reactive. If improperly

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managed, this waste can pose dangerous health and environmental consequences. The other major generators of industrial solid wastes are the thermal power plants, producing coal ash; the integrated Iron and Steel mills, producing blast furnace slag and steel melting slag; non-ferrous industries like aluminum, zinc and copper, producing red mud and tailings; sugar industries, generating press mud, pulp; and paper industries, producing lime and fertilizer; and allied industries, producing gypsum.

7.1.3 Biomedical Waste Biomedical waste, also known as infectious waste or medical waste, is defined as solid waste generated during the diagnosis, testing, treatment, research or production of biological products for humans or animals. Biomedical waste includes syringes, live vaccines, laboratory samples, body parts, bodily fluids and waste, sharp needles, cultures and lancets. The main sources of biomedical waste are hospitals, medical clinics and laboratories. Because biomedical waste can be detrimental to human health, the law requires such facilities to follow procedures that protect the public from coming into contact with it. Agencies that regulate different aspects of biomedical waste include Occupational Safety and Health Administration (OSHA), Food and Drug Administration (FDA) and Nuclear Regulatory Commission. Biomedical wastes may be categorized as follows:

• Animal Waste: Animals carcasses, tissues and body parts, blood and bodily fluids and infectious bedding.



• Biological Laboratory Waste: Cultures, stocks or specimens of microorganisms, live or attenuated vaccines, human or animal cell cultures and laboratory material that has come into contact with these (solid and liquid).



• Human Anatomical Waste: any part of the human body, including tissues and organs but excluding extracted teeth, hair, and nail clippings.



• Human Blood and Body Fluid Waste: Human fluid blood and blood products, items saturated or dripping blood, body fluids contaminated with blood and body fluids removed for diagnosis during surgery, treatment or autopsy. This does not include urine or feces. Material with minimal amounts of non-infectious blood (i.e., does not release blood, if compressed) are not considered biomedical waste.



• Sharps: Needles, syringes with needles, lancets, scalpels, razor blades, and precision knives. Contaminated broken glass, pipettes, test tubes, microscope.



• Biohazardous waste: Waste, that is known or suspected to contain infectious material or, which because of its physical or biological nature may be harmful to humans, animals, plants or the environment.

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• Infectious waste: Waste, which contains microorganisms in sufficient quantity which could result in the multiplication and growth of those microorganisms in a host. • Pathological waste: Any waste which contains microorganisms capable of causing disease. Surveys carried out by various agencies show that the health care establishments in India are not giving due attention to their waste management. After the notification of the Biomedical Waste (Handling and Management) Rules, 1998, these establishments are slowly streamlining the process of waste segregation, collection, treatment, and disposal. Many of the larger hospitals have either installed the treatment facilities or are in the process of doing so.

7.2

SOLID WASTE DISPOSAL AND MANAGEMENT:

Solid Waste Management (SWM) is the collection, transport, processing, disposal, managing and monitoring of waste materials. The term usually relates to materials produced by human activity, and the process is generally undertaken to reduce their effect on health, the environment or aesthetics. • Waste management is a distinct practice from resource recovery, which, focuses on delaying the rate of consumption of natural resources. • The management of wastes treats all materials as a single class, whether solid, liquid, gaseous or radioactive substances, and tries to reduce the harmful environmental impacts of each through different methods. • Waste management practices differ for developed and developing nations, for urban and rural areas, and for residential and industrial producers. • Management for non-hazardous waste—residential and institutional waste in metropolitan areas—is usually the responsibility of local government authorities, while management for non-hazardous commercial and industrial waste, is usually the responsibility of the generator. Disposal is the discharge, deposit, injection, dumping, spilling, leaking, or placing of any solid waste or hazardous waste into, or on any land, or water, so that such solid waste or hazardous waste or any constituent thereof, may enter the environment or be emitted into the air or discharged into any waters, including ground waters. Within our modern scheme of waste management, disposal is the last phase. As cities are growing in size with a rise in the population, the amount of waste generated is increasing becoming unmanageable. • As waste management issues gain public awareness, concern has risen about the appropriateness of various disposal methods. However, in most places, disposal of waste is the most neglected area of Solid Waste Management (SWM) services and the current practices are grossly unscientific.

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• Almost all municipal authorities deposit solid waste at a dump yard situated within or outside the city haphazardly and do not bother to spread and cover the waste with inert material. These sites emanate foul smell and become breeding grounds for flies, rodent, and pests.

Following are different methods for the disposal of waste – open dumps, landfills, sanitary landfills, incineration plants, pyrolysis and recycling. One of the important methods of waste treatment is composting. Open dumps or Non-engineered disposal: Open dumps refer to uncovered areas that are used to dump solid waste of all kinds. The waste is untreated, uncovered, and not segregated. This is the most common method of disposal in low-income countries, which have no control, or with only slight or moderate controls. They tend to remain for longer time and environmental degradation could be high, including mosquito, rodent and water pollution, and degradation of the land. In some countries, open dumps are being phased out. Landfills: Historically, landfills have been the most common methods of organized waste disposal, and remain so, in many places around the world.

• A landfill site is a site for the disposal of waste materials by burial and is the oldest form of waste treatment.



• Landfills may include internal waste disposal sites (where a producer of waste carries out their own waste disposal at the place of production) as well as sites used by many producers.



• Many landfills are also used for waste management purposes, such as the temporary storage, consolidation and transfer, or processing of waste material (sorting, treatment, or recycling).

While new methods of hazardous waste disposal are being developed, it appears that landfills will, at least for the time being, continue to be the most favoured technique. In many countries, land is a readily available commodity and often areas of non-productive or derelict land may be made available for waste disposal. In many instances, land can be utilized in the near vicinity or on the premises of industrial companies, thereby reducing transportation costs. The potential also exists to reclaim certain areas for recreational purposes.

• Land filling is still the major disposal method in many countries. Yet, in many instances, land filling sites are not properly chosen in terms of geophysical soil properties, hydrogeology, topography and climate.



• On a proposed site, there is a need to carefully consider the potential for ground or surface water contamination from pollution by leachate migration or surface run-off from the site.



• Nonetheless, even when a site appears to have the right geophysical properties, its selection, and use are not an absolute guarantee that contamination of groundwater can be avoided.

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• Hence, continuous surveillance of the site and its surroundings must be maintained to check that the disposal of hazardous wastes can continue without posing a threat to the environment and to the general public.



• To reduce this threat, landfill sites have been lined, for example, with plastic materials, in order to prevent leaching into groundwater supplies.

Sanitary landfill: Sanitary landfill is a fully engineered disposal option, which avoids harmful effects of uncontrolled dumping by spreading, compacting and covering the wasteland that has been carefully engineered before use. The four minimum requirements for setting up a sanitary landfill are: • full or partial hydrological isolation, • formal engineering preparation, • permanent control and • planned waste placement and covering. Land filling relies on containment rather than treatment (for control) of wastes. Appropriate liners for protection of the groundwater, leachate collection and treatment, monitoring wells and, appropriate final cover design are integral components of an environmentally sound sanitary landfill. Incineration (or) thermal treatment: Incineration is a disposal method in which solid organic wastes are subjected to combustion so as to convert them into residue and gaseous products. This method is useful for disposal of residue of both solid waste management and solid residue from wastewater management. • This process reduces the volumes of solid waste to 20 to 30 percent of the original volume. Incineration and other high temperature waste treatment systems are sometimes described as “thermal treatment”. • Incinerators convert waste materials into heat, gas, steam and ash. • Incineration is carried out both on a small scale by individuals and on a large scale by industry. It is used to dispose of solid, liquid and gaseous waste. • It is recognized as a practical method of disposing of certain hazardous waste materials (such as biological medical waste). Incineration is a controversial method of waste disposal, due to issues such as emission of gaseous pollutants. • Incineration is common in countries such as Japan where land is more scarce, as these facilities generally do not require as much area as landfills. • Waste-to-Energy (WtE) or Energy-from-Waste (EfW) are broad terms for facilities that burn waste in a furnace or boiler to generate heat, steam or electricity.

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• Combustion in an incinerator is not always perfect and there have been concerns about pollutants in gaseous emissions from incinerator stacks. Particular concern has focused on some very persistent organics such as dioxins, furans, PAHs which may be created and may have serious environmental consequences.



• Biomedical waste can be disposed of through incineration or decontamination by heating with steam under pressure in an autoclave. Trash chutes must not be used for the transfer or disposal of biomedical waste.

Pyrolysis/Gasification, Plasma Pyrolysis Vitrification (PPV)/Plasma Arc Process: Pyrolysis is a form of treatment that chemically decomposes organic materials by heat in the absence of oxygen. Pyrolysis, typically, occurs under pressure and at operating temperatures above 430°C.

• Pyrolysis gasification processes are established for homogenous organic matter like wood, pulp, etc., while plasma pyrolysis vitrification is a relatively new technology for disposal of particularly hazardous wastes, radioactive wastes, etc.



• Toxic materials get encapsulated in vitreous mass, which is relatively much safer to handle than incinerator/gasifier ash. These are now being offered as an attractive option for disposal of MSW also.



• In all these processes, besides net energy recovery, proper destruction of the waste is also ensured. These processes, therefore, have an edge over incineration.



• This process produces fuel gas/fuel oil, which replace fossil fuels and compared to incineration, atmospheric pollution can be controlled at the plant level. NO and SO gas emissions do not occur in normal operations due to the lack of oxygen in the system.



• It is a capital and energy intensive process and net energy recovery may suffer in case of wastes with excessive moisture and inert content.



• High viscosity of Pyrolysis oil maybe problematic for its transportation and burning. Concentration of toxic/hazardous matter in gasifier ash needs care in handling and disposal.

No commercial plant has come up in India or elsewhere for the disposal of Municipal Solid Waste (MSW). It is an emerging technology for MSW, yet to be successfully demonstrated for large-scale application. Recycling and reuse: Recycling involves the collection of used and discarded materials, processing these materials, and making them into new products. It reduces the amount of waste that is thrown into the community dustbins thereby making the environment cleaner and the air more fresh to breathe. Hence, recycling refers to the collection and reuse of waste materials such as empty beverage containers. The materials from which the items are made can be reprocessed into new products. Material for recycling may be

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7.9

collected separately from general waste using dedicated bins and collection vehicles, or sorted directly from mixed waste streams. Known as kerb-side recycling, it requires the owner of the waste to separate it into various different bins (typically wheelie bins) prior to its collection.

The Schematic diagram below depicts recycling of municipal wastes

The most common consumer products recycled include aluminum such as beverage cans, copper such as wire, steel food and aerosol cans, old steel furnishings or equipment, polyethylene and PET bottles, glass bottles and jars, paperboard cartons, newspapers, magazines and light paper, and corrugated fiberboard boxes.

• Different plastics (polymers) like PVC, LDPE, PP, and PS (see resin identification code) are also recyclable. These items are usually composed of a single type of material, making them relatively easy to recycle into new products. The recycling of complex products (such as computers and electronic equipment) is more difficult, due to the additional dismantling and separation required.



• The type of material accepted for recycling varies by city and country. Each city and country have different recycling programs in place that can handle the various types of recyclable materials. However, variation in acceptance is reflected in the resale value of the material, once it is reprocessed.

Surveys carried out by Government and non-government agencies in the country have all recognized the importance of recycling wastes. However, the methodology for safe recycling of waste has not been standardized. Studies have revealed that 7-15% of the waste is recycled. If recycling is done in a proper manner, it will solve the problems of waste or garbage. At the community level, a large number of NGOs (Non-Governmental Organizations) and private sector enterprises have taken an initiative in segregation and recycling of waste. Biomedical waste can be managed properly by ensuring proper segregation at the source, the use of accurate packaging (leak resistant, puncture resistant and not susceptible to degradation by cleaning agents in case the packaging is reused), appropriate color coding, proper in-house movement of waste

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(minimizing employee exposure to biomedical waste in a workplace), designating waste storage areas and ensuring safe disposal.

Waste to energy or Energy from waste (Energy recovery) The energy content of waste products can be harnessed directly by using them as a direct combustion fuel, or indirectly, by processing them into another type of fuel. • Thermal treatment ranges from using waste as a fuel source for cooking or heating and the use of the gas fuel, to fuel for boilers, to generate steam and electricity in a turbine. • Pyrolysis and gasification are two related forms of thermal treatment where waste materials are heated to high temperatures with limited oxygen availability. The process usually occurs in a sealed vessel under high pressure. • Pyrolysis of solid waste converts the material into solid, liquid and gas products. The liquid and gas can be burnt to produce energy or refined into other chemical products (chemical refinery). • The solid residue (char) can be further refined into products such as activated carbon. Gasification and advanced Plasma arc gasification are used to convert organic materials directly into a synthetic gas (syngas) composed of carbon monoxide and hydrogen. • The gas is then burnt to produce electricity and steam. An alternative to pyrolysis is high temperature and pressure supercritical water decomposition Even though the technology of waste to energy (WTE) projects has been proven worldwide, its viability and sustainability is yet to be to be demonstrated and established in India. The main factors that determine the techno-economic viability of WTE projects are quantum of investment, scale of operation, availability of quality waste, statutory requirements and project risks.

7.4

BIOLOGICAL REPROCESSING OF SOLID WASTE DISPOSAL

Solid waste materials that are organic in nature, such as plant material, food scraps, and paper products, can be recycled using biological composting and digestion processes to decompose the organic matter. The resulting organic material is then recycled as mulch or compost for agricultural or landscaping purposes. In addition, waste gas from the process (such as methane) can be captured and used for generating electricity and heat (CHP/cogeneration), maximizing efficiencies. The intention of biological processing in waste management is to control and accelerate the natural process of decomposition of organic matter.

• There is a large variety of composting and digestion methods and technologies varying in complexity from simple home compost heaps,

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7.11

to small town scale batch digesters, industrial-scale enclosed-vessel digestion of mixed domestic waste.

• Methods of biological decomposition are differentiated as being aerobic or anaerobic methods, though hybrids of the two methods also exist.



• Anaerobic digestion of the organic fraction of MSW has been found to be in a number of Life cycle analysis studies to be more environmentally effective, than landfill, incineration or pyrolysis.



• The resulting biogas (methane) though must be used for cogeneration (electricity and heat, preferably, on or close to the site of production) and can be used with a little upgrading in gas combustion engines or turbines.



• With further upgrading to synthetic natural gas, it can be injected into the natural gas network or further refined to hydrogen for use in stationary cogeneration fuel cells. Its use in fuel cells eliminates the pollution from products of combustion.

7.4.1 Composting Composting is a technology known in India since times immemorial. Composting is the decomposition of organic matter by microorganism in warm, moist, aerobic and anaerobic environment. Farmers have been using compost made out of cow dung and other agro-waste.

• The compost made out of urban heterogeneous waste is found to be of higher nutrient value as compared to the compost made out of cow dung and agro-waste.



• Composting of MSW is, therefore, the most simple and cost-effective technology for treating the organic fraction of MSW.



• Full-scale commercially viable composting technology is already demonstrated in India and is in use in several cities and towns. Its application to farm land, tea gardens, fruit orchards or its use as soil conditioner in parks, gardens, agricultural lands, etc., is however, limited on account of poor marketing.



• Main advantages of composting include improvement in soil texture and augmenting of micronutrient deficiencies.



• It also increases moisture-holding capacity of the soil and helps in maintaining soil health. Moreover, it is an age-old established concept for recycling nutrients to the soil.



• It is simple and straightforward to adopt, for source separated MSW. It does not require large capital investment, compared to other waste treatment options. The technology is scale-neutral.



• Composting is suitable for organic biodegradable fraction of MSW, yard (or garden) waste / waste containing high proportion of lignocelluloses

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materials, which do not readily degrade under anaerobic conditions, waste from slaughterhouse and dairy waste.

• This method, however, is not very suitable for wastes that may be too wet and, during heavy rains, open compost plants have to be stopped.



• Land required for open compost plants is relatively large. Also, issues of methane emission, odor, and flies from badly-managed open compost plants, remain. At the operational level, if waste segregation at source is not properly carried out, there is possibility of toxic material entering the stream of MSW.



• It is essential that compost produced be safe for application. Standardization of compost quality is, therefore, necessary.

A pilot system diagram for composting

Biotechnological processes for soil and land treatment: Enhanced bioremediation is a process in which indigenous or inoculated microorganisms (e.g., fungi, bacteria, and other microbes) degrade (metabolize) organic contaminants found in soil and/or ground water, converting them to innocuous end products.

• Nutrients, oxygen, or other amendments may be used to enhance bioremediation and contaminant desorption from subsurface materials.



• In the presence of sufficient oxygen (aerobic conditions), and other nutrient elements, microorganisms will ultimately convert many organic contaminants to carbon dioxide, water, and microbial cell mass.



• In the absence of oxygen (anaerobic conditions), the organic contaminants will be ultimately metabolized to methane, limited amounts of carbon dioxide, and trace amounts of hydrogen gas. Under sulfate-reduction conditions, sulfate is converted to sulfide or elemental sulfur, and under nitrate-reduction conditions, dinitrogen gas is ultimately produced.



• Sometimes contaminants may be degraded to intermediate or final products that may be less, equally, or more hazardous than the original contaminant. For example, TCE is anaerobically biodegrades to the

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7.13

persistent and more toxic vinyl chloride. To avoid such problems, most bioremediation projects are conducted in situ. Vinyl chloride can easily be broken down further, if aerobic conditions are created. • Enhanced bioremediation of soil, typically involves the percolation or injection of groundwater or uncontaminated water mixed with nutrients and saturated with dissolved oxygen. Sometimes, acclimated microorganisms (bioaugmentation) and/or another oxygen source such as hydrogen peroxide, are also added. An infiltration gallery or spray irrigation is typically used for shallow contaminated soils, and injection wells are used for deeper contaminated soils. • Although successful in situ bioremediation has been demonstrated in cold weather climate, low temperature slows the remediation process. For contaminated sites with low soil temperature, heat blankets may be used to cover the soil surface to increase the soil temperature and the degradation rate. Enhanced bioremediation may be classified as a long-term technology which may take several years for cleanup of a plume.

7.5

BIOTECHNOLOGICAL METHODS OF SOLID WASTE (AGRICULTURE, DOMESTIC AND INDUSTRIAL) DEGRADATION

In the present techno-economic era, the energy and environmental crises developed due to huge amount of cellulosic materials are disposed as “waste.” Municipal solid waste is composed of 40–50% cellulose, 9–12% hemicellulose, and 10—15% lignin on a dry weight basis. Annually, Asia alone generates 4.4 billion tons of solid wastes, and municipal solid waste comprises 790 million tons, of which about 48 million tons are generated in India. By the year 2047, municipal solid waste generation in India is expected to reach 300 million tons and land requirement for disposal of this waste would be 169.6 km2. Unscientific disposal causes an adverse impact on all components of the environment and human health. Microorganism performs their metabolic processes rapidly and with remarkable specificity under ambient conditions, catalyzed by their diverse enzyme-mediated reactions. An enzyme alternative to harsh chemical technologies has led to intensive exploration of natural microbial biodiversity to discover enzymes. There is a wide spectrum of microorganisms which can produce the variety of enzymes like cellulase under appropriate conditions. Cellulases are a consortium of free enzymes comprised of endoglucanases, exoglucanases, and cellobiases are found in many of the 57 glycosyl hydrolase families. Several studies were carried out to produce cellulolytic enzymes in organic waste degradation process by several microorganism, including fungi such as Trichoderma sp., Penicillium sp., and Aspergillus sp., respectively. Many fungi capable of degrading cellulose synthesize large quantities of

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Environmental Biotechnology

extracellular cellulases that are more efficient in depolymerising the cellulose substrate. Most commonly studied cellulolytic organisms include fungal species: Trichoderma, Humicola, Penicillium, and Aspergillus. Many cellulases produced by bacteria appear to be bound to the cell wall and are unable to hydrolyze native lignocellulose preparations to any significant extent. A wide variety of gram-positive and gram-negative species are reported to produce cellulose, including Clostridium thermocellum, Streptomyces spp., Ruminococcus spp., Pseudomonas spp., Cellulomonas spp., Bacillus spp., Serratia, Proteus, Staphylococcus spp., and Bacillus subtilis. Various biological studies have been carried out to identify the major microbiological agents responsible for biodegradation. Today, environmental policies and regulation progress lead to the development of biodegradation processes to turn organic wastes into a valuable resource by potential microbes because only few strains are capable of secreting a complex of cellulase enzymes, which could have practical application in the enzymatic hydrolysis of cellulose as well as biodegradation of organic municipal solid waste. Probably, the best and the most economical approach to the problem of—solid waste disposal, involve microbial degradation of solid wastes. Biodegradable wastes can be conveniently handled in this way. Recalcitrant and refractory solids like plastics, polymers and many plasticizers create problems. These materials are synthetic substances, and as such, are often too new to the biological system. The microbial system simply lacks the enzymatic machinery needed for their decomposition. Recent strides taken in the field of biotechnology has come to be of great help in this direction. Genetically engineered microbes have been produced which can decompose a number of organic compounds which were considered to be nondegradable earlier. It was only an Indian born American Scientist Chakrabarty A.N., who has patented for the first time a genetically engineered strain of bacteria, Pseudomonas, which decomposes a number of complex and toxic hydrocarbons. Efforts are also being made to produce biodegradable plastic and polymers through genetically engineered microbes. Biologically produced plastic and polymers shall create much less problem as microbial system shall decompose them quickly and effectively into simpler constituents.

7.6 IMPORTANCE, HEALTH IMPACTS AND AWARENESS OF WASTE MANAGEMENT Importance of waste reduction: In the affluent countries, the main motivations for waste reduction are frequently related to the high cost and scarcity of sites for landfills, and the environmental degradation caused by toxic materials in the deposited wastes. The same considerations apply to large metropolitan areas in developing countries that are surrounded by other populous jurisdictions. The places that currently do not have significant disposal pressures can still

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benefit from encouraging waste reduction. Their solid waste departments, already overburdened, cannot afford to spend more money and effort on the greater quantities of wastes that will inevitably be produced as consumption levels rise and urban wastes change. Solid waste managers in developing countries tend to pay little attention to the topic of reducing non-organic wastes because the wastes they collect are between 50 to 90% organics, dirt and ashes. These municipal wastes, however, are amenable to composting or digestion, provided they contain very low levels of synthetic materials. Solid waste departments thus have an interest in promoting diversion of synthetic recyclables from the waste stream. Each household generates garbage or waste, day in and day out. Items that are no longer needed or do not have any further use, fall in the category of waste and we tend to throw them away. There are different types of solid waste depending on their source. In today’s polluted world, learning the correct methods of handling the waste generated has become essential. Segregation is an important method of handling municipal solid waste. Segregation at source can be understood clearly by schematic representation. One of the important methods of managing and treating wastes is composting. As the cities are growing in size and in problems such as the generation of plastic waste, various municipal waste treatment and disposal methods are now being used to try and resolve these problems. One common sight in all cities is the rag picker who plays an important role in the segregation of this waste. Garbage generated in households can be recycled and reused to prevent creation of waste at source and reducing amount of waste thrown into the community dustbins.

7.7

HEALTH IMPACTS OF SOLID WASTE

Modernization and progress has had its share of disadvantages and one of the main aspects of concern is the pollution it is causing to the earth—be it land, air, and water. With increase in the global population and the rising demand for food and other essentials, there has been a rise in the amount of waste being generated daily by each household. This waste is ultimately thrown into municipal waste collection centers from where it is collected by the area municipalities to be further thrown into the landfills and dumps. However, either due to resource crunch or inefficient infrastructure, not all of this waste gets collected and transported to the final dumpsites. If, at this stage, the management and disposal is improperly done, it can cause serious impacts on health and problems to the surrounding environment. Waste that is not properly managed, especially excreta and other liquid and solid waste from households and the community, are a serious health hazard and lead to the spread of infectious diseases. Unattended waste lying around attracts flies, rats, and other creatures that, in turn, spread disease. Normally, it is the wet waste that decomposes and releases a bad odor. This

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leads to unhygienic conditions, and thereby, to a rise in the health problems. The plague outbreak in Surat is a good example of a city suffering due to the callous attitude of the local body in maintaining cleanliness in the city. Plastic waste is another cause for ill health. Thus, excessive solid waste that is generated should be controlled by taking certain preventive measures. • The group, at risk, from the unscientific disposal of solid waste include—the population in areas where there is no proper waste disposal method, especially the pre-school children; waste workers; and workers in facilities producing toxic and infectious material. Other high-risk group include, population living close to a waste dump and those, whose water supply has become contaminated either due to waste dumping or leakage from landfill sites. Uncollected solid waste also increases risk of injury, and infection. • In particular,  organic domestic waste  poses a serious threat, since they ferment, creating conditions favorable to the survival and growth of microbial pathogens. Direct handling of solid waste can result in various types of infectious and chronic diseases with the waste workers and the rag pickers being the most vulnerable. • Exposure to hazardous waste  can affect human health, children being more vulnerable to these pollutants. In fact, direct exposure can lead to diseases through chemical exposure, as the release of chemical waste into the environment leads to chemical poisoning. Many studies have been carried out in various parts of the world to establish a connection between health and hazardous waste. • Waste from agriculture and industries  can also cause serious health risks. Other than this, co-disposal of industrial hazardous waste with municipal waste can expose people to chemical and radioactive hazards. Uncollected solid waste can also obstruct storm water runoff, resulting in the forming of stagnant water bodies that become the breeding ground for disease. Waste dumped near a water source also causes contamination of the water body or the ground water source. Direct dumping of untreated waste in rivers, seas, and lakes results in the accumulation of toxic substances in the food chain through the plants and animals that feed on it. • Disposal of hospital and other medical waste  requires special attention since this can create major health hazards. This waste generated from the hospitals, health care centres, medical laboratories, and research centers such as discarded syringe needles, bandages, swabs, plasters, and other types of infectious waste are often disposed with the regular non-infectious waste.

• Waste treatment and disposal sites  can also create health hazards for the neighborhood. Improperly operated incineration plants cause air pollution and improperly managed and designed landfills attract all types of insects and rodents that spread disease. Ideally, these sites

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should be located at a safe distance from all human settlement. Landfill sites should be well-lined and walled to ensure that there is no leakage into the nearby ground water sources. • Recycling, too, carries health risks if proper precautions are not taken. Workers working with waste containing chemical and metals may experience toxic exposure. Disposal of health-care wastes require special attention since it can create major health hazards, such as Hepatitis B and C, through wounds caused by discarded syringes. Rag pickers and others, who are involved in scavenging in the waste dumps for items that can be recycled, may sustain injuries and come into direct contact with these infectious items. Diseases: Certain chemicals, if released untreated, e.g. cyanides, mercury, and polychlorinated biphenyls, are highly toxic and exposure can lead to disease or death. Some studies have detected excesses of cancer in residents exposed to hazardous waste. Many studies have been carried out in various parts of the world to establish a connection between health and hazardous waste. The role of plastics: The unhygienic use and disposal of plastics and its effects on human health has become a matter of concern. Colored plastics are harmful as their pigment contains heavy metals that are highly toxic. Some of the harmful metals found in plastics are copper, lead, chromium, cobalt, selenium, and cadmium. In most industrialized countries, colour plastics have been legally banned. In India, the Government of Himachal Pradesh has banned the use of plastics and so has Ladakh district. Other states should emulate their example. Preventive measures: Proper methods of waste disposal have to be undertaken to ensure that it does not affect the environment around the area or causes health hazards to the people living there. At the household-level proper segregation of waste has to be done and it should be ensured that all organic matter is kept aside for composting, which is undoubtedly the best method for the correct disposal of this segment of the waste. In fact, the organic part of the waste that is generated decomposes more easily, attracts insects and causes disease. Organic waste can be composted and then used as a fertilizer.

7.8

KEY CONCEPTS IN MUNICIPAL WASTE REDUCTION

Waste reduction: All means of reducing the amounts of waste that must be collected and disposed of by solid waste authorities. It ranges from legislation and agreements at the national level for packaging and product redesign to local programs to prevent recyclables and compostable organics from entering final waste streams. Source reduction: Any procedure to reduce wastes at the point of generation, in contrast to sorting out recyclable components after they have been mixed together for collection.

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Source separation: Keeping different categories of recyclables and organics separate at source, i.e., at the point of generation, to facilitate reuse, recycling, and composting. Waste recovery, materials recovery, or waste diversion: Obtaining materials/organics (by source separation or sorting out from mixed wastes) that can be reused or recycled. Reuse: Reusing a product for the same or a different purpose. Recycling: The process of transforming materials into secondary resources for manufacturing new products is called recycling. Redemption center: Waste trading enterprise that buys recyclable materials and sells to brokers. Sometimes also called “buy-back centre”. Producer responsibility: Producers of products or services accept a degree of responsibility for the wastes that result from the products/services they market, by reducing materials used in production, making repairable/ recyclable goods, and/or reducing packaging. Promoting waste reduction and materials recovery at the national and local levels: Action for waste reduction can take place at both national and local levels. At the national level, the main routes to waste reduction are: • redesign of products or packaging; • promotion of consumer awareness; and • promotion of producer responsibility for post-consumer wastes (this applies mostly to industrialized countries). At the local level, the main means of reducing waste are: • diversion of materials from the waste stream through source separation and trading; • recovery of materials from mixed waste; • pressure on national or regional governments for legislation on redesigning packaging or products; and • support of composting, either centralized or small-scale.

7.9 WASTEWATER Water is crucial for all aspects of life, the defining feature of our planet. Ninety seven and a half per cent of all water is found in the oceans; of the remaining freshwater only one per cent is accessible for extraction and use. Functioning and healthy aquatic ecosystems provide us with a dazzling array of benefits – food, medicines, recreational amenity, shoreline protection, processing our waste, and sequestering carbon. At the beginning of the 21st century, the world faces a water crisis, both of quantity and quality, caused by continuous population growth, industrialization, food production practices, increased living standards and poor water use strategies. Wastewater management or the lack of, has a direct impact on the biological diversity of aquatic ecosystems,

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disrupting the fundamental integrity of our life support systems, on which a wide range of sectors from urban development to food production and industry depend. It is essential that wastewater management is considered as part of integrated, ecosystem-based management that operates across sectors and borders, freshwater and marine.

What do we mean by wastewater? Wastewater can mean different things to different people with a large number of definitions in use. But in broad perspective wastewater may be defined as “a combination of one or more of: domestic effluent consisting of black water (excreta, urine and fecal sludge) and grey water (kitchen and bathing wastewater); water from commercial establishments and institutions, including hospitals; industrial effluent, storm water and other urban run-off; agricultural, horticultural and aquaculture effluent, either dissolved or as suspended matter.

7.9.1 Wastewater treatment and Management Wastewater treatment plants can be divided into two major types: 1. Biological and 2. Physical/Chemical. • Biological plants are more commonly used to treat domestic, or combined domestic and industrial, wastewater from a municipality. They use basically the same processes that would occur naturally in the receiving water, but give them a place to happen under controlled conditions, so that the cleansing reactions are completed before the water is discharged into the environment. • Physical/chemical plants are more often used to treat industrial wastewaters directly, because they often contain pollutants which cannot be removed efficiently by microorganisms— although industries that deal with biodegradable materials, such as food processing, dairies, breweries, and even paper, plastics and petrochemicals, may use biological treatment. And biological plants generally use some physical and chemical processes, too. A physical process usually treats suspended, rather than dissolved pollutants. It may be a passive process, such as simply allowing suspended pollutants to settle out or float to the top naturally—depending on whether they are more or less dense than water, Or, the process may be aided mechanically, such as by gently stirring the water to cause more small particles to bump into each other and stick together, forming larger particles which will settle or rise faster—a process known as flocculation. • Chemical flocculants may also be added to produce larger particles. To aid flotation processes, dissolved air under pressure may be added to cause the formation of tiny bubbles which will attach to particles.

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Filtration through a medium such, as sand as a final treatment stage, can result in a very clear water. Ultrafiltration, nanofiltration, and reverse osmosis are processes which force water through membranes and can remove colloidal material (very fine, electrically charged particles, which will not settle) and even some dissolved matter. Absorption (adsorption, technically) on activated charcoal is a physical process which can remove dissolved chemicals. Air or steam stripping can be used to remove pollutants that are gases or low-boiling liquids from water, and the vapors which are removed in this way, are also often passed through beds of activated charcoal, to prevent air pollution. These last processes are used mostly in industrial treatment plants, though activated charcoal is common in municipal plants, as well, for odor control. Some examples of chemical treatment processes, in an industrial setting, would be:

• converting a dissolved metal into a solid, settleable form by precipitation with an alkaline material like sodium or calcium hydroxide. Dissolved iron or aluminum salts or organic coagulant aids like polyelectrolytes can be added to help flocculate and settle (or float) the precipitated metal.



• converting highly toxic cyanides used in mining and metal finishing industries into harmless carbon dioxide and nitrogen by oxidizing them with chlorine



• destroying organic chemicals by oxidizing them using ozone or hydrogen peroxide, either alone or in combination with catalysts (chemicals which speed up reactions) and/or ultraviolet light.

In municipal treatment plants, chemical treatment in the form of aluminum or iron salts is often used for removal of phosphorus by precipitation. Chlorine or ozone (or ultraviolet light) may be used for disinfection, that is, killing harmful microorganisms before the final discharge of the wastewater. Sulfur dioxide or sulfite solutions can be used to neutralize (reduce) excess chlorine, which is toxic to aquatic life. Chemical coagulants are also used extensively in sludge treatment to thicken the solids and promote the removal of water. A typical treatment plant consists of a train of individual unit processes set up in a series, with the output (effluent) of one process becoming the input (influent) of the next process. The first stages will usually be made up of physical processes that take out easily removable pollutants. After this, the remaining pollutants are generally treated further by biological or chemical processes. These may—

• convert dissolved or colloidal impurities into a solid or gaseous form, so that they can be removed physically, or



• convert them into dissolved materials which remain in the water, but are not considered as undesirable as the original pollutants.

The solids (residuals or sludges) which result from these processes form a side stream which also has to be treated for disposal.

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A common set of processes that might be found at a municipal treatment plant would be:

• Preliminary treatment to remove large or hard solids that might clog or damage other equipment. These might include grinders (comminuters), bar screens, and grit channels.



— The first chops up rags and trash;



— the second simply catches large objects, which can be raked off;



— the third allows heavier materials, like sand and stones, to settle out, so that they will not cause abrasive wear on downstream equipment. Grit channels also remove larger food particles (i.e., garbage).



• Primary settling basins, where the water flows slowly for up to a few hours, to allow organic suspended matter to settle out or float to the surface. Most of this material has a density not much different from that of water, so it needs to be given enough time to separate. Settling tanks can be rectangular or circular.

In either type, the tank needs to be designed with some type of scrapers at the bottom to collect the settled sludge and direct it to a pit from which it can be pumped for further treatment— and skimmers at the surface, to collect the material that floats to the top (which is given the rather inglorious name of “scum”.)

— Secondary treatment, usually biological, tries to remove the remaining dissolved or colloidal organic matter. Generally, the biodegradation of the pollutants is allowed to take place in a location where plenty of air can be supplied to the microorganisms. This promotes formation of the less offensive, oxidized products. Engineers try to design the capacity of the treatment units so that enough of the impurities will be removed to prevent significant oxygen demand in the receiving water after discharge.

There are two major types of biological treatment processes: attached growth and suspended growth. In an attached growth process, the microorganisms grow on a surface, such as rock or plastic. Examples are:

(1) open trickling filters, where the water is distributed over rocks and trickles down to underdrains, with air being supplied through vent pipes,



(2) enclosed biotowers, which are similar, but more likely to use shaped, plastic media instead of rocks, and



(3) so-called rotating biological contacters, or RBCs, which consist of large, partially submerged discs which rotate continuously, so that the microorganisms growing on the disc’s surface are repeatedly exposed alternately to the wastewater and to the air.

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The most common type of suspended growth process is the so-called activated sludge system. This type of system consists of two parts, an aeration tank and a settling tank, or clarifier. The aeration tank contains a “sludge” which is what could be best described as a “mixed microbial culture”, containing mostly bacteria, as well as protozoa, fungi, algae, etc. This sludge is constantly mixed and aerated either by compressed air bubblers located along the bottom, or by mechanical aerators on the surface. The wastewater to be treated enters the tank and mixes with the culture, which uses the organic compounds for growth — producing more microorganisms — and for respiration, which results mostly in the formation of carbon dioxide and water. The process can also be set up to provide biological removal of the nutrients nitrogen and phosphorus. After sufficient aeration time to reach the required level of treatment, the sludge is carried by the flow into the settling tank, or clarifier, which is often of the circular design (An important condition for the success of this process is the formation of a type of culture which will flocculate naturally, producing a settling sludge and a reasonably clear upper, or supernatant layer. If the sludge does not behave this way, a lot of solids will be remain in the water leaving the clarifier, and the quality of the effluent wastewater will be poor). The sludge collected at the bottom of the clarifier is then recycled to the aeration tank to consume more organic material. The term “activated” sludge is used, because by the time the sludge is returned to the aeration tank, the microorganisms have been in an environment depleted of “food” for some time, and are in a “hungry”, or activated condition, eager to get busy biodegrading some more wastes. Since the amount of microorganisms, or biomass, increases as a result of this process, some must be removed on a regular basis for further treatment and disposal, adding to the solids produced in primary treatment.

Activated sludge process

Variations Sequencing Batch Reactor (SBR):The type of activated sludge system, described above, is a continuous flow process. There is a variation in which the entire activated sludge process take place in a single tank, but at different times.

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Steps include filling, aerating, settling, drawing off supernatant, etc. A system like this can provide more flexibility and control over the treatment, including nutrient removal, and is amenable to computer control. Membrane Bioreactor (MBR): In this more recent innovation, treated water is pumped out of the aeration tank through banks of microfiltration membranes. Clarifiers are not needed. The sludge concentration can be higher than in a conventional system, which allows treatment in a smaller volume; and the sludge’s ability to flocculate well is no longer a consideration. Low effluent solids concentrations can be achieved, which can help in phosphorus removal and disinfection (see below).

Removal of heavy metals from waste streams

Nutrient removal: Nutrient removal refers to the treatment of the wastewater to take out nitrogen or phosphorus, which can cause unnecessary growth of algae or weeds in the receiving water. • Nitrogen is found in domestic wastewater mostly in the form of ammonia and organic nitrogen. • These can be converted to nitrate by bacteria, if the plant is designed to provide enough oxygen and a long enough “sludge age” to develop these slow-growing types of organisms. • The nitrate which is produced, may be discharged; it is still usable as a plant nutrient, but it is much less toxic than ammonia. • If more complete removal of nitrogen is required, a biological process can be set up which reduces the nitrate to nitrogen gas (and some nitrous oxide). • There are also physical/chemical processes which can remove nitrogen, especially ammonia; they are not as economical for domestic wastewater, but might be suited for an industrial location where no other biological processes are in use (These methods include alkaline air stripping, ion exchange, and “breakpoint” chlorination). Phosphorous removal: Phosphorous removal is most commonly done by chemical precipitation with iron or aluminum compounds, such as ferric chloride or alum (aluminum sulfate).

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• The solids which are produced can be settled along with other sludges, depending upon where the treatment process takes place. (“Lime”, or calcium hydroxide, also works, but makes the water very alkaline, which has to be corrected, and produces more sludge.). • There is also a biological process for phosphorus removal, which depends on designing an activated sludge system in such a way as to promote the development of certain types of bacteria which have the ability to accumulate excess phosphorus within their cells. These methods mainly convert dissolved phosphorus into particulate form. • For treatment plants which are required to discharge only very low concentrations of total phosphorus, it is common to have a sand (or other type of) filter as a final stage, to remove most of the suspended solids which may contain phosphorus. Disinfection: Disinfection, usually the final process before discharge, is the destruction of harmful (pathogenic) microorganisms, i.e., disease-causing germs. The object is not to kill every living microorganism in the water, which would be sterilization, but to reduce the number of harmful ones to levels appropriate for the intended use of the receiving water. The most commonly used disinfectant is chlorine, which can be supplied in the form of a liquefied gas which has to be dissolved in water, or in the form of an alkaline solution called sodium hypochlorite, which is the same compound as common household, chlorine bleach. Chlorine is quite effective against most bacteria, but a rather high dose is needed to kill viruses, protozoa, and other forms of pathogen. Chlorine has several problems associated with its use, among them being: (1) It reacts with organic matter to form toxic and carcinogenic chlorinated organics, such as chloroform, (2) Chlorine is very toxic to aquatic organisms in the receiving water— the USEPA recommends no more than 0.011 parts per million (mg/L) and (3) It is hazardous to store and handle. Hypochlorite is safer, but still produces problems 1 and 2. Problem 2 can be dealt with by adding sulfur dioxide (liquefied gas) or sodium sulfite or bisulfite (solutions) to neutralize the chlorine. The products are nearly harmless chloride and sulfate ions. This may also help somewhat with problem 1. A more powerful disinfectant is ozone, an unstable form of oxygen containing three atoms per molecule, rather than the two found in the ordinary oxygen gas which makes up about 21% of the atmosphere. Ozone is too unstable to store, and has to be made as it is used. It is produced by passing an electrical discharge through air, which is then bubbled through the water. While chlorine can be dosed at a high enough concentration so that some of it remains in the water for a considerable time, ozone is consumed very rapidly and leaves no residual. It may also produce some chemical byproducts, but probably not as harmful as those produced by chlorine.

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The other commonly used method of disinfection is ultraviolet light. The water is passed through banks of cylindrical, quartz-jacketed fluorescent bulbs. Anything which can absorb the light, such as fouling or scale formation on the bulbs’ surfaces, or suspended matter in the water, can interfere with the effectiveness of the disinfection. Some dissolved materials, such as iron and some organic compounds, can also absorb some of the light. Ultraviolet disinfection is becoming more popular because of the increasing complications associated with the use of chlorine.

7.9.2 Water Purification By Waterweeds And Membrane Filters Water Purification: Water purification is the process of removing undesirable chemicals, biological contaminants, suspended solids and gases from contaminated water. The goal is to produce water fit for a specific purpose. Most water is purified for human consumption (drinking water) but water purification may also be designed for a variety of other purposes, including meeting the requirements of medical, pharmacology, chemical and industrial applications. In general, the methods used include physical processes such as filtration and sedimentation, biological processes such as slow sand filters or activated sludge, chemical processes such as flocculation and chlorination and the use of electromagnetic radiation such as ultraviolet light.

• The purification process of water may reduce the concentration of particulate matter including suspended particles, parasites, bacteria, algae, viruses, fungi; and a range of dissolved and particulate material derived from the surfaces that water may have made contact with after falling as rain.



• The standards for drinking water quality are typically set by governments or by international standards. These standards will typically set minimum and maximum concentrations of contaminants for the use that is to be made of the water.



• It is not possible to tell whether water is of an appropriate quality by visual examination. Simple procedures such as boiling or the use of a household activated carbon filter are not sufficient for treating all the possible contaminants that may be present in water from an unknown source. Even natural spring water—considered safe for all practical purposes in the 19th century—must now be tested before determining what kind of treatment, if any, is needed. Chemical analysis, while expensive, is the only way to obtain the information necessary for deciding on the appropriate method of purification.

According to a 2007 World Health Organization report, 1.1 billion people lack access to an improved drinking water supply, 88% of the 4 billion annual cases of diarrheal disease are attributed to unsafe water and inadequate sanitation and hygiene, and 1.8 million people die from diarrheal diseases each year. The WHO estimates that 94% of these diarrheal cases are preventable

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through modifications to the environment, including access to safe water. Simple techniques for treating water at home, such as chlorination, filters, and solar disinfection, and storing it in safe containers could save a huge number of lives each year. Reducing deaths from waterborne diseases is a major public health goal in developing countries. Purification of water by water weeds: Water weeds or the aquatic plants, often treated as a problem for development of lakes actually help in rejuvenation of water bodies by absorbing sewage and heavy metals that affect most of the lakes. The water weeds cover the lake surface reducing foul smell, manufacture biomass, purify the water body and absorb all heavy metals including sulphur, phosphate, nitrate, etc.  Growing water weeds especially Hyacinth which has more absorbing capacity for sometime, harvesting it and replanting it again will rejuvenate the water. But, the growth of the weeds needs to be monitored well as they grow at a very rapid pace, and once they are harvested, they have to be disposed scientifically to ensure that they have no adverse effect on the environment. The harvested weeds can be dumped into a compost pit for being converted into manures. Purification of water by Membrane filters: Membrane technology has become a dignified separation technology over the past decennia. The main force of membrane technology is the fact that it works without the addition of chemicals, with a relatively low energy use and easy and well-arranged process conductions. Membrane technology is a generic term for a number of different, very characteristic separation processes. These processes are of the same kind, because in each of them a membrane is used. Membranes are used more and more often for the creation of process water from groundwater, surface water or wastewater. Membranes are now competitive for conventional techniques. The membrane separation process is based on the presence of semi-permeable membranes. Principle: The principle is quite simple: the membrane acts as a very specific filter that will let water flow through, while it catches suspended solids and other substances. There are various methods to enable substances to penetrate a membrane. Examples of these methods are the applications of high pressure, the maintenance of a concentration gradient on both sides of the membrane and the introduction of an electric potential. Membranes occupy through a selective separation wall. Certain substances can pass through the membrane, while other substances are caught. There are two factors that determine the affectivity of a membrane filtration process—selectivity and productivity.

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• Selectivity is expressed as a parameter called retention or separation factor. • Productivity is expressed as a parameter called flux. Selectivity and productivity are membrane-dependent.

Schematic representation of the water purification by membrane filters

Membrane filtration can be divided up between micro and ultra filtration, on the one hand and nano filtration and Reverse Osmosis (RO or hyper filtration), on the other hand. When membrane filtration is used for the removal of larger particles, microfiltration and ultrafiltration are applied. Because of the open character of the membranes, the productivity is high while the pressure differences are low. When salts need to be removed from water, nano filtration and Reverse Osmosis are applied. Nano filtration and RO membranes do not work according to the principle of pores; separation takes place by diffusion through the membrane. The pressure that is required to perform nano filtration and Reverse Osmosis is much higher than the pressure required for micro and ultrafiltration, while productivity is much lower.

Steps involved in membrane filter technology

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Membrane filtration has a number of benefits over the existing water purification techniques:

• It is a process that can take place while temperatures are low. This is mainly important because it enables the treatment of heat-sensitive matter. That is why these applications are widely used for food production.



• It is a process with low-energy cost. Most of the energy that is required is used to pump liquids through the membrane. The total amount of energy that is used is minor, compared to alternative techniques, such as evaporation.



• The process can easily be expanded.

However, no filtration can remove substances that are actually dissolved in the water such as phosphorus, nitrates and heavy metal ions.

7.9.3 Indicator Organisms The following microbiological parameters are particularly important from the health point of view:

Indicator Organisms Coliforms and Faecal Coliforms: The Coliform group of bacteria comprises mainly species of the genera Citrobacter, Enterobacter, Escherichia and Klebsiella and includes Faecal Coliforms, of which Escherichia coli is the predominant species. Several of the Coliforms are able to grow outside of the intestine, especially in hot climates, hence their enumeration is unsuitable as a parameter for monitoring wastewater reuse systems. The Faecal Coliform test may also include some non-faecal organisms which can grow at 44°C, so the E. coli count is the most satisfactory indicator parameter for wastewater use in agriculture. Faecal Streptococci: This group of organisms includes species mainly associated with animals (Streptococcus bovis and S. equinus), other species with a wider distribution (e.g. S. faecalis and S. faecium, which occur both in man and in other animals) as well as two biotypes (S. faecalis var liquefaciens and a typical S. faecalis, that hydrolyzes starch) which appear to be ubiquitous, occurring in both polluted and non-polluted environments. The enumeration of Faecal Streptococci in effluents is a simple routine procedure but has the following limitations: the possible presence of the non-faecal biotypes as part of the natural microflora on crops may detract from their utility in assessing the bacterial quality of wastewater irrigated crops; and the poorer survival of Faecal Streptococci at high than at low temperatures. Further studies are still warranted on the use of Faecal Streptococci as an indicator in tropical conditions and especially, to compare survival with that of Salmonellae. Clostridium perfringens: This bacterium is an exclusively faecal sporeforming anaerobe normally used to detect intermittent or previous pollution

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of water, due to the prolonged survival of its spores. Although this extended survival is usually considered to be a disadvantage for normal purposes, it may prove to be very useful in wastewater reuse studies, as Clostridium perfringens may be found to have survival characteristics similar to those of viruses or even helminth eggs. Pathogens: The following pathogenic parameters can only be considered, if suitable laboratory facilities and suitably trained staff are available: Salmonella spp. Several species of Salmonellae may be present in raw sewage from an urban community in a tropical developing country, including S. typhi (causative agent for typhoid) and many others. It is estimated that a count of 7000 Salmonellae/liter is typical in a tropical urban sewage with similar numbers of Shigellae, and perhaps 1000 Vibrio cholera/liter. Both Shigella spp. and V. cholera are more rapidly killed in the environment so; if removal of Salmonellae can be achieved, then the majority of other bacterial pathogens will also have been removed. Enteroviruses. May give rise to severe diseases, such as Poliomyelitis and Meningitis, or to a range of minor illnesses such as respiratory infections. Although there is no strong epidemiological evidence for the spread of these diseases via sewage irrigation systems, there is some risk and, it is desirable to know to what extent viruses are removed by existing and new treatment processes, especially under tropical conditions. Virus counts can only be undertaken in a dedicated laboratory, as the cell culture techniques required are very susceptible to bacterial and fungal contamination. Rotaviruses. These viruses are known to cause gastro-intestinal problems and, though usually present in lower numbers than enteroviruses in sewage, they are known to be more persistent, so it is necessary to establish their survival characteristics relative to enteroviruses and relative to the indicator organisms in wastewaters. It has been claimed that the removal of viruses in wastewater treatment occurs in parallel simultaneously or insimultaneity would such better with the removal of suspended solids, as most virus particles are solids-associated. Hence, the measurement of suspended solids in treated effluents should be carried out as a matter of routine. Intestinal Nematodes: It is known that nematode infections, in particular from the roundworm Ascaris lumbricoides, can be spread by effluent reuse practices. The eggs of A. lumbricoides are fairly large (45-70 mm × 35-50 mm) and several techniques for enumeration of nematodes have been developed.

7.9.4 Reclaim of Treated Wastewater Reclaimed water or recycled water, is former wastewater (sewage) that is treated to remove solids and certain impurities, and used in sustainable landscaping irrigation or to recharge groundwater aquifers. The purpose of these processes is sustainability and water conservation, rather than discharging the treated water to surface waters such as rivers and oceans. Cycled repeatedly through

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the planetary hydrosphere, all water on Earth is recycled water. But, typically when we hear the term “recycled water” or “reclaimed water”, it means wastewater that is sent from our home or business through a pipeline system to a treatment facility where is treated to a level consistent with its intended use. It is then routed directly to a recycled water system for uses such as irrigation or industrial cooling.

• The recycling and recharging is often done by using the treated wastewater for designated municipal sustainable gardening irrigation applications. In most locations, it is intended to only be used for nonpotable uses, such as irrigation, dust control, and fire suppression.



• Reclaimed water is highly engineered for safety and reliability so that the quality of reclaimed water is more predictable than many existing surface and groundwater sources. Reclaimed water is considered safe when appropriately used.



• Reclaimed water planned for use in recharging aquifers or augmenting surface water receives adequate and reliable treatment before mixing with naturally occurring water and undergoing natural restoration processes. Some of this water eventually becomes part of drinking water supplies.

7.9.5 Uses and benefits of reclaimed water

• The cost of reclaimed water exceeds that of potable water in many regions of the world, where a fresh water supply is plentiful. However, reclaimed water is usually sold to citizens at a cheaper rate to encourage its use. As fresh water supplies become limited from distribution costs, increased population demands, or climate change reducing sources, the cost ratios will also evolve.



• Using reclaimed water for non-potable uses, saves potable water for drinking, since less potable water will be used for non-potable uses.



• It sometimes contains higher levels of nutrients such as nitrogen, phosphorus and oxygen which may somewhat help fertilize garden and agricultural plants, when used for irrigation.



• The usage of water reclamation decreases the pollution sent to sensitive environments. It can also enhance wetlands, which benefits the wildlife depending on that ecosystem.

In most locations, reclaimed water is not directly mixed with potable (drinking) water for several reasons:

• Utilities providing reclaimed water for non-potable uses do not treat the water to drinking water standards.



• Varying amounts of pathogens, pharmaceutical chemicals (e.g., hormones from female hormonal contraception) and other trace chemicals are able to pass through the treatment and filtering process,

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potentially causing danger to humans. Modern technologies such as reverse osmosis may help to somewhat overcome this problem. An experiment by the University of New South Wales reportedly showed a reverse osmosis system removed ethinylestradiol and paracetamol from the wastewater, even at 1000 times the expected concentration.

• Drinking water standards were developed for natural ground water, and are not appropriate for identifying contaminants in reclaimed water. In addition to pathogens, and organic and endocrine disrupting chemicals, a large number of compounds may be present in reclaimed water. They cannot all be tested for, and there is a paucity of toxicity information on many of the compounds.

Because of this, state regulatory agencies do not allow reclaimed water to be used for drinking, bathing, or filling swimming pools. They also warn those, who use reclaimed water for irrigation, to place a sign on their property, warning people not to drink from the irrigation system, and to not use it directly on fruits or vegetables.

CHAPTER

8

Biological Methods of Pest Management

Biological methods of pest management in agriculture is a method of controlling pests (including insects, mites, weeds and plant diseases) that relies on predation, parasitism, herbivory, or other natural mechanisms. The use of biological control is a fundamental tactic for pest suppression within an effective Integrated Pest Management (IPM) program, and biological control refers to the use of natural enemies against a pest population to reduce the pest’s density and damage to a level lower than would occur in their absence. IPM is an ecologically-based pest management strategy that forms a part of the overall crop production system. Ideally, it incorporates all appropriate methods from many scientific disciplines into a systematic approach to minimize pest damage. IPM control tactics include a variety of approaches including cultural control, resistant plant varieties, chemical control, and biological control.

• Biological control as a management tool dates back over 1,000 years when ancient Chinese citrus growers used ants to control caterpillar larvae infesting their trees. It is one of the safest methods of control since it is not toxic, pathogenic or injurious to humans.



• Biological control has the advantage of being self-perpetuating once established and usually does not harm non-target organisms found in the environment. In addition, it is not polluting or as disruptive to the environment as chemical pesticides, nor does it leave residues on food, a concern to many people today.



• However, the use of biological control does require detailed knowledge of the pest’s biology and population dynamics, as well as the natural enemies associated with the pest and their impact.



• Control is usually not complete with this IPM method since a residual population of the pest is often necessary for the natural enemies to remain in the environment, so some non-economic population levels of pests must be acceptable or tolerated.

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• Biological control also fits well in combination with other IPM strategies. There are many factors (crop, pest complex, and environment) that can influence the success of beneficial organisms in reducing pest densities to manageable levels. In many situations, the biological control method will need to be utilized in concert with other tactics.



• Selecting the least disruptive management tactic is recommended by IPM and should help conserve natural enemies.



• The use of biological control to manage pests is divided into three types of approaches.

o Importation refers to the search for better natural enemies to introduce and permanently establish. The need for importation biological control occurs when a pest is accidentally introduced into an area and its natural enemies are left behind. An attempt is made to locate these enemies and introduce them to reestablish the control that often existed in the native range of the pest. This may be from another country or another region of the same country. o Augmentation is an attempt to reduce a pest’s population to noneconomic levels by temporarily increasing natural enemy numbers in an area through periodic releases. The natural enemies then identifies and attack the pest. In some cropping systems, technology has been developed to rear natural enemies artificially; so these releases can be made economically. A number of commercial companies have been created to produce a wide variety of natural enemies, both predators and parasites.

o The third approach, conservation, is concerned with protecting the natural enemies that are already present in an area. In conservation, an attempt is made to manipulate the environment or the farming practices to protect the natural enemies or provide needed resources (e.g. alternate prey or food for adults) for them to survive and build up populations to levels where they can manage the pest and prevent it from causing economic damage to crops. Naturallyoccurring or indigenous natural enemies prevent many plantfeeding insects from achieving pest status. Conservation of these natural enemies allows them to operate, near their full potential. Conserving natural enemies requires the use of farming practices that are less disruptive to natural enemy populations. Insecticide use destroys the target pest as well as many natural enemies that are present. Reduced or carefully-timed insecticide treatments lower the negative impact on beneficial organisms. Effective conservation of natural enemies depends on: understanding the agro ecosystem; use of selective pesticides; use of the least disruptive formulation of the chemical; application of the insecticide only when necessary and based on reasonable economic injury levels of the pest; and pesticide

Biological Methods of Pest Management

8.3

application at the time or place that is the least injurious to natural enemies. Natural enemies of insect pests fall basically into three types:

• Parasites (also called parasitoids) adults are free-living; the immature stage lives on or inside a host and kills the host before the host completes its development. Parasites lay one or more eggs on the outside of the host body or they insert the eggs inside their host. The immature parasite feeds on the host and requires only a single individual prey to complete its development. Free-living adults may feed on nectar from flowering plants or obtain nutrients by piercing the body of host insects and withdrawing fluids (host-feeding). Parasites attack a particular stage of the host, but all host stages are attacked by various parasites. Parasites are often small, easily overlooked and can be difficult to distinguish from other small non-parasitic flies and wasps. Parasites are not harmful to humans and tend to attack and parasitize one, or at most a few, closely related species of pest insects. Parasites are usually members of the order Hymenoptera and a few are members of the order Diptera. Parasites are often considered more effective natural enemies than predators because many have a narrower host range, require only one host to complete development, have an excellent ability to locate and kill their host and, can respond rapidly to increases in host populations.



• Predators include birds, fish, amphibians, reptiles, small mammals, and arthropods. Arthropods (insects, mites and spiders) are the most important predators in pest management and include lady beetles, ground beetles, syrphid flies, green lacewings, assassin bugs, predaceous bugs, minute pirate bugs, predatory mites, and spiders. Predators are usually larger than the prey which they capture and kill. They may use camouflage to “sit and wait” for prey or may be active hunters. Predators usually deposit their eggs near their prey, so the immature ones can immediately find their host and begin feeding. Immature stages are mobile, usually consume more than one prey during their development, are often generalist feeders (more than one species of host is attacked), and usually, both the adults and immature population feed on the prey insect.



• Diseases also occur among insects. Insect diseases are caused by fungi, viruses, bacteria, protozoans, and other microorganisms. Insectparasitic nematodes are also included in this group of natural enemies. Insect-parasitic nematodes are small worms that attack and kill insects that live in moist habitats. A few species are currently being sold commercially for insect control. Insect pathogens, including nematodes, are an important component in suppressing pest species. Some insect pathogens and nematodes are commercially available and can be

8.4

Environmental Biotechnology

manipulated to achieve biological control of specific pests. Both diseases and nematodes, like parasites, tend to be specific to certain species or groups of pests; they do not harm non-target organisms, such as beneficial insects, animals, humans, or plants. They can quickly spread through an insect population causing rapid mortality in a short period of time, and can be important in the natural control of pest populations. This phenomenon, called an epizootic, occurs when the insect pest population level is high or environmental conditions are especially suitable for the pathogen or disease-causing organism, enabling the disease organism to spread from insect to insect very quickly. In highvalue crops, the pest population usually cannot be allowed to reach a level where an epizootic can occur. However, epizootics can be an important natural control of pests of forests, rangeland, and certain types of field crops. Insect viral pathogens vary in how they attack and kill their host. Most insect viruses need to be ingested to successfully infect their host, though some can be transferred from the parent insect to the offspring through the egg. Symptoms usually occur within a few days after the virus is ingested. The infected insect will appear sluggish, feeding will stop, and the cuticle will have a pale discoloration and will often hang from its legs.

Caterpillar killed by viral infection

The infected insect will die one to two days after the symptoms appear. The decomposing cadaver will burst, liberating the viral particles into the environment. Important groups of viruses that attack insects are the—

• nuclear polyhedrosis viruses (NPV),



• cytoplasmic polyhedrosis viruses (CPV) and



• granulosis viruses (GV).

Biological Methods of Pest Management

8.5

Viruses usually attack the caterpillar stage, such as the Helicoverpa NPV that invades the corn earworm larva or the codling moth GV that infects the codling moth larva. Baculoviruses is the most popular choice for microbial control as they are distinct from any type of virus recorded from vertebrates. They have been used regularly for pest control since the 1950s, particularly in forestry, where they have been highly effective at controlling sawflies. Baculoviruses can infect many species, mostly caterpillars and sawflies, but also some species of beetle and flies. Baculoviruses infect their hosts through ingestion. Virus particles invade the cells of the gut before colonizing the rest of the body. Infection reduces mobility and feeding and insects are killed in five to eight days. Mass production of baculoviruses can be done only in insects, but this is economically viable for larger hosts such as caterpillars, and formulation and application are straightforward. The bacteria most important in insect pest management are in the genus Bacillus. Species in this genus form spores that are toxic to the insect when ingested. Symptoms of infected insects include, a loss of appetite, sluggishness, discharge from the mouth and anus, discoloration and liquefaction and putrefaction of the body tissues.

Caterpillar killed by a bacterial infection

Bacillus thuringiensis (commonly called Bt) is the most widely-used bacterium for insect pest control. Different strains of Bt are specific against caterpillars, mosquito larvae and some beetles and their larvae. Bacillus popillae and B. lentimorbus cause “milky disease” of white grubs. “Milky disease” refers to the white discoloration of the insect blood. The spores of B. popillae and B. lentimorbus survive in the soil and are ingested by the grubs as they feed on

8.6

Environmental Biotechnology

roots of grasses. Bacillus thuringiensis, B. popillae, and B. lentimorbus have been formulated into microbial insecticides by several companies for application to crops in an augmentative manner.

Spores and bipyramidal crystals of Bacillus thuringiensis













• The major advantage of the Bacillus thuringiensis (Bt) toxin is that it is harmful to only a few species of insects, while it is essentially harmless to other animals and humans. These biological pesticides also degrade rapidly in the environment. Thus, the use of such biological pesticides appears to be a significantly more environmentally safe solution to pest control than the classical (synthetic chemical) pesticides. • Features of Bt biopesticides limit their use in insect control. In contrast to contact insecticides, Bt insecticides must be ingested by the target insect. The timing of Bt sprays is critical to attaining economic levels of insect control. Usually Bt is applied when early instar larvae are present, as older larvae are more tolerant. Bt sprays persist only a few days on the leaf surface. • The chemistry of the leaf surface, proteinases and sunlight contribute to the degradation of CRY proteins. It is rare for a Bt insecticide to have greater efficacy than the best available chemical control. Hence, Bt adoption suffers at the hand of more efficacious chemical insecticides. • Bt biopesticides have inherent advantages in certain pest control applications. They are used as a resistance management tools in insect control. Due to their distinct mode-of-action, they are alternated or combined with chemical pesticides. • Bt is especially suited for specialty or ‘high value crops.’ The tightening of registration procedures for new chemical pesticides has led many of the larger crop protection companies to take the decision not to register products for use on specialty crops. • Increased usage of Bt biopesticides will occur as organic markets expand and consumer demand for eco-friendly pest control alternatives in home gardens and treatments for high-value alternative crops.

Biological Methods of Pest Management

8.7

Most recently, genes that produce Bt toxins have been genetically engineered into crop plants (corn, cotton, tobacco and potato) for season-long protection against larval pests and some adult insects. Larval and adult pests ingest the toxin after they have fed on the foliage and subsequently die. A beneficial soil bacterium, Saccharopolyspora spinosa, produces a natural metabolite, Spinosad, when cultured under aerobic fermentation conditions. Spinosad has been formulated as a microbial insecticide. Insects become poisoned with Spinosad when they ingest treated foliage or come into contact with the microbial sprays of the metabolite. Sickened insects stop feeding, become limp and are unable to move, and may appear to have weak tremors. Spinosad is effective against a wide spectrum of insect pests, including armyworms, European corn borer, diamondback moth, leaf miners, and Colorado potato beetle. Examples of Bacterial Insecticides: Pathogen: Bacillus thuringiensis var. kurstaki (Bt) Host range: Caterpillars (larvae of moths and butterflies) Uses and comments: Effective for foliage-feeding caterpillars (and Indian meal moth in stored grain). Deactivated rapidly in sunlight; apply in the evening or on overcast days and direct some spray to lower surfaces of leaves. Does not cycle extensively in the environment. Available as liquid concentrates, wettable powders, and ready-to-use dusts and granules. Active only if ingested. Pathogen: Bacillus thuringiensis var, israelensis (Bti) Host range: Larvae of Aedes and Psorophora mosquitoes, black flies, and fungus gnats Uses and comments: Effective against larvae only. Active only if ingested. Culex and Anopheles mosquitoes are not controlled at normal application rates. Activity is reduced in highly turbid or polluted water. It does not cycle extensively in the environment. Applications generally made over wide areas by mosquito and black-fly abatement districts. Not all Bti products are labeled for use against fungus gnats. Pathogen: Bacillus thuringiensis var. san diego Host range: Larvae of Colorado potato beetle, elm leaf beetle adults Uses and comments: Effective against Colorado potato beetle larvae and the elm leaf beetle. Like other bacteria, it must be ingested. It is subject to breakdown in ultraviolet light and does not cycle extensively in the environment. Pathogen: Bacillus thuringiensis var. tenebrionis Host range: Larvae of Colorado potato beetle

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Environmental Biotechnology

Uses and comments: Very similar to Bacillus thuringiensis var. san diego. Foil contains a bacterial conjugate that produces toxins that act against these same beetles and caterpillars. Pathogen: Bacillus thuringiensis var. aizawa Host range : Wax moth Caterpillars Uses and comments : Used only for the control of wax moth infestations in honeybee hives. Pathogen: Bacillus popilliae and Bacillus lentimorbus Host range : Larvae (grubs) of Japanese beetle Uses and comments : The main Illinois lawn grub (the annual white grub, Cyclocephala sp.) is not susceptible to milky spore disease. The disease is very effective against Japanese beetle grubs and cycles effectively for years in the soil. Pathogen: Bacillus sphaericus Host range : Larvae of Culex, Psorophora, and Culiseta mosquitoes, larvae of some Aedes spp. Uses and comments : Active only if ingested. Under development for use against Culex, Psorophora, and Culiseta species; also effective against Aedes vexans. Remains effective in stagnant or turbid water. Commercial formulations will not cycle to infect subsequent generations. Insect pathogenic fungi produces spores that germinate when they come in contact with the insect cuticle during favorable conditions. Germinating spores penetrate the insect cuticle and invade the body cavity. Hyphae rapidly grow, filling the body cavity with a fungal mass, killing the insect. The fungus also may produce a toxin. Hyphae penetrate outward through the softer parts of the insect and under favorable moisture conditions, produce spores that ripen and are released into the environment to complete the life cycle. • Insects that are attacked by fungi often retain their shape, but usually become hardened or “mummy-like” and appear “fuzzy” from the fungal growth. There are many genera of fungi that attack insects. • The most important ones are Metarhizium, Beauveria, Entomophthora and Zoopthora. Metarhizium anisopliae and B. bassiana attack a wide range of insects, such as grasshoppers, true bugs, aphids, caterpillars, and beetles. • Entomophthora muscae attacks many types of adult flies including the seed corn maggot and hover fly. Zoophthora radicans attacks the potato leafhopper and many aphids, and Z. phytonomi infects the alfalfa weevil. Fungi used as insecticides include the following: • Beauveria bassiana: This common soil fungus has a broad host range that includes many beetles. It infects both larvae and adults of many

Biological Methods of Pest Management

8.9

species. Understanding the interactions between Beauveria bassiana and other soil microorganisms may be the key to successful use of this fungus.

• Nomuraea rileyi: In soybeans, naturally occurring epidemic infections of Nomuraea rileyi cause dramatic reductions in populations of foliagefeeding caterpillars. Research, directed at predicting disease outbreaks caused by this fungus, may help in determining the need for application of insecticides.



• Verticillium lecanii: This fungus has been used in greenhouses to control aphids and whiteflies.



• Lagenidium giganteum: This aquatic fungus is highly infectious to larvae of several mosquito genera. It cycles effectively in the aquatic environment (spores produced in infected larvae persist and infect larvae of subsequent generations), even when mosquito density is low. Its effectiveness is limited by high temperatures.



• Hirsutella thompsonii: Hirsutella thompsonii is a pathogen of the citrus rust mite.

8.1

ADVANTAGES OF MICROBIAL INSECTICIDES

Individual products differ in important ways, but the following list of beneficial characteristics applies to most microbial insecticides.

• The organisms used in microbial insecticides are essentially non-toxic and non-pathogenic to wildlife, humans, and other organisms not closely related to the target pest.



• The toxic action of most microbial insecticides is specific to a single group or species of insects and this specificity means that most microbial insecticides do not directly affect beneficial insects (including predators or parasites of pests) in treated areas. If necessary, most microbial insecticides can be used in conjunction with synthetic chemical insecticides, because in most cases, the microbial product is not deactivated or damaged by residues of conventional insecticides.



• Because their residues present no hazards to humans or other animals, microbial insecticides can be applied even when a crop is almost ready for harvest.



• In some cases, the pathogenic microorganisms can become established in a pest population or its habitat and provide control during subsequent pest generations or seasons.

Microorganisms that are pathogenic to insects provide a wealth of biological material that can be exploited by humans to control insect pests. Innovative applications of a few such entomopathogens are found throughout the world, but widespread commercial production of microbial insecticides awaits further studies of the biology, ecology, and pathogenicity of the agents.

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Environmental Biotechnology

Genetic engineering techniques may be used to increase the virulence of these microorganisms, as well as, to make them more tolerant of physical and chemical conditions and perhaps, to broaden their host ranges. The use of microbial insecticides could decrease our dependence on chemical pesticides.

8.2

DISADVANTAGES OF MICROBIAL INSECTICIDES

The limitations or disadvantages listed below do not prevent the successful use of microbial insecticides.

• Understanding how these limitations affect specific microorganisms, will help users to choose effective products and take necessary steps to achieve successful results.



• Because a single microbial insecticide may be toxic to only a specific species or group of insects, each application may control only a portion of the pests present in a field, garden, or lawn. If other types of pests are present in the treated area, they will survive and may continue to cause damage. Conventional insecticides are subject to similar limitations because, they too, are not equally effective against all pests. Nonetheless, the negative aspect of selectivity is often more noticeable for microbials.



• Heat, desiccation (drying out), or exposure to ultraviolet radiation reduces the effectiveness of several types of microbial insecticides. Consequently, proper timing and application procedures are especially important for some products.



• Special formulation and storage procedures are necessary for some microbial pesticides. Although these procedures may complicate the production and distribution of certain products, storage requirements do not seriously limit the handling of microbial insecticides that are widely available. (Store all pesticides, including microbial insecticides, according to label directions.)



• Because several microbial insecticides are pest-specific, the potential market for these products may be limited. Their development, registration, and production costs cannot be spread over a wide range of pest-control sales. Consequently, some products are not widely available or are relatively expensive (several insect viruses, for example).

8.3

INSECT GROWTH REGULATORS

Several features of Insect Growth Regulators (IGRs) make them attractive as alternatives to broad-spectrum insecticides. Because they are more selective, they are less harmful to the environment and more compatible with pest management systems that include biological controls. Compared with conventional pesticides, insect growth regulators are:

Biological Methods of Pest Management



• More selective



• Less harmful to the environment



• More compatible with biological controls



• Less likely to be lost because of resistance

8.11

Insects have demonstrated a propensity to develop resistance to insecticides. Broad-spectrum insecticides that are used routinely will eventually be lost because of resistance. Intelligent use of IGRs should reduce the likelihood of resistance-developing. IGRs show good potential on pears because their selectivity preserves the natural enemies that can help control pear psylla. Because of its ability to rapidly develop resistance to insecticides, it is important that psylla be controlled by an integrated system, incorporating several control factor. The selectivity of IGRs is due to the different way they act on insects, compared with most conventional insecticides. Virtually, all chemicals used to control insects fall into one of three categories: neurotoxins, growth regulators and behavior modifiers. Neurotoxins: Most chemicals used to control insects are neurotoxins which interfere with normal nerve function. Organophosphate insecticides were derived from nerve gases that were first exploited for military purposes. Other insecticides were discovered by testing chemicals to find those that killed pests quickly. About the only thing that kills quickly is a neurotoxin; so chemicals that acted on neurotransmissions were sought and developed as insecticides. In the early discovery and development of insecticides, efforts were focused on chemistry rather than biology. Because all animals share, basically the same neurochemical systems, neurotoxins are toxic to all animals. Insect growth regulators: The origin of IGRs was entirely different. Their discovery was based on knowledge of how insects grow, develop, function and behave. They have been discovered in two ways.

• One way was to expose an insect to IGRs and observe abnormalities in how it develops, functions or behaves. Chemicals that produce desired effects were developed.



• Another was to find out what processes in the insects’ development involve hormones and to use those hormones as models to synthesize chemical analogs that will interfere with normal insect growth and development. Because IGRs act on systems unique to insects, or shared with close relatives, they are less likely to affect other organisms.

Behavior modifiers: Behavior-affecting chemicals, such as pheromones, are discovered in the same way as IGRs but tend to be even more specific. Pheromones aid the sexes of a single species to find each other so that effort is not wasted chasing mates of a different species. How insect growth regulators work? : Insects wear their skeletons on the outside. The skeletons are called exoskeletons. As the insect grows, a new exoskeleton must be formed inside the old exoskeleton and the old one

8.12

Environmental Biotechnology

shed. The new one then swells to a larger size and hardens. The process is called molting. The changes from larval to adult form, a process called metamorphosis, also take place during molting. Hormones control the phases of molting by acting on the epidermis, which is part of the exoskeleton. Chitin synthesis inhibitors: These prevent the formation of chitin, a carbohydrate that is an important structural component of the insect’s exoskeleton. When treated with one of these compounds, the insect grows normally until, the time to molt. When the insect molts, the exoskeleton is not properly formed and it dies. Death may be quick, but in some insects it may take several days. As well as disrupting molting, chitin synthesis inhibitors can kill eggs by disrupting the normal development of the embryo. Role of Juvenile hormones and its analogues for pest management: Juvenile hormone, also called Neotenin, is a hormone in insects, secreted by endocrine glands near the brain (corpora allata), that controls the retention of juvenile characters in larval stages. The hormone affects the process of molting, the periodic shedding of the outer skeleton during development, and in adults, it is necessary for normal egg production in females.

Juvenile hormone is produced in the corpora allata of insects

• Juvenile Hormones (JHs) are a group of acyclic sesquiterpenoids that regulate many aspects of insect physiology. JHs regulate development, reproduction, diapause, and polyphenisms. • Juvenile Hormone Analogs (JHAs) represent a class of insecticides that were designed specifically to disrupt endocrine-regulated processes relatively unique to insects. Synthetic analogues of the juvenile hormone are used as an insecticide, preventing the larvae from developing into adult insects. Since the early 1970s, numerous analogs of JH have been tested for insecticidal activity JH itself is expensive to synthesize and is unstable in light. At high levels of JH, larva can still molt, but the result will only be a bigger larva, not an adult. Thus, the insect’s reproductive cycle is broken. • One JH analogue, methoprene, is approved by the WHO for use in drinking water cisterns to control mosquito larvae. Methoprene is a growth regulator and prevents the immature insect from developing

Biological Methods of Pest Management



8.13

normally. Juvenile hormone has several functions within an insect. If the hormone is present when the larva molts the insect will remain a larva. If the hormone is absent, the larva will become a pupa and begin to develop into an adult insect. Since methoprene mimics juvenile hormone, the larva is prevented from developing into an adult. • Pyriproxyfen, 4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether, is a juvenile hormone analog with relatively low mammalian toxicity that was first registered in Japan in 1991 for controlling public health pests. It has been used for controlling a variety of insect pests including mosquitoes and other flies, whiteflies, scale insects and aphids, cockroaches, lepidopteron, ants, fleas, locusts and grasshoppers, and thrips.

Chemical structures of the juvenile hormones, terpenoidal (methoprene), and nonterpenoidal (fenoxycarb, phyproxyfen and diofenolan) juvenile harmone analogs

Juvenile hormone analogs and mimics: When applied to an insect, these abnormal sources of juvenilizing agent can have striking consequences. • For example, if the normal course of events calls for a molt to the pupal stage, an abnormally high level of juvenilizing agent will produce another larval stage or produce larval-pupal intermediates. Juvenoid insect growth regulators (IGRs) can also act on eggs. They can cause sterilization, disrupt behavior and, disrupt diapause—the process that triggers dormancy before the onset of winter. In theory, all insect systems influenced by juvenile hormone are potential targets for a juvenoid IGR.

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Environmental Biotechnology

The early juvenoid IGRs were true analogs of juvenile hormone and were unstable when exposed to ultraviolet light. This seriously limited their use in plant protection. Another group of juvenoid IGRs, called juvenile mimics, was discovered. Entomologist found that extracts of many plant tissues have juvenilizing effects, but they have different chemical structures from juvenile hormones and are much more stable. They have been used as models to synthesize some highly effective and stable juvenile hormone mimics which have potential to control tree fruit pests. Anti-juvenile hormone agents: Anti-juvenile hormone agents cancel the effect of juvenile hormone by blocking juvenile hormone production.

• For example, an early instar, treated with an anti-juvenile hormone agent, molts prematurely into a non-functional adult. A disadvantage of these chemicals is that, they are so selective that, they may not be economic for a manufacturer to develop.

8.4  USE OF PHEROMONES FOR PEST MANAGEMENT With increasing public concern about the use of toxic pesticides to control insects and other pestiferous organisms, resource managers are turning toward other techniques of integrated pest management. Some of these techniques are common-sense approaches, such as completing sanitation or clean-up activities before the season when the damaging stages of an insect pest are present. Other tools are more “hi-tech”, such as the use of odors called semiochemicals, and in particular, pheromones, to manipulate the behavior of insect pests. With these non-toxic and biodegradable chemicals, insects can be lured into traps or foiled into wasting energy that they normally need for locating food and mates. Semiochemicals are chemical signals that are produced by a plant or animal and are detected by a second plant or animal and cause a response in the second organism. Many species depend on these chemical signals for survival. Pheromones: Pheromones are a class of semiochemicals that insects and other animals release to communicate with other individuals of the same species. The key to all of these behavioral chemicals is that they leave the body of the first organism, pass through the air (or water) and reach the second organism, where they are detected by the receiver.

• In insects, these pheromones are detected by the antennae on the head. The signals can be effective in attracting faraway mates, and in some cases, can be very persistent, remaining in place and active for days.



• Long-lasting pheromones allow marking of territorial boundaries or food sources. Other signals are very short-lived, and are intended to provide an immediate message, such as a short-term warning of danger or a brief period of reproductive readiness.

Biological Methods of Pest Management









8.15

• Pheromones can be of many different chemical types, to serve different functions. As such, pheromones can range from small hydrophobic molecules to water-soluble peptides. • Over the last 40 years, scientists have identified pheromones from over 1,500 different species of insects. Pheromones have also been isolated from many higher animals such mammals and reptiles. Human pheromones remain elusive. • Scientists have found certain chemical effects associated with the human reproductive cycle, but have not identified any powerful attractants for humans, so far. • With insects, though, pheromones have found wide application in the fields of agriculture, forestry, and urban pest management, and there are companies that specialize in the discovery, manufacturing, and sales of pheromone-related products.

8.5  PHEROMONES: INSECT PEST MANAGEMENT There are three main uses of pheromones in the integrated pest management of insects.

• The most important application is in monitoring a population of insects to determine if they are present or absent in an area or to determine if enough insects are present to warrant a costly treatment. This monitoring function is the keystone of integrated pest management. Monitoring is used extensively in urban pest control of cockroaches, in the management of stored grain pests in warehouses or distribution centers, and to track the nationwide spread of certain major pests such as the gypsy moth, Medfly, and the Japanese beetle. With major increases in worldwide trade, exotic pests are being brought into ports of entry in cargo containers and packaging materials (ship dunnage). Sometimes, containers from ships are transferred uninspected to semitrailers and trucked far inland. When the containers are opened and packaging materials are removed, the exotic insect pests are able to disperse without the usual level of scrutiny provided at ports of entry. Pheromone traps are currently in use to monitor the movement of such exotic insect pests into most major North American ports of entry.



• A second major use of pheromones is to mass trap insects to remove large numbers of insects from the breeding and feeding population. Massive reductions in the population density of pest insects, ultimately, help to protect resources such as food or fiber for human use. Mass trapping has been explored with pine bark beetles and has resulted in millions of insects attracted specifically into traps and away from trees. Relatives of bark beetles, called ambrosia beetles, have been mass-trapped from log sorting and timber processing areas throughout British Columbia. These trapping operations have reduced damage to

8.16

Environmental Biotechnology

the wood in raw logs and newly cut boards. Mass trapping has also been used successfully against the codling moth, a serious pest of apples and pears. Another common example of mass trapping involves yellow jackets, which can become bothersome at the end of the summer season. However, mass trapping of yellow jackets in colorful yellowgreen traps is carried out with a food attractant, rather than pheromone bait.

• A third major application of pheromones is in the disruption of mating in populations of insects. This has been most effectively used with agriculturally-important moth pests. In this scenario, synthetic pheromone is dispersed into crops and the false odor plumes attract males away from females that are waiting to mate. This causes a reduction of mating, and thus, reduces the population density of the pests. In some cases, the effect has been so great that the pests have been locally eradicated.

In summary, pheromones are species-specific chemicals that affect insect behavior, but are not toxic to insects. They are active (e.g. attractive) in extremely low doses (one millionth of an ounce) and are used to bait traps or confuse a mating population of insects. Pheromones can play an important role in integrated pest management for structural, landscape, agricultural, or forest pest problems.

8.6  BIOLOGICAL CONTROL OF WEEDS Biological control is a long-term solution which is most effective as part of an integrated weed management approach. Plants that have become weeds in Australia are rarely invasive and troublesome in their home country. This is often because populations in the home country are regulated by a variety of natural enemies such as insects and pathogens (disease-causing organisms like fungi and bacteria) that attack the seeds, leaves, stems and roots of the plant. If plants are introduced to a new country without these natural enemies, their populations may grow unchecked to the point where they become so prevalent that they are regarded as weeds. The biological control approach makes use of the invasive plant’s naturally-occurring enemies, to help reduce  the invasive plant’s impact on agriculture and the environment. It simply aims to reunite weeds with their natural enemies and achieve sustainable weed control. These natural enemies of weeds are often referred to as biological control agents. It is critical that the biological control agents do not become pests themselves. Considerable host-specificity testing is done prior to the release of biological control agents to ensure they will not pose a threat to non-target species such as native and agricultural plants. Not all weeds are suitable for biological control. A biological control agent is generally only used when the cost of conventional control methods such as herbicides, mechanical control or fire is

Biological Methods of Pest Management

8.17

so great, both in dollar terms and impact on the environment, that there is little option than to pursue the biological control avenue.

8.6.1  The process A weed becomes a problem in the introduced range because its population density fluctuates around an equilibrium that is above a threshold at which the weed begins to affect the economic or ecological sustainability of the ecosystem.

The process of biological control of weeds

Best case scenario of the course of events in classical biological control programs targeting weeds invading habitats such as rangeland, pasture, or natural ecosystem.  Following their introduction and establishment, populations of biological control agents build up to very high levels due to the abundance of their host plant. Eventually, their attack on the plant causes a decline in the weed population. This, in turn, leads to a decline in the numbers of biological control agents until an equilibrium is reached between the amount of damage caused by an agent and regeneration by the weed. In a successful biological control program, this new equilibrium is below the damage threshold that the ecosystem can tolerate. The advantages of biological control of weeds are: • It is inexpensive. • It poses little threat to non-target organisms. • Once established, biological control agents are self-perpetuating and can spread on their own. • Little additional effort is required once a biological control organism is established, while other control methods require action or inputs, periodically.

• The environmental impact is generally low.

8.18

Environmental Biotechnology

The main drawbacks to biological control of weeds are: • There is always some risk and concern with introducing an exotic organism into the environment. The main concern is a host shift of a biological control agent that results in the agent feeding on a desirable plant. • Biological control agents are not available for all target pests. Some of the target weeds are closely related to plants that are desirable, so the risk of introducing an agent is too great. • Research time and money is needed to locate biological agents and screen them for host range before the agents are released. It generally takes years of research and testing, before agents are released. • Biological control takes place slowly, in most cases. Localized weed problems may not be eliminated quickly enough to satisfy public opinion. • Biological control relies on populations of the weed and the agent to maintain the system. Thus, weeds are not completely eliminated. Also, there is often highs and lows in population levels of the weed and the agent both in time and space. Thus, there are some situations where biological control does not seem to work well. If there are advantages and disadvantages, how is the decision to use biological control determined? The decision is made on a case-by-case basis. The potential impact of the weed, the alternative control measures available, the risk to the environment, and the consequences of doing nothing are all considered. The scientific information and the social values, all influence the decision. Once the organism is released, there may be little good alternative to reverse the decision, so this decision is taken seriously. This is further complicated by the fact that social values change through time and, that the scientific information available will also change as new information becomes available. There are a number of regulations in place that impact the release of organisms. Biocontrol and the environment: • Biological control is attractive because it has negligible environmental impact. Most biocontrol organisms are very selective to their target pests thus posing little danger of damage to non-target species. • They do not cause contamination of groundwater or leave residues behind. • Biocontrol also has its limitations. It may not consistently provide a high level of pest control because environmental conditions may not always be suitable for the growth and reproduction of the biocontrol organism. • Therefore, biological control is well adapted for use within an integrated pest management program where other cultural practices will complement and boost the level of control.

Biological Methods of Pest Management



8.19

• Since biological control uses living organisms, the user must be educated on how to handle the product and under what circumstances it will work best.

8.7  CHROMOSOMAL MANIPULATION Methods to manage invasive species range from ignoring them and hoping, they will go away through to a variety of options for physical removal, biocides and biological control. For well-established and widely distributed pests, the only realistic options currently available are augmentative and classical biological control, and sterile male release programs, both of which have significant constraints on their application. As a result, most invasive pests remain uncontrolled except at small scales and for short periods. In the 1960s, entomologists speculated that genetic techniques could be a powerful means of controlling pest populations, based on the observation that meiotic drive (a genetic sex ratio distorter) had apparently driven some insect populations to extinction. Practical development of such techniques lied fallow, however, until recent developments in recombinant genetics stimulated renewed interest in the field. Currently, at least three recombinant methods for pest control are being tested in the laboratory. They are:

• repressible male sterility,



• virally vectored immuno contraception, and



• female-biased sex ratio distortion.

Another widely publicized study has speculated that the escape of even one carrier of a “Trojan gene” (a construct, that pleiotropically enhances mating advantage, while otherwise, reducing fitness) could cause species extinction, an issue of considerable concern with regards to accidental escapement of genetically modified (GM) organisms, but also, one, that offers options for pest control, if properly managed. A number of recent studies have modeled the potential for pest control of methods that have been proposed, incorporating varying degrees of ecological reality. All conclude that pest control using genetic methods is feasible, at least under the conditions specified in the models. Three broadly different approaches for using recombinant genetics to control pests have been investigated:

• genetically engineered viruses that, when incorporated into a bait, act as a species-specific toxin or sterilizer;



• self-disseminating engineered viral diseases; and



• Non-disseminating (i.e., sexually transmitted) “autocidal” genes.

In the last, include as a special case, chromosomal modifications, designed to achieve the same outcomes, as the recombinant approaches Release of Insects carrying a Dominant Lethal: A method using recombinant DNA technology to create genetically modified insects called

8.20

Environmental Biotechnology

RIDL (Release of Insects carrying a Dominant Lethal) is under development by a company called Oxford Insect Technologies (Oxitec), UK. • The method works by introducing a repressible “Dominant Lethal” gene into the insects. This gene kills the insects but it can be repressed by an external additive, which allows the insects to be reared in manufacturing facilities. • This external additive is commonly administered orally, and so can be an additive to the insect food. The insects can also be given genetic markers, such as DsREDfluorescence, that make monitoring the progress of eradication easier, preferably under the field conditions. • There are potentially several types of RIDL, but the more advanced forms have a female-specific dominant lethal gene. This avoids the need for a separate sex separation step, as the repressor can be withdrawn from the final stage of rearing, leaving only males. • These males are then released in large numbers into the affected region. The released males are not sterile, but any female offspring their mates produce will have the dominant lethal gene expressed, and so will die. The number of females in the wild population will therefore decline, causing the overall population to decline. • Using RIDL means that the males will not have to be sterilized by radiation before release, making the males healthier when they need to compete with the wild males for mates. • Progress towards applying this technique to mosquitos has been made by researchers at Imperial College London who created the world’s first transgenic malaria mosquito. • A similar technique is the daughterless carp, a genetically modified organism produced in Australia by the CSIRO in the hope of eradicating the introduced carp from the Murray River system. • As stated earlier, biotechnological approaches based on genetically modified organism (transgenic organisms) are still under development. However, since no legal framework exists to authorize the release of such organisms in the nature, sterilization by irradiation remains the most used technique.

8.8  STERILE MALE TECHNOLOGY Screwworm was the first pest successfully eliminated from an area through the sterile male technique by the use of an area-wide approach. The sterile insect technique is a method of ‘biological control, whereby overwhelming numbers of sterile insects are released’. The released insects are normally male, as it is the female that causes the damage, usually by laying eggs in the crop, or, in the case of mosquitoes, taking a blood-meal from humans. The sterile males compete with the wild males for female insects. If a female mates with a sterile male then it will have no offspring, thus reducing the next generation’s

Biological Methods of Pest Management

8.21

population. Repeated release of insects can eventually wipe out a population, though it is often more useful to consider controlling the population rather than eradicating it. • The technique has successfully been used to eradicate the Screw-worm fly (Cochliomyia hominivorax) in areas of North America. There have also been many successes in controlling species of fruit flies, most particularly, the Medfly (Ceratitis capitata), and the Mexican fruit fly (Anastrepha ludens). • Insects are mostly sterilized with radiation, which might weaken the newly sterilized insects, if doses are not correctly applied, making them less able to compete with wild males. However, other sterilization techniques are under development which would not affect the insects’ ability to compete for a mate. • The technique was pioneered in the 1950s by American entomologists Dr. Raymond C. Bushland and Dr. Edward F. Knipling. For their achievement, they jointly received the 1992 World Food Prize. Drawbacks • As with insecticide treatment, repeated treatment is sometimes required to suppress the population before the use of sterile insects. • Sex separation could be difficult for some species, though this can be easily performed on Medfly and screwworm, for example. • Radiation treatment in some cases affects the health of males, so sterilized insects in such cases are at a disadvantage when competing for females. • The technique is species-specific. For instance, there are 22 species of Tsetse fly in Africa, and the technique must be implemented separately for each. • Standard operating procedures of mass rearing and irradiation do not leave room for mistakes. Since the Fifties, when SIT was first used as a means for pest control, several failures have occurred in different places around the world where non-sterilized artificially produced insects were released before the problem was spotted. • Application to large areas should be long-lasting, otherwise, migration of wild insects from outside the control area could repopulate. • The major drawback to this technique is that the cost of producing such a large number of sterile insects is often prohibitive in poorer countries.

8.9  ENVIRONMENTAL EPIDEMIOLOGICAL SURVEYS AS INDICES OF HEALTH HAZARDS FOR ENVIRONMENTAL POLLUTION Epidemiology is the study of the distribution and determinants of disease frequency in human populations and the application of this study to control health problems.

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Environmental Biotechnology



• The term study includes both, surveillance, whose purpose is to monitor aspects of disease occurrence and, spread—that are related to effective control, and epidemiologic research, whose goal is to collect valid and precise information about the causes, preventions, and treatments for disease.



• The term ‘disease’ refers to a broad array of health-related states and events including diseases, injuries, disabilities, and death.

Epidemiologic research encompasses several types of study designs, including experimental studies and observational studies such as cohort and case-control studies. Each type of epidemiologic study design simply represents a different way of harvesting information. The selection of one design over another depends on the particular research question, concerns about validity and efficiency, and practical and ethical considerations. For example, experimental studies, also known as trials, investigate the role of some factor or agent in the prevention or treatment of a disease. In this type of study, the investigator assigns individuals to two or more groups that either receive or do not receive the preventive or therapeutic agent. Because experimental studies closely resemble controlled laboratory investigations, they are thought to produce the most scientifically rigorous data of all the designs. However, because experimental studies are often infeasible because of difficulties in enrolling participants, high costs, and thorny ethical issues, most epidemiologic research is conducted using observational studies. Observational studies are considered “natural” experiments because the investigator lets nature take its course. Observational studies take advantage of the fact that people are exposed to noxious and/or healthy substances through their personal habits, occupation, place of residence, and so on. The studies provide information on exposures that occur in natural settings, and they are not limited to preventions and treatments. Furthermore, they do not suffer from the ethical and feasibility issues of experimental studies. For example, although it is unethical to conduct an experimental study of the impact of drinking alcohol on the developing fetus by assigning newly-pregnant women to either a drinking or non-drinking group, it is perfectly ethical to conduct an observational study by comparing women who choose to drink during pregnancy with those, who decide not to do so. The two principal types of observational studies are cohort and casecontrol studies. A classical cohort study examines one or more health effects of exposure to a single agent. Subjects are defined according to their exposure status and followed over time to determine the incidence of health outcomes. In contrast, a classical case-control study examines a single disease in relation to exposure to one or more agents. Cases who have the disease of interest and controls, who are a sample of the population that produced the cases,

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Biological Methods of Pest Management

are defined and enrolled. The purpose of the control group is to provide information on the exposure distribution in the population that gave rise to the cases. Investigators obtain and compare exposure histories of cases as well as controls. Additional observational study designs include cross-sectional studies and ecologic studies. A cross-sectional study examines the relationship between a disease and an exposure among individuals in a defined population at a point in time. Thus, it takes a snapshot of a population and measures the exposure prevalence in relation to the disease prevalence. An ecologic study evaluates an association using the population rather than the individual as the unit of analysis. The rates of disease are examined in relation to factors described on the population level. Both the cross-sectional and ecologic designs have important limitations that make them less scientifically rigorous than cohort and case-control studies. An overview of these study designs is provided in the following table: Main Types of Epidemiologic Studies Type of study

Characteristics

Experimental

Studies preventions and treatments for diseases; investigator actively manipulates which groups receive the agent under study.

Observational

Studies causes, preventions, and treatments for diseases: investigator passively observes, as nature takes its course.

Cohort

Typically examines multiple health effects of an exposure: subjects are defined according to their exposure levels and followed for disease occurrence.

Case-control

Typically examines multiple exposures in relation to a disease; subjects are defined as cases and controls, and exposure histories and compared.

Cross-sectional

Examines relationship between exposure and disease prevalence in a defined population at a single point in time.

Ecological

Examines relationship between exposure and disease with population-level rather than individual-level data.

The goal of all these studies is to determine the relationship between an exposure and a disease with validity and precision using a minimum of resources. Validity is defined as the lack of bias and confounding. Bias is an error committed by the investigator in the design or conduct of a study that leads to a false association between the exposure and disease. Confounding, on the other hand, is not the fault of the investigator, but rather, reflects the

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Environmental Biotechnology

fact that epidemiologic research is conducted among free-living humans with unevenly distributed characteristics. As a result, epidemiological studies that try to determine the relationship between an exposure and disease are susceptible to the disturbing influences of extraneous factors, known as confounders. Precision is the lack of random error, which leads to a false association between the exposure and disease just by “chance,” an uncontrollable force that seems to have no assignable cause. Several factors help epidemiologists determine the most appropriate study design for evaluating a particular association, including the hypothesis being tested, state of knowledge, the frequency of the exposure and the disease, and the expected strength of the association between the two.

8.10 TOXICITY Toxicity is the degree to which a substance can damage a living or non-living organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell (cytotoxicity) or an organ (organ toxicity), such as the liver (hepatotoxicity). By extension, the word may be metaphorically used to describe toxic effects on larger and more complex groups, such as the family unit or society at large.

Symbol of Toxicity

A central concept of toxicology is that effects are dose-dependent; even water can lead to water intoxication when taken in too many doses, whereas, for even a very toxic substance such as snake venom, there is a dose below which there is no detectable toxic effect. Toxicity is species-specific, lending cross-species analysis, problematic. Newer paradigms and metrics are evolving to bypass animal-testing, while maintaining the concept of toxicity endpoints. • Toxicology: Toxicology is the study of the nature, effects, detection, and mitigation of poisons and the treatment or prevention of poisoning. • Toxin: A toxin is a toxic substance.

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8.25

8.10.1  Types of toxicity There are generally three types of toxic entities; chemical, biological, and physical:

• Chemical toxicants include inorganic substances such as lead, mercury, asbestos, hydrofluoric acid, and chlorine gas, organic compounds such as methyl alcohol, most medications, and poisons from living things.



• Biological toxicants include bacteria and viruses that can induce disease in living organisms. Biological toxicity can be difficult to measure because the “threshold dose” may be a single organism. Theoretically, one virus, bacterium or worm can reproduce to cause a serious infection. However, in a host with an intact immune system the inherent toxicity of the organism is balanced by the host’s ability to fight back; the effective toxicity is then a combination of both parts of the relationship. A similar situation is also present with other types of toxic agents.



• Physical toxicants are substances that, due to their physical nature, interfere with biological processes. Examples include coal dust and asbestos fibers, both of which can ultimately be fatal if inhaled.

8.10.2  Factors influencing toxicity Toxicity of a substance can be affected by many different factors, such as the pathway of administration (whether the toxin is applied to the skin, ingested, inhaled, injected), the time of exposure (a brief encounter or long-term), the number of exposures (a single dose or multiple doses, over time), the physical form of the toxin (solid, liquid, gas), the genetic makeup of an individual, an individual’s overall health, and many others. Several of the terms used to describe these factors have been included here.

• Acute exposure: A single exposure to a toxic substance which may result in severe biological harm or death; acute exposures are usually characterized as lasting no longer than a day.



• Chronic exposure: Continuous exposure to a toxin over an extended period of time, often measured in months or years; it can cause irreversible side effects.

8.11  MEASURING TOXICITY (Use of LC50 and LD50 of organisms for evaluation of toxicity)

Toxicity can be measured by its effects on the target (organism, organ, tissue or cell). Because individuals typically have different levels of response to the same dose of a toxin, a population-level measure of toxicity is often used which relates the probabilities of an outcome for a given individual in a population. LC50: Lethal Concentration 50. An LC50 value is the concentration of a material in air that will kill 50% of the test subjects (animals, typically mice

8.26

Environmental Biotechnology

or rats) when administered as a single exposure (typically 1 or 4 hours). This value gives you an idea of the relative toxicity of the material. This value applies to vapors, dusts, mists and gases. Solids and liquids use the closely related LD50 value (50% lethal dose). LD50: Lethal Dose 50. It’s the amount of a solid or liquid material that it takes to kill 50% of test animals (for example, mice or rats) in one dose. Toxicologists can use many kinds of animals, but most often, testing is done with rats and mice. It is usually expressed as the amount of chemical administered (e.g., milligrams) per 100 grams (for smaller animals) or per kilogram (for bigger test subjects) of the body weight of the test animal. The LD50 can be found for any route of entry or administration, but dermal (applied to the skin) and oral (given by mouth) administration methods are the most common. Common procedures to do LD50/LC50 tests: • In nearly all cases, LD50 tests are performed using a pure form of the chemical. Mixtures are rarely studied. • The chemical may be given to the animals by mouth (oral); by applying on the skin (dermal); by injection at sites such as the blood veins (i.v.intravenous), muscles (i.m. - intramuscular) or into the abdominal cavity (i.p. - intraperitoneal). • The LD50 value obtained at the end of the experiment is identified as the LD50 (oral), LD50 (skin), LD50 (i.v.), etc., as appropriate. • Researchers can do the test with any animal species but they use rats or mice most often. Other species include dogs, hamsters, cats, guineapigs, rabbits, and monkeys. • In each case, the LD50 value is expressed as the weight of chemical administered per kilogram body weight of the animal and it states the test animal used and route of exposure or administration; e.g., LD50 (oral, rat) - 5 mg/kg, LD50 (skin, rabbit) - 5 g/kg. So, the example “LD50 (oral, rat) 5 mg/kg” means that 5 milligrams of that chemical for every 1 kilogram body weight of the rat, when administered in one dose by mouth, causes the death of 50% of the test group. • If the lethal effects from breathing a compound are to be tested, the chemical (usually a gas or vapor) is first mixed in a known concentration in a special air chamber where the test animals will be placed. • This concentration is usually quoted as parts per million (ppm) or milligrams per cubic meter (mg/m3). In these experiments, the concentration that kills 50% of the animals is called an LC50 (Lethal Concentration 50) rather than an LD50. • When an LC50 value is reported, it should also state the kind of test animal studied and the duration of the exposure, e.g., LC50 (rat) - 1000 ppm/ 4 hr or LC50 (mouse) - 5mg/m3/ 2hr.

Biological Methods of Pest Management

8.27

8.11.1  Toxic Toxic is defined by OSHA (Occupational Safety and Health Administration) as, a chemical which falls in any of these three categories:

• A chemical that has a median lethal dose (LD50) of more than 50 milligrams per kilogram but not more than 500 milligrams per kilogram of body weight when administered orally to albino rats weighing between 200 and 300 grams each.



• A chemical that has a median lethal dose (LD50) of more than 200 milligrams per kilogram but not more than 1,000 milligrams per kilogram of body weight when administered by continuous contact for 24 hours (or less, if death occurs within 24 hours) with the bare skin of albino rabbits weighing between two and three kilograms each.



• A chemical that has a median lethal concentration (LC50) in air of more than 200 parts per million but not more than 2,000 parts per million by volume of gas or vapor, or more than two milligrams per liter but not more than 20 milligrams per liter of mist, fume, or dust, when administered by continuous inhalation for one hour (or less, if death occurs within one hour) to albino rats weighing between 200 and 300 grams each.

8.11.2  Highly toxic Highly toxic is defined by OSHA (Occupational Safety and Health Administration) as:

• A chemical that has a median lethal dose (LD50) of 50 milligrams or less per kilogram of body weight when administered orally to albino rats weighing between 200 and 300 grams each.



• A chemical that has a median lethal dose (LD50) of 200 milligrams or less per kilogram of body weight when administered by continuous contact for 24 hours (or less, if death occurs within 24 hours) with the bare skin of albino rabbits weighing between two and three kilograms each.



• A chemical that has a median lethal concentration (LC50) in air of 200 parts per million by volume or less of gas or vapor, or 2 milligrams per liter or less of mist, fume, or dust, when administered by continuous inhalation for one hour (or less, if death occurs within one hour) to albino rats weighing between 200 and 300 grams each.

CHAPTER

9

Algae-Biotechnology

Algae, belonging to the kingdom Protista, are simple photosynthetic organisms. Based on the pigment and food reserve, algae are classified into different types, namely, blue green algae (BGA), green algae, red algae and brown algae.

• Algae are simple, autotrophic organisms that can synthesize their own food by means of photosynthesis. The taxonomy of algae is very confusing. Previously, algae were classified under the kingdom Plantae, as they possess chlorophyll for photosynthesis. However, algae are mostly aquatic and lack true roots, stem and leaves, which are not so in plants. Hence, in the modern classification, they are excluded from Plantae and categorized under Protists. The size of algae may range from few micrometers to several meters. For example, the freshwater alga Micromonas is about 1 micrometer, whereas the giant marine kelp can grow to about 60 meters in length. The branch of science that deals with the study of algae is called Phycology. Those who specialize in the study of algae are known as phycologists.

Types of Algae: Algae are classified based on the type of pigments and food reserves present in the particular species. The difference in the pigments play a major role in determining the habitat distribution of the particular algal species. Regarding the distribution of algae, they can adapt in diverse environmental conditions. Majority of algae are found in aquatic habitats, either in freshwater or marine water. Some of the species are found in extreme environment like snow and ice, whereas some are adapted in hot springs. Blue Green Algae (BGA): Blue green algae (BGA), also referred to as Cyanobacteria, are the simplest forms of algae. Examples of BGA are Nostoc and Calothrix. As the name suggests, they are blue green in color, ranging from single-celled organization to colonial forms. BGA contain chlorophyll a, b and phycobilins. They are prokaryotic in cellular organization, that resembles

9.2

Environmental Biotechnology

bacteria. BGA are considered to be an intermediate between bacteria and plants. Hence, the name cyanobacteria is assigned to these algal species. Since BGA lack specialized organelles, they photosynthesize directly through the cytoplasm. Green Algae: Green algae, belonging to the phylum Chlorophyta, contain chlorophyll a, b, carotenoids and xanthophylls. The main food reserve of green algae is starch. Some examples of green algae are Ulva, Codium and Caulerpa. As of now, about 7000 species of green algae are identified. They may be either unicellular or multicellular. Most of them are freshwater algae, while a few species are found in the marine water. Red Algae: Red algae, belonging to Rhodophyta, contain chlorophyll a, d, carotenoids, xanthophylls and phycobilins. The food reserve of red algae is floridean starch. The examples of red algae are Chondrus and Gelidiella species. Majority of red algae are marine species. More than 6500 species of red algae have been identified, out of which about 200 are freshwater species. The red pigment, phycobilin helps in harvesting light at a greater depth, hence some members of red algae are found in such a depth in the ocean floor, where no other photosynthetic organisms can adapt. Brown Algae: Brown algae, belonging to the class Paeophyceae, contain chlorophyll a, c and fucoxanthin pigment. Due to the green color, chlorophyll and brown pigment, fucoxanthin, the members belonging to phaeophyta exhibit a typical greenish-brown coloration. The food reserve of brown algae are complex carbohydrate polymers, called laminarin. Laminaria  and  Macrocystis are the examples of brown algae. Similar to red algae, majority of these algal groups are adapted in marine water. Brown algae are the most complex algae, in which some species are adapted at certain depths in the seas and oceans. The giant kelps, found in the ocean floor are brown algae belonging to the order Laminarales. Kelps are the only algae with tissue differentiation. Algal species are very sensitive to the changes in the environmental conditions. Hence, they are used as biological indicators to determine any modification in the environment. For example, simple freshwater algae like Euglena and Chlorella are used to indicate the extent of water pollution. Products Obtained from Algae (Industrial uses of algae) : Humans use algae as food, for production of useful compounds, as biofilters to remove nutrients and other pollutants from wastewaters, to assay water quality, as indicators of environmental change, in space technology, and as laboratory research systems. Algae are commercially cultivated for Pharmaceuticals, Nutraceuticals, Cosmetics and Aquaculture purpose. Agar: Agar, a substance made from algae, is gelatinous in nature. It is an ingredient in many different Japanese desserts. The Japanese red bean jelly, called Mizuyôkan, is made from agar, and is a very popular delicacy. Agar is

Algae-Biotechnology

9.3

commercially produced with the help of Gelidium amansii—a species of red algae. Agar is used very commonly as a laxative, and has also been used as a vegetarian substitute for gelatin. Sometimes, it is also used as a soup thickener. In Southeast Asia, agar is commonly used in jellies, ice creams and desserts. It is also used as an industrial clarifying agent for brewing and paper sizing fabrics. Alginic Acid: Another very useful form of algae is alginic acid. Alginic acid is a viscous, gum-like substance, derived from algae. It is used as an additive in dehydrated products. It is also a very important ingredient in the manufacturing of papers and textiles. As it possess most of the properties of gum, it is also used in the water-proofing and fire-proofing industry. It is especially helpful in making fabrics that are fire and water-resistant. In the pharmaceutical industry, it is used in the manufacture of Gaviscon, Asilone, Bisodol, etc. It is used as a mold-making material in life casting, prosthetics and dentistry. Like most of the forms or products of algae, alginic acid is used extensively in the food processing industry as an ingredient of soups and jellies. Carrageenan: The carrageenan is also one of the substances that has been derived from the red algae, found near the Irish coastline. Like many algae products, it is used as an ingredient in food products such as ice creams, milkshakes, and sauces, to increase the viscosity of the delicacy. In many parts of Europe, local beer and alcoholic drink manufacturers use carrageenan as a protein remover. Manufacturers of processed and canned meats use it as a substitute for fats. The presence of carrageenan also increases the water retaining capacity of meat products. Apart from that, carrageenan has a wide range of uses. It is prominently used in shampoos, toothpaste, diet sodas, pet food and soy milk. Fuels from Algae: The energy crisis that hit the world in the recent decades has triggered off the race for invention of effective as well as cheap biofuels. Algae oleum is one of the 3rd generation biofuel that has been derived from algae. The concept of algae culture or algae farming has been derived as a result, and many forms of algae fuels like cooking oil, biodiesel, bioethanol, biogasoline, etc. are in the process of development. Algae is one of the best example of putting eco-friendly resources to use, as none of the products derived from algae are considered to be pollutants. On the upside, many products of algae can be used to curb pollution. Algae can also be used to treat sewage, and is an excellent alternative for chemical fertilizers. It can also be used to curb and arrest the toxic chemicals that are present in water bodies. Algae is also the ideal substitute for chemical dyes and pigments. In many industries, algae bioreactors are used to curb the emission of carbon and carbon compounds. Humans use algae as food, for production of useful compounds, as biofilters to remove nutrients and other pollutants from wastewaters, to assay water quality, as indicators of environmental

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Environmental Biotechnology

change, in space technology, and as laboratory research systems. Algae is commercially cultivated for Pharmaceuticals, Nutraceuticals, Cosmetics and Aquaculture purpose.

Food supplement

• It is a complete protein with essential amino acids (unlike most plant foods) that are involved in major metabolic processes such as energy and enzyme production.



• It contains high amounts of simple and complex carbohydrates which provide the body with a source of additional fuel. In particular, the sulfated complex carbohydrates are thought to enhance the immune system’s regulatory response.



• It contains an extensive fatty acid profile, including Omega 3 and Omega 6. These essential fatty acids also play a key role in the production of energy.



• It has an abundance of vitamins, minerals, and trace elements in naturally-occurring synergistic design.

Stabilizing agent: Chondrus crispus (probably confused with Mastocarpus stellatus, common name: Irish moss), is also used as “carrageen”. It is an excellent stabiliser in milk products—it reacts with the milk protein caesin other products include: petfoods, toothpaste, icecreams and lotions, etc. Alginates in creams and lotions are absorbable through the skin. Fertilizer: Algae are used by humans in many ways. They are used as fertilizers, soil conditioners and are a source of livestock feed. Because many species are aquatic and microscopic, they are cultured in clear tanks or ponds and either harvested or used to treat effluents pumped through the ponds

Role of Algae in Pollution control

• Algae are used in Wastewater Treatment facilities, reducing the need for greater amounts of toxic chemicals than are already used.



• Algae can be used to capture fertilizers in runoff from farms. When subsequently harvested, the enriched algae itself can be used as fertilizer.



• Algae Bioreactors are used by some power plants to reduce CO2 emissions. The CO2 can be pumped into a pond, or some kind of tank, on which the algae feed. Alternatively, the Bioreactor can be installed directly on top of a smokestack.

9.1  ALGAE AS A SOURCE OF FOOD AND FEED Algae are primitive eukaryotic organisms that do not have true stems, roots and leaves. They are primary producers in aquatic ecosystems and marine organisms. Most of these are edible and have been consumed in different forms

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Algae-Biotechnology

since ancient times. The foods we consume, often dictate our physical and mental health. A healthy diet will always help fight illness-causing infections, whereas, a poor diet will make us more susceptible to illness. 

9.1.1  Microalgae nutritional composition As for higher plants, the nutritional composition of microalgae is made up mainly of proteins, carbohydrates, lipids and trace nutrients, including vitamins, antioxidants, and trace elements. Their content varies with different species, even strains and growing conditions, including nutrient supplies, temperature, sunlight, etc. Following Table illustrates general composition of some animal feeds and foods, and microalgae species (% of dry matter of maximum values achieved in each commodity). General composition of some animal feeds and foods, and microalgae Crude Protein

Carbohydrates

Lipids

Meat

Commodity

43

1

34

Fish

55

—

38

Egg

49

3

45

Milk

26

38

28

]Rice

8

77

2

Soyabean

37

30

20

Corn

10

85

4

Wheat

14

84

2

Fish-meal

60–72

—

6–10

Anaboena cylindrical

43–56

25–30

4–7

Chlamydomonos rheinhordff Chlorella vulgoris Chlorella pyrenoidoisa

48

17

21

51–58

12–17

14–22

57

26

2

Dunatlela salina

57

32

6

Euglena gracitis

39–61

14–18

14–20

Porphyridium cruentum

28–39

40–57

9–14

Scenedesmus obliquus

50–56

10–17

12–14

Arthrospira maxima

60–71

13–16

6–7

Spirutina platensis

46–63

8–14

4–9

Spirogyro sp.

6–20

33–64

11–21

Synechococcus sp.

73

15

11

Tetraselmis suecica

41

Isochrysis golbana

39

Dunolietta tertiolecta

54

Chlorella stigmatophora

39

9.6

Environmental Biotechnology

It can be noted that there is a large variation in compositions among conventional feeds / foods and microalgae. Indeed, depending on growth conditions, microalgae even the same strain—can exhibit large variations in composition, with protein, carbohydrate and lipid contents, each ranging from about 15 to over 50% of dry weight. This plasticity in composition is an important attribute in their use as animal feeds. About six decades ago, the mass production of certain protein-rich microalgae was considered as a possibility to close the predicted “food gap”. Although this “gap” failed to materialize, as a result of the “green revolution”, catalyzed in large part by the enormous amounts of fertilizers produced by the Harber Bosch process, the early interest in microalgae resulted in many comprehensive nutritional studies in both animals and humans. These nutritional studies continued during the 60s and 70s, due in part to the following interest in microalgae for space applications, and the early commercial production of two algae species, Chlorella and Spirulina. However, by the 1970s, the scale-up of commercial production to reduce production costs was not achieved, and the field of large-scale production is delaying this potential. Essential Amino Acids: These early and extensive nutritional studies of microalgae as foods and feeds demonstrated that algal proteins are of high quality, comparable to conventional vegetable proteins in terms of their content of essential amino acids, which mainly determine the nutritional quality of a protein source. The amino acid profile of various algae are compiled in the following table and compared with some basic conventional food items and a reference pattern of a well-balanced protein, recommended by WHO/FAO. List of Amino acids present in conventional foods and some microalgae Leu WHO/FAO

Val

Arg

Lys

He

Phe

Thr

Met

Try

His

7

5

—

5.5

4

6.0

—

3.50

1

—

Egg

8,8

7,2

6,2

5,3

6,6

5,8

5

3,2

1,7

2,4

Meat

7,8

5,3

6,6

8,2

5,1

4,2

4,5

2,4

—

3,2

Milk

9,2

5,7

3,3

7,8

4,3

5,6

4,5

2,5

—

2,6

Soyabean

7,7

5,3

7,4

6,4

5,3

5

4

1,3

1,4

2,6

Fish-meal

4,48

2,77

3,82

4,72

2,66

4,35

2,31

2,31

0,57

1,45

Chlorella vulgaris

8,8

5,5

6,4

8,4

3,8

5

4,8

2,2

2,1

2

Dunatella bordawil

11

5,8

7,3

7

4,2

5,8

5,4

2,3

0,7

1,8

Scenedesmus obliquus

7,3

6

7,1

5,6

3,6

4,8

5,1

1,5

0,3

2,1

Arthrospira maxima

8

6,5

6,5

4,6

6

4,9

4,6

1,4

1,4

1,8

Spirutinep latensis

9,8

7,1

7,3

4,8

6,7

5,3

6,2

2,5

0,3

2,2

Tetraselmis suecica

9,3

5,6

7,6

9,8

4,1

5,9

5,3

1,5

—

2,5

9.7

Algae-Biotechnology

Leu

Val

Arg

Lys

He

Phe

Thr

Met

Try

His

Isochrysis galbena

10,5

6

8,7

12,3

4,9

6,1

6,1

0,7

—

2,5

Dunaliela

10,7

5,3

7,2

13,6

4,2

6,6

2,6

0,8

—

2,5

Chlorella

9,3

5,7

8,6

13,4

3,8

5,5

4,9

1,4

—

2,3

Most of the early work in determining which amino acids could be classed as indispensable for humans and animals was carried out with rats fed on purified diets. The following ten indispensable amino acids are required for growth in the rat: Arginine, Histidine Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan and Valine. Some animals differ slightly in these requirements (the pig can synthesize arginine, for example), but the content of these essential amino acids determines in large measure the nutritional value of these feeds. Vitamins and Trace Elements: A similar comparison can be made for vitamins, demonstrating that microalgae contain high levels of essential vitamins, similar to the best food sources such as baker’s yeast and liver and are vastly superior to all commodity feeds (such as soybeans, corn, fishmeal, etc.) which have little, if any, vitamin content. List of vitamins and trace elements present in conventional foods and some algae Vit A

Vit B1

Vit B2

Vit B6

Vit B12

Vit C

Vit E

Nicotinate

Blotin

Folic Acid

Panthotenic Acid

1.7

1.5

2.0

2.5

0.005

50.0

30.0

18.0

—

0.6

8.0

Liver

60.0

3.0

29.0

9.0

0.65

310.0

10.0

136.0

1.0

2.9

73.0

Spinach

130.0

0.9

1.8

1.8

—

470.0

—

5.5

0.007

0.7

2.8

RDI (mg/d)

Baker’s yeast

trace

9.1

16.5

21.0

—

trace

112.0

4.0

5.0

53.0

Spiruling platenis

840.0

44.0

39.0

3.0

9.0

80

120.0

—

0.3

0.4

13.0

Chlorella pyrenoldolsa

480.0

10.0

36.0

23.0

—

—

—

240.0

0.15

—

20.0

Scenedesmus quadricauda

554.0

11.5

29.0

—

1.1

396.0

—

108.0

—

—

46.0

The nutritionally necessary trace elements, of which, there are about two dozen, have similarly high levels in microalgae to those of vitamins, compared to most feed sources. However, data on trace elements is more limited, contents are even more variable between and even within species, and composition more plastic, depending on growing conditions, than is the case even for the major constituents, essential amino acids or vitamins. Indeed, microalgae with desired high concentrations of trace elements could be produced on demand by adjusting the trace elements in their growth medium. In the case of both vitamins and trace elements, but also for essential amino acids, the bioavailability of these trace nutrients is often more important and decisive in terms of feed quality than just their bulk constituents. Bioavailability

9.8

Environmental Biotechnology

studies are more difficult than just simple analytical measurements, but, overall, bioavailability of all these components is good to high for various microalgae biomass sources studied; in most cases; the microalgae already being produced commercially (e.g. Chlorella and Spirulina). Health Benefits Associated with Algae: Over the years, research has proven many health benefits associated with algae. It is commonly used as a dietary supplement to improve general health and wellness. Some of the important health benefits of algae include: • It is known to have a high concentration of beta-carotene that helps fight some types of cancer and cardiovascular diseases. • Full of antioxidants, algae restricts the growth of free radicals and toxins. Antioxidants also aid in production of necessary enzymes needed to keep the body’s function smooth. • As it is organic in nature and full of enzymes, it is easily digestible and light on the stomach. It is known to facilitate smooth bowel movements, thereby preventing abdominal muscle associated diseases. • Algae is considered a complete source of protein, as it contains amino acids, minerals and many vitamins, essential for the growth of hair, skin and nails. • Algae contains immune boosting and stimulating properties such as amino acids, minerals, proteins and vitamins. These help fight infections, detoxify the immune system, and aid in building the body’s resistance to infections. • There are only two sources of GLA found; mother’s milk and spirulina, a blue-green algae. GLA is essential for the development and growth of babies. Deficiency in nutrition reduces GLA in the mother’s milk, which results in poor baby health. • Algae is known to treat diabetes, anemia, liver disease, ulcers, allergies, radiation and chemical poisoning. Its concentrated sugar stabilizes blood sugar levels in people with high or low blood sugar. • Algae  ensures a healthy nutrient level for people who diet, as well as people who detoxify their bodies regularly. The amino acids present in algae are known to influence neurotransmitters in the brain that control appetite. • A balanced diet consists 80% alkaline food and 20% acidic food. An acidic body is vulnerable to diseases. Algae is considered as a natural source of alkaline food. Other benefits of Algae: • It contains calcium and magnesium, which are essential for strong bones. • Has high levels of iron, useful for anemic people and pregnant or lactating women.

Algae-Biotechnology

9.9



• It helps to reduce anxiety and treat sleep disorders. • It is popular in weight reduction diets. • Algae contains the zeaxanthin nutrient, which is good for the eyes. • Regular consumption of algae aids in enhancing memory and increasing concentration and focus. Consuming algae as a regular health supplement helps improve the overall development and growth of the body, as it contains almost all nutrients, minerals, vitamins, proteins, amino acids and nucleic acids, chlorophyll, fiber, etc. There are many types of algae available, like Spirulina and Chlorella. It is mostly available as powders, capsules, tablets, etc. While algae health benefits are enormous, it is best to consult a medical practitioner on the type and the amount one needs to consume to derive maximum health benefits.

9.2  MASS CULTIVATION OF COMMERCIALLY VALUBLE MARINE MICROALGAE FOR AGAR AGAR, ALGINATES Microalgae were one of the first organisms to come into existence in the Earth’s ocean more than 3 billion years ago, when the Earth’s environment formed. They are also called phytoplankton. These unicellular organisms have chlorophyll and produce oxygen (O2) by immobilizing carbon dioxide (CO2) in the atmosphere through photosynthesis. There are about 100,000 different types of microalgae living not only in the oceans but also in fresh water (lakes, ponds, and rivers). Marine microalgae, the largest primary biomass, have been attracting attention as resources for new metabolites and biotechnologically useful genes. The diversified marine environment harbors a large variety of microalgae. Industrial culture technique: Along with rising expectations for microalgae, it has become necessary to develop technology to industrially and efficiently produce the required amounts of microalgae. In particular, it is essential to establish culture techniques ranging from small-to large-scale culturing. It is also necessary to enhance productivity based on individual culture methods and accumulate know-how with regard to ensuring quality. In the case of producing relatively small amounts along with rising expectations for microalgae, it has become necessary to develop technology to industrially and efficiently produce the required amounts of microalgae. In particular, it is essential to establish culture techniques ranging from smallto large-scale culturing. It is also necessary to enhance productivity based on individual culture methods and accumulate know–how with regard to ensuring quality. In the case of producing relatively small amounts of medicine, food and feed, which require purity and safety, an enclosed culture system (Enclosed System) is used. In the case of biofuel and biomass, large-scale-yet low-cost, production methods are required. Currently, as a large-scale culture method, culturing in open spaces such as ponds (Open Pond System) has been employed.

9.10

Environmental Biotechnology

At present, such methods are a long way from being efficient. In particular, the development of a culture method to industrially produce low-priced products in large quantities is still at the study stage. A study is now under way to develop an enclosed culture system that utilizes light more effectively than open culture methods and is suitable for low-priced, mass culture. Macroalgae have long been used for the production of phycocolloids such as alginates, carrageenans or agars. These polymers are either located in cell walls or within the cells serving as storage materials. A characteristic of marine algae is the abundance of sulphated polysaccharides in their cell walls. AGAL-AGAL: Agars are 1,3-b-1,4-b-galactans from cell walls of red algae that are substituted with sulphate groups. Like the carrageenans, the agals are extracted with hot water. The genera Gelidium and Gracilaria supply most of the raw material for agar production. Gelidium used for commercial agal production is harvested from the wild, whereas Gracilaria species have also been cultivated in Chile, China and Indonesia, in protected bays in the ocean, on line ropes or nets, or in ponds on land. Like carrageenans, agals are used as stabilisers for emulsions and suspensions and as gelling agents. About 90% of the agal produced was for food applications and the remaining 10% were used for bacteriological and other biotechnological uses. The production process of agal-agal from red algae contains the following parts:

• Drying of algae: Once collected, the algae are dried in the sun.



• Alkalinization of algae: Once dried, they are placed in a container with very hot water in which an alkali product, such as caustic soda, has been poured.



• Washing and bleaching with cold water: Then, the algae are washed with cold water to remove all impurities. Sulfuric acid is added to dealkalinize them and sodium hypochlorite for bleaching them . Finally, they are cleaned with cold water.



• Extraction by cooking: The product is extracted by subjecting the washed and bleached algae to a process of cooking for two hours.



• Filtering: The filtering aims to separate the product from other waste such as rocks, shells, dirt, etc. This is accomplished by passing the water with the algae thorough different filtering tanks.



• Gelling: A process intended to bring the product to the texture of a gel. To do this, the algae are cooled by different processes from 80°C to 25°C.



• Pressing and drying: After this, the product is compacted by a press and dried with hot air.



• Grinding, sieving and packaging: Finally, the algae are ground and sieved before being packed.

Algae-Biotechnology

9.11

The properties of agar agar are numerous. It is used primarily in the following areas: • Food Industry: The food industry uses it as a food additive with the code E-406. It is a natural thickener, non-toxic, with no flavor, not degradable by acid or proteolytic enzymes, and with no many calories. The thickening ability of agar agar comes from its power to make the liquid more viscous so that it produces, what is called, a gelling effect, that is to say, it makes a liquid to become a gel. Therefore, it is very suitable for the manufacture of canned fish and meat because it compacts them and provides them a better texture. Moreover, this product forms a layer that protects these foods from contact with the metal walls of the container. By doing this, it prevents canned food to become rusted (cooked meat, ham, shoulder, sausage, canned chicken, etc.) The beverage industry (beer, wine, etc.) uses it to purify and clarify the liquids because it settles the impurities to the bottom. Other packaged products such as fruit juices, sauces, soups, yogurt, curds, ice cream, cakes ... also contain agar-agar as a thickener or stabilizer. It is the natural substitute for animal gelatin obtained from bones and skin. In this regard, agar agar is highly valued within the vegetarian people who use it in many recipes. • Metalworking: Used to improve the coating of metal objects made out of lead or zinc or to encourage the stretching of certain metals. • Paper industry: Its use provides waterproofing properties to paper. • Leather industry: Used to promote leather tanning. • Textile industry: It gives strength and stiffness to clothes and facilitates printing. • Pharmaceuticals: The pharmaceutical industry includes it in the composition of many drugs and excipients, to improve taste, color, texture, etc. It is also used to form the cover of some preparations such as suppositories, pessaries or gels. It is one of the basic ingredients in the preparation of drugs for constipation. • Natural medicine: In natural medicine, is mainly used as a laxative and in slimming cures. • Cosmetics industry: It becomes a part of the manufacture of skin creams. • Microbiological research: agar agar is one of the main culture media for microbiological research. It presents a greater capacity than any animal gelatine to resist microorganisms and remain stable at a suitable temperatures for incubating them. On the other hand, it can be solidified or become, easily a gel, allowing the microorganisms to be mixed in it easily.

9.12

Environmental Biotechnology

• Traditionally, agar agar is produced in Asia, mainly China, Korea, Japan and Indonesia. Although it has been used since ancient times in the East, it is documented for the first time in the seventeenth century in Japan. In Europe it was not used until the nineteenth century. • At present, there are many countries that produce it. These include, for example, United States, Korea, Spain, France, Morocco, Chile, South Africa and Portugal. Among all the varieties, the Atlantic agar agar stands out. This is obtained form the alga Gelidium sesquipedale. Spanish production of this type of alga in Galicia is very well known. • Most of agar agar is produced for human consumption, and only about 10% is used for other applications. Most of the agar agar is derived from the following two kinds of algae: Gelidium: It inhabits rocky places of eulitoral and sublitoral zone. Eulitoral area (= interdital) is that part of the coast that lies between tides, so it is devoid of water at low tide and is covered with it when the tide rises. The sublitoral zone extends from eulitoral area and is characterized by being permanently covered by water. In general, the species of this genus do not like excessive sun exposure and prefer moderate temperatures between 15 and 20°C. Formerly, there were plenty of productive areas of these algae in Japan, but industrial pollution has extinguished most populations in this area. The main collection areas are currently on the north coast of Spain, the east coast of Portugal, the west coast of Morocco and the southwest coast of France. To a lesser proportion, they are also found in Indonesia, on the southwest coast of Mexico and the southeastern coast of Korea. Gracilaria: They inhabit the eulitoral areas of sandy soils. They need higher temperatures, although there are species adapted to colder waters, and more easily resist external aggressions, such as freshwater input or fertilizer. The species that prefer warmer areas live in coastal areas of Indonesia and southern China. Some of these species are grown in ponds and estuaries. Other places with lower production are the west coast of South Africa and the coast of Namibia. Species adapted to cold areas can be found in places such as southern Chile and eastern Canada. Other genres used extensively in some regions are Gelidiella which is the most commonly used in India, Pterocladia in Russia and Ahnfeltia in New Zealand and the Azores. Collection and cultivation of agal agal: Although most of the agal agal is obtained from collecting it naturally in coastal areas, it is increasingly spreading in cultivation. Among the species grown, Gracilaria is the most used and the only one that, in most cases, can be considered economically viable. There are three main methods for the cultivation of Gracilaria: • Open cultivation in estuaries or bays: Sandy soils are required. On these types of soils, algae are placed for them to take root and develop new algae.

Algae-Biotechnology

9.13



• On ropes or nets: They form a sort of row with filaments or nylon or polypropylene ropes to which algae are attached so that they can thrive. Each line is held on stakes that are inserted and fastened to the bottom of the water. Sometimes they are fixed on nets that, in turn, are placed into the sea ground with bamboo poles.



• On ponds or pools: In this case, a pond is sown so that the algae become entrenched to the bottom. Since they are not anchored as in the previous cases, we need to do so in not very windy ponds.

Each pond should be about 60 or 70 cm deep and must contain a whole surface of one or half hectare. Salinity and temperature must be controlled, something which is done by means of a water supply from the outside, so that each pool or pond should be connected with a salt water source and another source of fresh water. To get a proper salinity, water should be added or removed. Since it requires a constant temperature of about 15 to 30°C, more water should be added when the outdoor temperature is higher so that the sun does not heat the water from the bottom too much. On the contrary, some water should be removed when the temperature is lower to allow that sunlight to access the deeper level of water and heat it. Alginates: Alginates are polymers from the cell walls of a wide variety of species of the brown algae, particularly species of Laminaria, Macrocystis and Ascophyllum. They are polymers composed of D-mannuronic acid and L-guluronic acid monomers. The alginates are extracted from the cell walls using hot alkali (sodium carbonate). Alginates are commonly used in the food and pharmaceutical industries as stabilizers for emulsions and suspensions, e.g. ice cream, jam, cream, custard, creams, lotions, tooth paste, as coating for pills. They are also used in the production of paint, construction material, glue and paper, oil, photo and textile industry. Brown seaweeds for alginate production are harvested from the wild, and not cultivated, for this purpose. Although these seaweeds are cultivated to produce food in China, their cultivation to provide raw material for industrial uses would be too expensive. Production: Biotechnological and Traditional: There has been significant progress in the understanding of alginate biosynthesis over the last few years. The fact that the alginate molecule enzymatically undergoes a postpolymerization modification with respect to chemical composition and sequence opens up the possibility for in vitro modification and tailoring of commercially available alginates. Isolation from Natural sources/fermentative production: All commercial alginates today are produced from marine brown agar. Alginates with more extreme composition can be isolated from the bacterium Azotobacter vinelandii, which in contrast to Pseudomonas species, produces polymer containing G-blocks. Production by fermentation, therefore, is technically feasible at the moment.

9.14

Environmental Biotechnology

Molecular Genetics and in vitro Modification: Alginate with a high content of guluronic acid can be prepared from special algal tissues by chemical fractionation or by in vitro using mannuronam C-5 epimerases from A. vinelandii. These epimerases, which convert M and G in the polymer chain, recently have allowed for the production of highly programmed alginates with respect to chemical composition and sequence. Commercial application of alginates: The uses of alginates are based on three main properties. • The first property of alginates is their ability to thicken the solution when dissolved in water (more technically, described as their ability to increase the viscosity of aqueous solutions). • The second is their ability to form gels; gels form, when a calcium salt is added to a solution of sodium alginate in water. The gel forms by chemical reaction, the calcium displaces the sodium from the alginate, holds the long alginate molecules together and a gel is the result. No heat is required and the gels do not melt, when heated. This is in contrast to the agar gels where the water must be heated to about 80°C to dissolve the agar and the gel forms, when cooled below about 40°C. • The third property of alginates is the ability to form films of sodium or calcium alginate and fibers of calcium alginates. Alginate molecules are long chains that contain two different acidic components, abbreviated here for simplicity to M and G. The way in which these M and G units are arranged in the chain and the overall ratio, M/G, of the two units in a chain, can vary from one species of seaweed to another. In other words, all “alginates” are not necessarily the same. So some algae may produce an alginate that gives a high viscosity when dissolved in water, others may yield a low viscosity alginate. The conditions of the extraction procedure can also affect viscosity lowering it, if conditions are too severe. All of this results in sellers, normally—offering a range of alginates with differing viscosities. Similarly, the strength of the gel formed by the addition of calcium salts can vary from one alginate to another. Generally, alginates with a higher content of G will give a stronger gel; such alginates are said to have a low M/G ratio. Some examples: Macrocystis can gives a medium-viscosity alginate, or a high viscosity with a careful extraction procedure (lower temperature for the extraction). Sargassum, usually, gives a low viscosity product. Laminaria digitata gives a soft to medium strength gel, while Laminaria hyperborea and Durvillaea give strong gels. These are some of the reasons why alginate producers like to have a variety of seaweed sources, to match the alginate to the needs of particular applications. Textile printing: In textile printing, alginates were used as thickeners for the paste containing the dye. These pastes may be applied to the fabric by either screen—or roller printing equipment. Alginates became important

Algae-Biotechnology

9.15

thickeners with the advent of reactive dyes. These combine chemically with cellulose in the fabric. Many of the usual thickeners, such as starch, react with the reactive dyes, and this leads to lower colour yields and sometimes byproducts, that are not easily washed out. Alginates do not react with the dyes, they easily wash out of the finished textile and are the best thickeners for reactive dyes. Alginates are more expensive than starch and recently starch manufacturers have made efforts to produce modified starches that do not react with the reactive dyes, so it is becoming a more competitive market. This use of alginate represents a large market, but it is affected by economic recessions when there is often a fall in demand for clothing and textiles. The types of alginate required vary from medium-to-high viscosity with older screen printing equipment, to low viscosity, if modern, high speed, roller printing is used. Textile printing accounts for about 50 per cent of the global alginate market. Food: The thickening property of alginate is useful in sauces and in syrups and toppings for ice cream. By thickening pie fillings with alginate, softening of the pastry by liquid from the filling is reduced. Addition of alginate can make icings non-sticky and allow the baked goods to be covered with plastic wrap. Water-in-oil emulsions such as mayonnaise and salad dressings are less likely to separate into their original oil and water phases if thickened with alginate. Sodium alginate is not useful when the emulsion is acidic, because insoluble alginic acid forms; for these applications propylene glycol alginate (PGA) is used, since this is stable in mild acid conditions. Alginate improves the texture, body and sheen of yoghurt, but PGA is also used in the stabilization of milk proteins under acidic conditions, as found in some yoghurts. Some fruit drinks have fruit pulp added and it is preferable to keep this in suspension; addition of sodium alginate, or PGA in acidic conditions, can prevent sedimentation of the pulp. In chocolate milk, the cocoa can be kept in suspension by an alginate/phosphate mixture, although in this application, it faces strong competition from carrageenan. Small amounts of alginate can thicken and stabilize whipped cream. Alginates have some applications that are not related to either their viscosity or gel properties. They act as stabilizers in ice cream; addition of alginate reduces the formation of ice crystals during freezing, giving a smooth product. This is especially important when ice cream softens between the supermarket and the home freezer; without alginate or similar stabilizer, the refrozen ice cream develops large ice crystals, giving it an undesirable crunchy mouth feel. Alginate also reduces the rate at which the ice cream will melt. Beer drinkers prefer some foam on the top of a newly-poured glass, and poor foam leads to a subjective judgment that the beer is poor quality. Addition of a very low concentration of propylene glycol alginate will provide a stable, longer-lasting beer foam. A variety of agents are used in the clarification of wine and removal of unwanted coloring—wine fining—but in more difficult cases, it has been found that the addition of sodium alginate can be effective.

9.16

Environmental Biotechnology

The gelling properties of alginate were used in the first production of artificial cherries in 1946. A flavored, colored solution of sodium alginate was allowed to fall, in large drops, into a solution of a calcium salt. Calcium alginate immediately formed as a skin on the outside of the drop and when the drop was allowed to sit in the solution, the calcium gradually penetrated the drop converting it all into a gel that hardened with further standing. Because the cherry-flavored gels did not melt, they became very popular in bakery products. Fruit substitutes can now be made by automated and continuous processes that are based on similar principles. Either the calcium can be applied externally, as above, or internally. In the latter case, a calcium salt that does not dissolve is added to the fruit puree, together with a weak acid; the weak acid slowly attacks the calcium salt and releases water-soluble calcium that then reacts with the alginate and forms the gel. Edible dessert jellies can be formed from alginate-calcium mixtures, often promoted as instant jellies or desserts because they are formed simply by mixing the powders with water or milk, no heat being required. Because they do not melt, alginate jellies have a different, firmer mouth feel when compared to gelatin jellies, which can be made to soften and melt at body temperature. Mixtures of calcium salts and sodium alginate can be made to set to a gel at different rates, depending on the rate at which the calcium salt dissolves. Gel formation can also be delayed even after everything is mixed together; this is done using a gel-retarder that reacts with the calcium before the alginate does, so, no calcium is available to the alginate until all the retarder is used. In this way, gel formation can be delayed for several minutes if desired, such as when other ingredients need to be added and mixed before the gel starts to set. Alginate gels are used in restructured or reformed food products. For example, restructured meats can be made by taking meat pieces, binding them together and shaping them to resemble usual cuts of meat, such as nuggets, roasts, meat loaves, even steaks. The binder can be a powder of sodium alginate, calcium carbonate, lactic acid and calcium lactate. When mixed with the raw meat, they form a calcium alginate gel that binds the meat pieces together. This is used for meats for human consumption, such as chicken nuggets; it has become especially useful in making loaves of meat for fresh pet food; some abattoir wastes are suitable as cheap ingredients. Up to 1 per cent alginate is used. Similar principles are applied to making shrimp substitutes using alginate, proteins such as soy protein concentrate, and flavors. The mixture is extruded into a calcium chloride bath to form edible fibers which are chopped, coated with sodium alginate and shaped in a mould. Restructured fish fillets have been made using minced fish and a calcium alginate gel. Onion rings are made from dried onion powder; pimento olive fillings are made using pimento pulp. In 2001, a new line of olives launched in Spain were stuffed with flavored pastes, such as garlic, herbs, hot pepper, lemon and cheese. Each of these is made with green manzanilla olives and an alginate-based paste containing the appropriate ingredient to provide the flavour.

Algae-Biotechnology

9.17

Calcium alginate films and coatings have been used to help preserve frozen fish. The oils in oily fish such as herring and mackerel can become rancid through oxidation even when quick frozen and stored at low temperatures. If the fish is frozen in a calcium alginate jelly, the fish is protected from the air and rancidity from oxidation is very limited. The jelly thaws with the fish, so they are easily separated. If beef cuts are coated with calcium alginate films before freezing, the meat juices released during thaw are reabsorbed into the meat and the coating also helps to protect the meat from bacterial contamination. If desired, the calcium alginate coating can be removed by redissolving it with sodium polyphosphate. Immobilized biocatalysts: Many commercial chemical syntheses and conversions are best carried out using biocatalysts such as enzymes or active whole cells. Examples include, the use of enzymes for the conversion of glucose to fructose, the production of L-amino acids for use in foods, the synthesis of new penicillin after hydrolysis of penicillin G, the use of whole cells for the conversion of starch to ethanol (for beer brewing), and the continuous production of yoghurt. To carry out these processes on a moderate to large scale, the biocatalysts must be in a concentrated form and be recoverable from the process for reuse. This can be achieved by “immobilizing” the enzymes or cells by entrapping them in a material that will still allow penetration by the substance to be converted or changed. Originally, single enzymes were isolated and used for a specific conversion, but now similar or better results can be obtained using whole cells, and this is more economical. An added advantage of immobilization is, that the cells last longer. Ordinary suspended cells may have good activity for only 1-2 days, while immobilized cells can last for 30 days. Beads made with calcium alginate were one of the first materials to be used for immobilization. The whole cells are suspended in a solution of sodium alginate and this is added dropwise to a calcium chloride solution. The beads form in much the same way as described for artificial cherries. In use, they are packed into a column and a solution of the substance to be converted is fed into the top of the column and allowed to flow through the bed of beads containing the immobilized biocatalyst in the cells. The conversion takes place and the product comes out at the bottom. A simple example is to immobilize yeast cells, flow a solution of sugar through the beads, and the sugar is converted to alcohol. Pharmaceutical and medical uses: If a fine jet of sodium alginate solution is forced into a bath of a calcium chloride solution, calcium alginate is formed as fibers. If low viscosity alginates are used, a strong solution can be used without any viscosity problems and the calcium bath is not diluted as rapidly. The fibres have very good strength when both wet and dry. As with most polymer fibers formed by extrusion, stretching, while forming, increases the linearity of the polymer chains and the strength of the fiber. Good quality stable fibers

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have been produced from mixed salts of sodium and calcium alginate, and processed into non-woven fabric that is used in wound dressings. They have very good wound healing and haemostatic properties and can be absorbed by body fluids because the calcium in the fiber is exchanged for sodium from the body fluid to give a soluble sodium alginate. This also makes it easy to remove these dressings from large open wounds or burns since they do not adhere to the wound. Removal can be assisted by applying saline solutions to the dressing to ensure its conversion to soluble sodium alginate. Recently, the consumer division of a multinational pharmaceutical company launched a new line of adhesive bandages and gauze pads based on calcium alginate fibers. They are being promoted as helping blood to clot faster—twice as fast as their older, well established, product. Alginic acid powder swells when wetted with water. This has led to its use as a tablet disintegrant for some specialized applications. Alginic acid has also been used in some dietary foods, such as biscuits; it swells in the stomach and, if sufficiently taken, it gives a “full” feeling so the person is dissuaded from further eating. The same property of swelling has been used in products such as Gavisconä tablets, which are taken to relieve heartburn and acid indigestion. The swollen alginic acid helps to keep the gastric contents in place and reduce the likelihood of reflux irritating the lining of the oesophagus. Alginate is used in the controlled release of medicinal drugs and other chemicals. In some applications, the active ingredient is placed in a calcium alginate bead and slowly released as the bead is exposed in the appropriate environment. More recently, oral controlled-release systems involving alginate microspheres, sometimes coated with chitosan to improve the mechanical strength, have been tested as a way of delivering various drugs. Pronova Biomedical AS, a leading supplier of ultra-pure alginates and chitosans for controlled release and other medical materials applications, was acquired by FMC Bioploymer in early 2002; FMC had previously acquired Pronova Biopolymer, producer of food and technical grade alginates.

9.2.1  Other applications Paper: The main use for alginate in the paper industry is in surface sizing. Alginate added to the normal starch sizing gives a smooth continuous film and a surface with less fluffing. The oil resistance of alginate films give a size with better oil resistance and enhances grease-proof properties. An improved gloss is obtained with high gloss inks. If papers or boards are to be waxed, alginate in the size will keep the wax mainly at the surface. They give better coating runability than other thickeners, especially in hot, on-machine coating applications. Alginates are also excellent film–formers and improve ink holdout and printability. The quantity of alginate used is usually 5-10 per cent of the weight of starch in the size. Alginate is also used in starch adhesives for making corrugated boards because it stabilizes the viscosity of the adhesive and allows control of its rate

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of penetration. One per cent sodium alginate, based on the weight of starch used, is usually sufficient. Paper coating methods and equipment has developed significantly since the late 1950s with the demand for a moderately priced coated paper for high quality printing. Trailing blade coating equipment runs at 1,000 m/minute or more, so the coating material, usually clay plus a synthetic latex binder, must have consistent rheological properties under the conditions of coating. Up to 1 per cent alginate will prevent change in viscosity of the coating suspension under the high shear conditions where it contacts the roller. The alginate also helps to control water loss from the coating suspension into the paper, between the point where the coating is applied and the point where the excess is removed by the trailing blade. The viscosity of the coating suspension must not be allowed to increase by loss of water into the paper because this leads to uneven removal by the trailing blade and streaking of the coating. Medium to high viscosity alginates are used, at a rate of 0.4-0.8 per cent of the clay solids. Because of the solvent resistance of alginate films, the print quality of the finished paper is improved. Welding rods: Coatings are applied to welding rods or electrodes to act as a flux and to control the conditions in the immediate vicinity of the weld, such as temperature or oxygen and hydrogen availability. The dry ingredients of the coating are mixed with sodium silicate (water glass) which gives some of the plasticity necessary for extrusion of the coating onto the rod; it also acts as the binder for the dried coating on the rod. However, the wet silicate has no binding action and does not provide sufficient lubrication to allow effective and smooth extrusion. An additional lubricant is needed, and a binder that will hold the damp mass together before extrusion and maintain the shape of the coating on the rod during drying and baking. Alginates are used to meet these requirements. The quantities of alginates used are very dependent on the type of welding rod being coated and the extrusion equipment being used. Alginate manufacturers are the best source of information for using alginates in welding rod applications. Binders for fish feed: The worldwide growth in aquaculture has led to the use of crude alginate as a binder in salmon and other fish feeds, especially, moist feed made from fresh waste fish mixed with various dry components. Alginate binding can lower consumption by up to 40 per cent and pollution of culture ponds is sharply reduced. Release agents: The poor adhesion of films of alginate to many surfaces, together with their insolubility in non-aqueous solvents, have led to their use as mould, release agents, originally for plaster moulds and later in the forming of fiber glass plastics. Sodium alginate also makes a good coating for anti-tack paper, which is used as a release agent in the manufacture of synthetic resin decorative boards. Films of calcium alginate, formed in situ on a paper, have been used to separate decorative laminates after they have been formed in a hot-pressing system.

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Future prospects: The overall annual growth rate for alginates is 2-3 per cent, with textile printing applications accounting for about half of the global market. However, the textile industry is flat at present, as it rides a trough in the cycle of peaks and troughs, and it is 90 per cent based in Asia and the Near East (Turkey). Pharmaceutical and medical uses are about 20 per cent by value of the market and have stayed buoyant, with 2-4 per cent annual growth rates, driven by ongoing developments in controlled release technologies and the use of alginates in wound care applications. Food applications are worth about 20 per cent of the market. That sector has been growing only slowly, and recently has grown at only 1-2 per cent annually. The paper industry takes about 5 per cent and the sector is very competitive, not increasing but just holding its own. The alginate industry faces strong competition from Chinese producers, whose prices do not reflect the real expense of cultivating Laminaria japonica, even in China, yet they do not appear to import sufficient wild seaweeds to offset those costs. The result is low profitability for most of the industry, with the best opportunities lying in the high end of the market, such as pharmaceutical and medical applications.

9.3  COMMERCIAL APPLICATION OF MICROALGAE AND THEIR PRODUCTS The use of microalgae by humans dates back thousands of years to the Chinese who used Nostoc to survive famine. Other species of microalgae including, the blue green algae species, Arthrospira (Spirulina) and Aphanizomenon, have also been used by humans for thousands of years. Spirulina has been exploited by ancient peoples in both Chad and Mexico as a source of food. Although, microalgae have been used for food by humans for thousands of years, microalgae culture is one of the modern biotechnologies. Unialgal cultures were first achieved in 1890, with Chlorella vulgaris and, the use of this type of cultures was used for the study of plant physiology in the early 1900. In 2004, the global market for microalgal biomass was estimated to be 5,000 t of dry matter per year and generated a turnover of US$ 1,250 million. Microalgae contain about 50% carbon in their biomass and this carbon is obtained in most cases from ‘‘atmospheric’’ carbon dioxide, and therefore, microalgae are attracting interest as vehicles for carbon sequestration for industrial processes. The use of algae for therapeutic purposes has a long history, but the search for biologically-active substances from algae, especially, examination for antibiotic activity began only in the 1950s. Much of that laboratory work up until the 1980s, focused on macroalgae. Approximately, 15,000 natural marine products have now been screened for biological activity and 45 marine derived natural products have been tested to be used as medical drugs in preclinical and clinical trials. Only 2 have been developed into registered drugs—one from a marine snail and the other from a sea squirt, although, none, as yet, from microalgae. It is reported that preclinical trials are currently being undertaken on an anti-cancer drug, Curacin, derived from the blue-green algae, Lyngbya majuscula. There have also been reports of the use of products derived from algae being used to combat HIV infection.

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Microalgae are responsible for over 50% of primary photosynthetic productivity on earth and are budding sunlight factories for a wide range of potentially useful products, but are barely used. In the early 1950s, the increase in world population lead to the search for new alternative food and protein sources and algae, appeared at the time, a good. Although, algae have and are still used for food, to some extent around the world, the large scale production of algae to solve the world’s food calorie and protein shortage has not materialized. The large-scale cultivation of microalgae and the use of its biomass for the production of useful products were first considered seriously in Germany during World War II. Commercial large–scale modern algae culture started in the early 1960s in Japan with the culture of Chlorella. In the early 1970s, culturing and harvesting of Spirulina began in Mexico. The third major area of commercialisation of algae occurred, in Australia, with the growth of Dunaliella salina for the production of b-carotene. Plants were then subsequently built in the USA and Israel and production of blue-green algae also commenced in India. Plants have recently been built in the USA and India for the growth of Haematococcus pluvialis as a source of astaxanthin, approved as food-coloring and also a powerful anti-oxidant. A common feature of most of the algal species currently produced commercially (i.e. Chlorella, Spirulina and Dunaliella) is that they grow in highly selective environments which means that they can be grown in open air cultures and still remain relatively free of contamination by other algae and protozoa. Thus, Chlorella grows well in nutrient-rich media, Spirulina requires a high pH and bicarbonate concentration and Dunaliella salina grows at very high salinity. Those species of algae, which do not have environmental-selective advantages, may need to be grown in closed systems. This includes most of the marine algae grown as aquaculture feeds (e.g. Skeletonema, Chaetoceros, Thalassiosira, Tetraselmis and Isochrysis) and the dinoflagellate, Crypthecodinium cohnii, grown as a source of long-chain polyunsaturated fatty acids, as well as, almost all other species being considered for commercial mass culture.

9.3.1  Current commercial uses of algae ‘Health’ foods: Chlorella is produced by more than 70 companies with the largest producer, Taiwan Chlorella Manufacturing and Co, producing 400t of dry algal biomass per year. Chlorella is sold as a health food or dietary supplement. Several reports from Japan have described various potential therapeutic effects of Chlorella, and although, these are encouraging findings, the investigations were initiated by the Chlorella producing companies and must be viewed as such. Suggested health benefits include, efficacy on gastric ulcers, wounds and constipation, together with, preventive action against both atherosclerosis and hyper-cholesterol and antitumor activity. The suggested most important active substance is b-1,3-glucan which is believed to be an active immune-stimulator, free radical scavenger and a reducer of blood lipids. Unfortunately, the American Cancer Society concluded, ‘‘however, available

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scientific studies do not support its effectiveness for preventing or treating cancer or any other disease in humans’’. Spirulina (Arthrospira) is used in human nutrition because of it high protein content and excellent nutrient value. It is also a valuable source of the essential fatty acid, linolenic acid, that cannot be synthesised by humans. A wide range of medical benefits for a broad range of conditions have been claimed, but the full validations of many of these claims is still awaited. Many companies are producing ‘‘nutraceuticals’’ (food supplements with claimed nutritional and medicinal benefits) made from Spirulina. DIC claims to be the biggest manufacture of Spirulina in China. Production of Spirulina in Hainan by DIC was estimated at 300 t per annum. The largest plant is Earthrise Farms in California, USA, covering over 444,000 m2, producing algal tablets and powder, sold in over 20 countries, is owned by DIC in Japan. Cyanotech, in Kona, Hawaii, produces a powder under the name Spirulina Pacifica. The market for dried Spirulina was estimated to be US$ 40 million, in 2005. Carotenoids: Algae contain carotenoids, yellow orange or red pigments, that include b-carotene, a substance converted by the body to Vitamin A. There are over 400 known carotenoids, but only a few are used commercially, the two main compounds being b-carotene and astaxanthin. The most important uses of carotenoids are as food colourants and as supplements for human and animal feeds. The average concentration of carotenoids in most algae is only 0.1–2%, but Dunaliella, when grown under the right conditions of high salinity and light intensity, will produce up to 14% b-carotene. Dunaliella is, therefore, well suited to the commercial production of b-carotene and several industrial production plants are in operation around the world including Australia, Israel, USA and China. The major producer is Cognis Nutrition and Health, whose farms cover 800 ha in Western Australia and produce b-carotene extracts together with algal powder for human and animal use. Until 1980, production of b-carotene was synthetic. Natural carotenoids, although more expensive than synthetic, have the advantage of supplying the natural isomers in their natural ratio and the natural isomers of b-carotene are considered superior to the trans-synthetic form. In 1994, algal b-carotene was sold in small quantities due to the cost and the majority of production was synthetic, however, it was concluded that the increasing number of algal plants producing b-carotene rich Dunaliella, at that time, may change the situation. It has since been reported that b-carotene from Dunaliella, is a substantial growing industry and commercial utilisation is economically viable. Astaxanthin is another carotenoid that can be derived from algae and is principally used in fish farming and as a dietary supplement or anti-oxidant. The annual worldwide aquaculture market for this pigment in 2004 was estimated to be US$ 200 million with an average price of US$ 2500/kg, but the market is dominated by the synthetic form. Astaxanthin can be produced by Haematococcus, a freshwater alga that normally grows in puddles, birdbaths

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and other shallow fresh water depressions. Haematococcus can contain up to 3% astaxanthin, but it requires a two-stage culture process which is not suited to open pond cultivation. The first stage of the process is designed to optimize algal biomass (green thin-walled flagellated stage with optimum growth at a temperature 22–25°C) and the second stage (thick-walled resting stage) under intense light and nutrient-poor conditions during which astaxanthin is produced. Due to its high price, the astaxanthin produce from Haematococcus cannot compete with synthetic forms. However, for some applications, natural astaxanthin is preferred, for example, in carp, chicken and red sea bream diets due to enhanced natural pigment deposition, regulatory requirements and consumer demand for natural products. Commercial production is being carried out in Hawaii, India and Israel, where Algatech sell a crushed Haematococcus biomass on the pharmaceutical market. Cyanotech, in Hawaii, claimed a market share of over 95% of the animal nutrition market for algaebased astaxanthin products, but subsequently stopped selling into the animal nutrition market in March, 2008. Phycobiliproteins: In addition to chlorophyll and the lipophilic pigments, such as the carotenoids, certain types of microalgae especially red algae, or rhodophyta, contain phycocyanin and Phycoerythrin. These photosynthetic accessory pigments, collectively known Phycobiliproteins, are deeply colored (red or blue), water soluble, complex, proteinaceous compounds. These algae pigments have the potential as natural colorants for food, cosmetics and pharmaceuticals. Dainippon Ink and Chemicals produces a blue food colorant from Spirulina, called Lina blue, that is used in chewing gum, ice slush, sweets, soft drinks, dairy products and wasabi. Phycobiliproteins can be commercially produced from Spirulina and the red microalgae Porphyridium and Rhodella. Phycobiliproteins are widely used in clinical or research immunology because they have very powerful and highly sensitive fluorescent properties. In 1997, the global market for Phycobiliproteins colorants was estimated at US$ 50 million and prices vary from US$ 3 to US$ 25/mg. Fatty acids: Humans and animals lack the requisite enzymes to synthesize polyunsaturated fatty acids (PUFAs) of more than 18 carbon atoms and they must obtain them from food and are, therefore, often known as essential fatty acids. A group of essential fatty acids which are attracting a lot of attention currently are known as omega-3, a group of unsaturated fatty acids where a carbon double bond is in the third position from the methyl or omega end. Oily Fish and fish oils are well known sources of PUFAs, but issues have been raised concerning possible accumulation of toxins in fish. Fish obtain PUFAs from algae, with marine algae, such as diatoms, being a particularly rich source and therefore, algae have been proposed as a Commercial source. Docosahexaenoic acid (DHA) produced by heterotrophic (using plant or animal materials as an energy source rather than light in photosynthesis

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(autotrophic)) culture of the dinoflagellate, Crypthecodinium cohnii, is the only currently commercial produced fatty acid from algae. DHA (22:6) is a 22-carbon chain with six cis double bonds; the first double bond is located at the third carbon from the omega end (an omega-3 fatty acid). It is used as a supplement in infant formulas and as a dietary supplement. It is essential for the proper functioning of our brains as adults, and for the development of our nervous system and visual abilities during the first 6 months of life. In addition, omega-3 fatty acids are part of a healthy diet that helps lower risk of heart disease. Although, infants that are breastfed should receive enough DHA if the mother has an adequate intake of this fatty acid, many organizations have suggested that DHA should be added to baby milk formula. The world wholesale market for infant formula in 2005 was estimated to be about US$ 10 billion per annum. Martek produce DHA using Crypthecodinium cohnii for baby formula, and in 2003, production was 240 tons and it is now a company with more than 525 employees and revenue of more than US$ 300 million. Martek acquired, OmegaTech, another producer of a DHA, oil, known as DHA gold an adult dietary supplement from Schizochytrium—Nutrinova, formerly known as Hoechst until 1997, when it was taken over by Celanese, produced an oilcontaining DHA from Ulkenia. In 2005, Lonza, based in Switzerland, acquired Nutrinova’s DHA business and sells the DHA oil as a vegetarian source of omega-3. A process for producing high-purity Eicosapentaenoic acid, EPA, another omega-3 fatty acid (20:5), from Phaeodactylum tricornutum, has been developed by the University of Almeria in Spain. An economic analysis, on a potential facility producing 430 kg 96% pure EPA per year, estimated the total cost of production at US$ 4,602/kg, with 60% of the cost arising from the recovery process and 40% from the biomass production. It is believed that the cost needs to be reduced by 80% to be economically viable. The residual biomass following the extraction of the EPA contains too much residual solvent to be sold for animal feed, and therefore, must be incinerated. The annual worldwide demand of EPA is 300 t. The production of EPA from Nannochloropsis and, the diatom, Nitzschia, is reportedly under study. It is reported that studies are being undertaken to produce omega-6 PUFAs; Linolenic acid (18:3) from Arthrospira and Arachidonic acid (20:4) from Porphyridium. Stable isotopic biochemicals: Microalgae are wellsuited as a source of isotopically labelled compounds due to their ability to incorporate stable isotopes from relatively inexpensive inorganic molecules into high value isotopic organic chemicals. The market for these chemicals is in excess of US$ 13 million. Spectra Stable Isotopes, now part of Cambridge Isotope Laboratories, sells marked amino acids at up to US$ 5900/g and marked nucleic acids at US$ 28/mg. Animal feed: Microalgae are an important food source and feed additive in the commercial rearing of many aquatic animals. Over 30% of the current

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world algal production is sold for animal feed and over 50% of the world production of Spirulina is used as feed supplements. In 1999, the production of microalgae for use in aquaculture reached 1000 t. Approximately, a dozen types are produced in relatively small quantities for the aquaculture industry. Many studies have shown the suitability of algae as a potential animal feed and as a replacement for conventional protein sources such as soybean and fish-meal. Algae are the normal natural food for many animals used in aquaculture and, it is not surprising that, they are considered the best food source for aquaculture. Unfortunately, the trend is to avoid using live algae due to their high cost and production difficulties. The cost of producing dry algal biomass feed, in Australia, varies from US$ 80/kg to US$ 800/kg. Other cost estimates have given costs between US$ 50/kg to US$ 150/kg with a peak value of US$ 1000/kg. Yeast can be used as a replacement feed, but the omission of algae from aquaculture may give less predictable performance and the total replacement of algae in aquaculture diets are not yet considered sufficiently advanced for widespread utilisation. In addition to direct feed, microalgae can be used as a feed source for zooplankton which can, in turn, be used as a feed for fish. One problem that has been encountered is, with the notable exception of Spirulina, poor digestibility due to the high content of cellulose cell wall material. Ruminates, such as sheep and cattle, are capable of digesting cellulose material and it is therefore, possible to feed algae direct to them, but this has not gained much commercial favor yet. Poultry can be fed up to 5–10% algae and, this can have a positive effect on the development of colour within the skin and egg yolk due to the carotenoids. Higher concentrations of algae in the feed can lead to adverse effects. Human food: Although, microalgae are eaten as a food in China and Chad and, had been considered as a solution to the world’s food shortage, their use on a global scale appears limited to health food and food supplements. The current state of microalgal production Alga

Annual production

Producer country

Applications and products

Spindina

3,000 t dry wt

China, India, USA, Myanmar, Japan

Human and animal nutrition, phycobiliproteins, cosmetics

Chlorella

2,000 t dry wt

Taiwan, Germany, Japan

Human nutrition, aquaculture, cosmetics

Davabella

1,200 t dry wt

Australia, Israel, USA, China

Human nutrition, cosmetics, b-carotene

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Apyani

500 t dry wt

USA

Human nutrition

Haemodo

300 t dry wt

USA, India, Aquaculture, Israel Astaxanthin

Crypthiodinium

240 t DHA oil

USA

DHA oil

Schizohytrium

10 t DHA oil

USA

DHA oil

9.3.2  Future Development of Microalgal Applications Market potential: Microalgae have been exploited by man for millennia and the BEAM network, supported by Murdoch University, Australia, believes that microalgae biotechnology has grown and diversified significantly over the past 30 years. Although, some progress has been made and there are commercial microalgae applications, including pigments, fatty acids and health foods, only a few hundred of the thousands of species of microalgae have been studied and, just a handful are cultivated on an industrial scale. It would appear that the potential of commercial applications of microalgae are enormous, but, despite development over last 50 years, the number of commercially available products are still fairly limited and, although there are a number of types of closed bioreactor, being investigated and available, the majority of microalgal production is in open ponds. The main commercial product, despite the enormous range of biochemicals potentially available from microalgae, appears to be ‘‘health food’’ that may produce health benefits, but may be subject to fashion and fad. The second current key area of commercial application of microalgae appears to be food additives in form of carotenes, pigments and fatty acids. These can have functional advantages over synthetic products or products from other natural sources, but can be at a cost disadvantage. The challenge in the application of microalgae for commercial ends is to focus only on those products with a large market and/ or profit potential where the use of microalgae leads to clear competitive advantages. Fuel and food would appear to offer the largest markets. The growth of microalgae to solve the world food shortage has, in the past, been considered, but the wide-scale commercial growth of algae for human food is restricted mainly to health foods, food supplements and food additives. The health food market is the branch of algae production with the highest sales, but the market is dependent on a number of claims of health benefits without the necessary scientific proof of efficacy. There has also been some market sentiment that Spirulina was being overproduced. Food additives, such as b-carotene, pigments and fatty acids, from microalgae, can be superior to synthetic products and products from other natural sources, such as fish oil, but unfortunately, they are often also considerably more expensive. There appears to be very considerable potential

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for the discovery of new therapeutic biochemicals from microalgae, but an immediate breakthrough for algae-based products does not appear imminent. Current commercially viable exploitation of microalgae products is limited to products other than fuel and, the immediate future for the commercialization of microalgae may be with non-fuel products, but the ‘‘lessons’’ learned from microalgal non-fuel products, together with their potential coproduction with fuel, may lead to the more rapid commercial realization of microalgal biofuel. Growth systems: The commercial growth of algae in open ponds, despite over 30 years of research, is still only currently viable for three taxi, Spirulina, Dunaliella and Chlorella, mainly due to the suppression of the growth of competitive species by use of highly selective environments. Currently, the majority of microalgal production occurs in outdoor ponds. Concerns have also been expressed about the possible contamination of food from microalgae grown in open ponds. Bioreactors are considerably more expensive than open ponds and their use will probably be restricted to very high value products. It has been argued that lower extraction costs, due to the higher algae content, can make bioreactors more competitive. A study on the production of the valuable fatty acid, EPA, found that 60% of the costs arise from the recovery process and the cost of algae biomass production in bioreactors is high and needs to be reduced. It has also been concluded that, although some products are now being produced in bioreactors commercially, the development of microalgae biotechnology has been slowed by the limited performance of bioreactors. Open ponds are likely to remain the major means of production, but efficient closed bioreactors may be viable in the production for high value products where purity is essential or a ‘sensitive’ algae species is required. Species selection: Many consider that the genetic modification of microalgae is the best way to improve the yield of valuable products at reduced cost. The absence of cell differentiation in microalgae can make genetic manipulation simpler than in higher plants, but progress on the genetic engineering of microalgae was relatively slow, until recently. The NREL in the USA spent considerable time, effort and resources on the genetic modification of microalgae. The NREL work on genetic modification may have diverted their resources from algae selection and process optimization that may have yielded more commercial benefit. Genetic modification has a considerable image problem, particularly in Europe and, genetically modified algae may also be seen as a potential environmental threat, particularly, if open pond systems are to be used. Part of the appeal of microalgae substances for food and therapeutic use is their ‘‘natural image” and genetic modification may have a negative effect on this. Algae strain selection and process optimization, rather than genetic engineering, may be the key areas for future development.

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9.4  MASS CULTIVATION OF MICROALGAE AS A SOURCE OF PROTEIN AND FEED Microalgae are microscopic photosynthetic organisms that are found in both marine and freshwater environments. Their photosynthetic mechanism is similar to landbased plants, but being due to a simple cellular structure, and submerged in an aqueous environment where they have efficient access to water, CO2 and other nutrients, they are generally more efficient in converting solar energy into biomass. In terms of biomass, microalgae form the world’s largest group of primary producers and they occur in benthic, epithelic, symbiotic and pelagic forms. The microalgal biomass contains all the essential amino acids, unsaturated fatty acids, carbohydrates, dietary fiber, and a whole range of vitamins and other bioactive compounds, so that, it can be a highly suitable alternative in livestock feeding, human nutrition and perhaps, also in biofuel industry. Photo bioreactors: Photo bioreactors are different types of tanks or closed systems in which microalgae are cultivated. Microalgal cultures consist of a single or several specific strains optimized for producing the desired product. Water, necessary nutrients and CO2 are provided in a controlled way, while oxygen has to be removed. Microalgae receive sunlight either directly through the transparent container walls or, via light fibers or tubes, that channel it from sunlight collectors. A great amount of developmental work to optimize different photobioreactor systems for algae cultivation has been carried out by many researchers. It has also been suggested to grow heterotrophic algae in conventional fermenters instead of photobioreactors for production of high-value products. Instead of light and photosynthesis, heterotrophic algae are relying on utilizable carbon sources in the medium for their carbon and energy generation. Open pond systems: Open pond systems are shallow ponds in which algae are cultivated. Nutrients can be provided through runoff water from nearby land areas or by channeling the water from sewage/water treatment plants. The water is typically kept in motion by paddle wheels or rotating structures, and some mixing can be accomplished by appropriately designed guides. Algal cultures can be defined (one or more selected strains), or are made up of an undefined mixture of strains. • The high capital cost associated with producing microalgae in closed culture systems is the main challenge for commercialization of such systems. Open systems do not require expenses associated with sterilization of axenic algal cultures. However, this leads to high risk of contamination of the culture by bacteria or other unwanted microorganisms. A common strategy, therefore, to achieve monocultures in an open pond system is to keep them at extreme culture conditions such as high salinity, nutrition or alkalinity. Consequently, this strictly limits the species of algae that can be grown in such systems. From the public literature, currently only Dunaliella (high salinity), Spirulina

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(high alkalinity) and Chlorella (high nutrition) have been successfully grown in commercial open pond systems. • The necessity for a large cultivation area has been pointed out as a limitation in using open ponds to grow microalgae for mitigating the CO2 released from power generating plants. It has been estimated that a raceway pond requires 1.5 km2 to fix the CO2 emitted from a 150 MW thermal power plant. The large area requirements are partly due to the comparable lower productivity of open pond systems. It was pointed out that improving the control of limiting parameters in open ponds such as culture medium temperature and contamination, and thereby, increasing productivity, could be accomplished by using a transparent cover over the ponds, such as a greenhouse. • Selection of a suitable production system clearly depends on the purpose of the production facility. For example, closed bioreactors will not be suitable for wastewater treatment, because the costs for treating wastewater in this system will be too high in relation to the low value added during the production process. On the other hand, high quality/ value products that are produced only in small amounts might require production in bioreactors. Harvesting of microalgae: Conventional processes used to harvest microalgae include concentration through centrifugation, foam fractionation, flocculation, membrane filtration and ultrasonic separation. Harvesting costs may contribute 20 – 30% to the total cost of algal biomass. The micro-algae are typically small with a diameter of 3–30 mm, and the culture broths may be quite dilute at less than 0.5g L–I. Thus, large volumes must be handled. The harvesting method depends on the species, on the cell density, and often also on the culture conditions. Applications of microalgae: Microalgae find uses as food and as live feed in aquaculture for production of bivalve molluscs, for juvenile stages of abalone, crustaceans and some fish species and, for zooplankton used in aquaculture food chains. Therapeutic supplements from microalgae comprise an important market in which compounds such as b-carotene, astaxanthin, polyunsaturated fatty acid (PUFA) such as DHA and EPA and polysaccharides such as b-glucan, dominate. Exploitation of microalgae for bioenergy generation (biodiesel, biomethane, biohydrogen), or combined applications for biofuels production and CO2-mitigation, by which CO2 is captured and sequestered, are under research. The dominating species of micro-algae in commercial production includes Isochrysis, Chaetoceros, Chlorella, Arthrospira (Spirulina) and Dunaliella.

9.5  MICROALGAE AS A SOURCE OF FEED Microalgae aquaculture feeds: The largest current application of microalgae feeds is in aquaculture. Microalgae are used fresh (e.g. live, or at least not

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dried) in bivalve, shrimp and fish fry and fingerling production (in the latter case, via an intermediate food source, such as zooplankton or brineshrimp). Several companies produce aquaculture feeds using Chlorella and Spirulina, or a mixture thereof. Some examples of the use of microalgae for aquaculture:

• Microalgae species Hypneacervicornis and Cryptonemia crenulata particularly, rich in protein, were tested in shrimp diets. Algae were collected, rinsed, dried and ground up for the feed formulations. Larvae shrimps were fed daily with one of four diets prepared with different percentages of seaweed powder: 39%, 26%, 13%, 0%. The results suggest that there is an increase in feed conversion when the levels of algae are increased. Amount of algae in fish meal resulted in significant increase in shrimp growth rates.



• A large number of marine nitrogen-fixing cyanobacteria have been tested for their nutritional value with the hybrid Tilapia fish fry; a majority were acceptable as single ingredient feeds. Very high growth rates of Tilapia fish using marine cyanobacteria, with indoor and outdoor cultures, have been reported. The marine cyanobacterium Phormidium valderianum was shown to serve as a complete aquaculture feed source, based on the nutritional qualities and non-toxic nature with animal model experiments.



• More than 40 species of microalgae are used in aquaculture worldwide, depending on the special requirements of local seafood production.



• Apart from feeding larvae and zooplankton, often with special microalgal species, the addition of Spirulina and Chlorella to common fish feed compositions seems to be a promising market. Initially, the colour-enhancing effects of phycocyanin-containing Spirulina biomass or carotenoides from Dunaliella were exploited in ornamental fish.



• In recent years, questions of feed utilization and health status in the dense aquacultural fish populations became more important. Here, the addition of microalgae can, depending on concentration, directly enhance the immune system of fish, as investigations on carp have shown.

The addition of microalga-derived astaxanthin to feed formulations enhances the colour of the muscles of salmonids. This has a high biotechnological potential and culture techniques for Haematococcus pluvialis are well developed for this purpose. Microalgae as poultry feeds: In poultry rations, algae up to a level of 5-10% can be used safely as partial replacement for conventional proteins. The yellow colour of broiler skin and shanks, as well as of egg yolk, is the most important characteristics that can be influenced by feeding algae. Moreover, the Institut für Getreideverarbeitung (Bergholz-Rehbrücke, Germany) produces a natural feed with the algae Chlorella and Arthrospira, called Algrow.

Algae-Biotechnology

9.31



• Ginzberg and his group in the year 2000 studied role of algae, Porphyridium sp. as feed supplement on metabolism of chicken. Earlier results in the same laboratory showed a reduction in serum cholesterol and triglyceride-levels in rodents fed with red algal biomass. In this study, lyophilized algae biomass was fed to chickens at a proportion of 5% or 10% of the standard chicken diet. Chickens fed with algae biomass consumed 10% less food and their serum cholesterol levels were significantly lower (by 11% and 28%, for the groups fed with 5% and 10% supplement, respectively) as compared with the respective values of the control group (with unsupplemented diet).



• Egg yolk of chickens fed with algae tended to have reduced cholesterol levels (by 10%) and increased linoleic acid and arachidonic acid levels (by 29% and 24%, respectively). In addition, the colour of egg yolk became darker, indicating that higher carotenoid was produced (2.4 fold higher).



• Other poultry feeding studies with Spirulina (up to 30%) showed that, both protein and energy efficiency of this alga were similar to other conventional protein carriers up to a level of 10%. Significantly higher growth rates and lower non-specific mortality rate were observed in turkey poults fed with Spirulina at the level of 1-10 g.kg -1 diet.

Microalgae as swine feeds: Abril and his group studied the potential toxicity of DHA-rich microalgae (DRM) from Schizochytrium sp., administered in the diet of growing swine. The only DHA-rich microalgae treatmentrelated changes were higher weight gain and feed conversion efficiency. The administration of DRM (at up to five times the anticipated commercial dose) did not produce any treatment-related adverse effects in commercial strains of swine. Microalgae trials in pet feeds: Another very promising application for microalgal biomass is the pet food market, where not only the healthpromoting effects but also, effects on the external appearance of the pet (shiny hair, beautiful feathers) are of consumer importance. Studies on minks and rabbits provide evidence of such effects for pets. Other microalgae trials: Belay and his group assessed potential of Arthrospira (Spirulina) in animal feed. About 30% of the current world production of 2000 tõn  Spirulina is sold for animal feed applications. Some of these positive effects of Spirulina like, increased growth rate, colour enhancement and general tissue quality may be nutritional effects. However, the fact that growth rates are improved even at 0.1% Spirulina supplementation may suggest the presence of substances that may mimic the effects of or stimulate production of growth hormones. The most promising application may be its immune enhancement effects and through this, its anti-viral and anti-bacterial properties, since these effects are exhibited at very low supplemental concentrations in the feeds.

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• Spirulina or its extracts may accelerate development of the immune system of many animals, especially, during the early stages of their lives. Presently, Arthrospira is widely used as food additive and can replace 50% of protein diets in existing feeds.



• Astaxanthin, from H. pluvialis, is therefore, of interest comparing with other sources. More than 80% of astaxanthin from this microalgae, is found in an esterified form, whereas in synthetic astaxanthin, it is in the free form. In birds, astaxanthin in an esterified form has been shown to be more efficiently absorbed than free astaxanthin.



• In mice infected with Helicobacter pylori, treatment with H. pluvialis algal meal, significantly reduced the bacterial load of H. pylori in the stomach. This was explained by the effects of astaxanthin on cytokines produced by H. pylori-specific T- cells.

CHAPTER

10

Concepts and Scope of Plant Biotechnology

Plant biotechnology may be defined as the application of knowledge obtained from study of the life sciences to create technological improvements in plant species. By this very broad definition, plant biotechnology has been conducted for more than ten thousand years.

• The roots of  plant biotechnology  can be traced back to the time when humans started collecting seeds from their favorite wild plants and began cultivating them in tended fields. It appears that when the plants were harvested, the seeds of the most desirable plants were retained and replanted the next growing season.



• While these primitive agriculturists did not have extensive knowledge of the life sciences, they evidently did understand the basic principles of collecting and replanting the seeds of any naturally-occurring variant plants with improved qualities, such as those with the largest fruits or the highest yield, in a process that we call artificial selection. This domestication and controlled improvement of plant species was the beginning of plant biotechnology. 



• This very simple process of selectively breeding naturally-occurring variants with observably improved qualities served as the basis of agriculture for thousands of years and resulted in thousands of domesticated plant cultivars that no longer resembled the wild plants from which they descended. The second era of plant biotechnology began in the late 1800s as the base of knowledge derived from the study of the life sciences increased dramatically. 



• In the 1860s Johannes Gregor Mendel, using data obtained from controlled pea breeding experiments, deduced some basic principles of genetics and presented these in a short monograph modestly titled “Versuche über Pflanzenhybriden” (in Verhandlungen des naturforschenden Vereins, 1866; Experiments with Plant-Hybridisation, 1910). 

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• In this publication, Mendel proposed that heritable genetic factors segregate during sexual reproduction of plants and that factors for different traits assort independently of each other.  • Mendel’s work suggested a mechanism of heritable factors that could be manipulated by controlled breeding of plants through selective fertilization and, also suggested that, the pattern of inheritance for these factors could be analyzed or, in some cases, predicted by the use of mathematical statistics. • These findings complemented the work of Charles Darwin, who expounded the principles of descent with modification and selection as the chief factor of evolutionary change in his 1859 book on the Origin of Species by Means of Natural Selection. The application of these principles to agriculture resulted in deliberatelyproduced hybrid varieties for a large number of cultivated plants via selective fertilization. These artificially selected hybrids soon began to benefit humankind with tremendous increases in both the productivity and the quality of food crops.

10.1 APPLICATIONS OF GENETIC ENGINEERING TECHNOLOGY FOR CROP IMPROVEMENT The third era of plant biotechnology involves a drastic change in the way crop improvement may be accomplished, by direct manipulation of genetic elements (genes). This process is known as genetic engineering and results in plants that are called genetically modified organisms (GMOs), to distinguish them from plants that are produced by conventional plant-breeding methods. Genetically modified plants can contribute desirable genes from outside traditional breeding boundaries. Even genes from outside the plant kingdom can now be brought into plants. For example, animal genes, including human genes, have been transferred into plants, a feat not replicated in nature. • Our ancestors have been improving crops and livestock for thousands of years through selective breeding or crossbreeding to produce desired traits. Biotechnology is just an extension of this process. Genes are added, deleted or temporarily silenced to produce desired results. • Genetic engineering involves cutting and moving snippets of DNA from one plant to another. Permanently integrating new DNA into a plant’s original DNA forms what’s known as a transgenic plant or genetically modified organism (GMO). Steps in Genetic Engineering: The first genetically engineered plants, tobacco plants, were reported in the scientific literature in 1984. Since 1984, there have been thousands of genetically engineered plants produced in laboratories worldwide. The process of genetically engineering a plant involves several key steps: • Isolating the genetic sequence (gene) to be placed from its biological source.

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10.3



• Placing the gene in an appropriate vehicle to facilitate insertion into plant cells.



• Inserting the gene into the plant by a process known as plant transformation.



• Selecting the few plant cells that contain the new gene (transformed cells) out of all the plant cells in the explant.



• Multiplying the transformed cells in sterile tissue culture.



• Regenerating the transformed cells into a whole plant that can grow outside the tissue culture vessel.

The gene or genes to be placed in the plant may be obtained from virtually any biological source: animals, bacteria, fungi, viruses, or other plants. Placing genes into an appropriate vehicle for transfer into a plant involves using various molecular biology techniques, such as restriction enzymes and ligation, to essentially “cut and paste” the gene or genes of interest into another DNA molecule, which serves as the transfer vehicle (vector). Major goals of genetic engineering of plants:

• Produce crops with less impact on environment.



• Reduce expense of food production.



• Produce crops less vulnerable to insects, diseases, weeds and harsh environments.



• Develop crops with more nutrients.



• Develop crops for production of medicines and vaccines. Major genetically engineered traits in plants:



• Insect resistance,



• Herbicide resistance,



• Virus resistance,



• Delayed fruit ripening,



• Altered oil content,



• Pollen control.

Public Concern: It is perhaps this lack of natural boundaries for genetic exchange that seems so foreign to conventional scientific thought and that makes plant genetic engineering controversial.

• The thought of taking genes from animals, bacteria, viruses, or any other organism and putting them into plants, especially plants consumed for food, has raised a host of questions among concerned scientists and public alike. 



• Negative public perception of genetically modified crops has affected the development and commercialization of many  plant biotechnology products, especially food plants. While there are dozens

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Environmental Biotechnology

of genetically engineered plants ready for field production, public pressure has delayed the release of some of these plants and has caused the withdrawal of others from the marketplace.

• This public concern also appears to be driving increased government review of products and decreased government funding for  plant biotechnology  projects in Europe. Negative public perceptions do not seem to be as strong in Asia, since the pressures of feeding large populations tend to outweigh the perceived risks. 



• The social climate of the United States toward biotechnology, although guarded, appears to be less apprehensive than that of most European countries. Therefore, many agricultural biotechnology projects have moved from European countries to U.S. laboratories.

Economic Goals: To what end are humans genetically engineering plants? This is an  essential question for researchers, executives of biotechnology companies, and consumers, at large.  Before addressing technical questions about how to apply biotechnology, the desired goals must be clearly defined. The general goals of  plant biotechnology appear to be: 1. economic improvement of existing products,  2. improvement of human nutrition, and  3. development of novel products from plants. Economic improvements include increases in yield, quality, pest resistance, nutritional value, harvest ability, or any other change that adds value to an established agricultural product.  Examples of this category include insect-protected tomatoes, potatoes, cotton, and corn; herbicide-resistant canola, corn, cotton, flax, and soybeans; canola and soybeans with geneticallyaltered oil compositions; virus-resistant squash and papayas; and improved ripening tomatoes. All these examples were introduced to agriculture in the later half of the 1990s. Nutritional Goals: Additionally, some products appearing in the scientific literature but awaiting commercialization have the potential to dramatically improve human nutritional deficiencies, which are especially prevalent in developing countries. 

• These products include “golden rice,” genetically modified rice that produces carotenoids, a dietary source of vitamin A. Golden rice has the potential to prevent vitamin A deficiency in developing countries, where this vitamin deficiency is a leading cause of blindness.



• Researchers are also using genetic engineering to increase the amount of the iron-storing protein ferritin in seed crops such as legumes. Iron deficiency, which affects 30 per cent of the human population, can impair cognitive development and cause other health problems. This

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10.5

proposed enhancement of iron content in consumable plant products could help more than a billion people who suffer from chronic iron deficiency.

10.2 PRODUCTION OF TRANSGENIC PLANTS WITH IMPROVED YIELDS AND NUTRITIONAL QUALITY During the last decades, a tremendous progress has been made in the development of transgenic plants using various techniques of genetic engineering. The plants, in which a functional foreign gene has been incorporated by any biotechnological methods, that generally are not present in the plant, are called transgenic plants. As per estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) land by about 5.5 million farmers. Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control, etc. Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High-lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insectresistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows: Stress tolerance: Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity, etc). Abiotic stress tolerance: The plants show their abiotic stress response reactions by the production of stress related osmolytes like sugars (e.g. trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline, glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic plants have been produced which, overexpress the genes for one or more of the above mentioned compounds. Such plants show increased tolerance to environmental stresses. Resistance to abiotic stresses includes stress induced by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone and intense light. These environmental stresses result in the destruction and deterioration of crop plants which leads to low crop productivity. Several strategies have been used and developed to build resitance in the plants against these stresses. Herbicide tolerance: Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g., the overproduction of herbicide-target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme

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Environmental Biotechnology

targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants, tolerant to herbicide—Roundup, is already being used commercially. The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) Overexpression of the target protein by integrating multiple copies of the gene or by using a strong promoter. (b) Enhancing the plant detoxification system which helps in reducing the effect of herbicide. (c) Detoxifying the herbicide by using a foreign gene, and (d) Modification of the target-protein by mutation. Some of the examples are: Glyphosate resistance—Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus, blocks the shikimic acid pathway. Certain strategies were used to provide glyphosate resistance to plants. • It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia and introduced into the other plants. These plants could tolerate glyphosate at a dose of 2–4 times higher than that required to kill wildtype plants. • By using mutant EPSPS genes—A single base substitution from C to T resulted in the change of an amino acid from proline to serine in EPSPS. The modified enzyme cannot bind to glyphosate and thus, provides resistance. • The detoxification of glyphosate by introducing the gene (isolated from soil organism—Ochrobactrum anthropi) encoding for glyphosate oxidase into crop plants. The enzyme glyphosate oxidase converts glyphosate to glyoxylate and amino methylphosponic acid. The transgenic plants exhibited very good glyphosate resitance in the field.

Another example is of Phosphinothricin resistance Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed weeds. It acts as a competitive inhibitor of the enzyme glutaminesynthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbance in the glutamine synthesis also inhibits the photosynthetic activity. The enzyme phosphinothricin acetyl transferase (which was first observed in Streptomyces sp. in natural detoxifying mechanism against phosphinothricin)

Concepts and Scope of Plant Biotechnology

10.7

acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin. Other abiotic stresses: The abiotic stresses due to temperature, drought, and salinity are collectively also known as water-deficit stresses. The plants produce osmolytes or osmo protectants to overcome the osmotic stress. The attempts are on to use genetic engineering strategies to increase the production of osmoprotectants in the plants. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding the key enzymes have been isolated. E.g., Glycine betaine is a cellular osmolyte which is produced by the participation of a number of key enzymes like choline dehydrogenase, choline monooxygenase, etc. The choline oxidase gene from Arthrobacter sp. was used to produce transgenic rice with high levels of glycine betaine giving tolerance against water-deficit stress. Scientists also developed cold-tolerant genes (around 20) in Arabidopsis, when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold-tolerant genes), expression of cold-tolerant genes was triggered, giving protection to the plants against the cold temperatures. Insect resistance: A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management. About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants. The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular group of insect pests, and are not harmful to other useful insects like butterflies and silk worms. Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal, cauliflower, cabbage, etc.) have been developed. This has proved to be an effective way of controlling the insect pests and has reduced the pesticide use. The most notable example is Bt cotton (which contains CrylAc gene) that is resistant to a notorious insect pest Bollworm (Helicoperpa armigera). There are certain other insectresistant genes from other microorganisms which have been used for this purpose. Isopentenyl transferase gene, from Agrobacterium tumefaciens, has been introduced into tobacco and tomato. The transgenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid. Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. One of the examples is the Cowpea

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Environmental Biotechnology

trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and oilseed rape for developing transgenic plants. Earlier it was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. It was observed that the insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera, etc. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsin, hence, it is non-toxic to mammals. Virus resistance: There are several strategies for engineering plants for viral resistance, and these utilize the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded gene, virus coat proteins, movement proteins, transmission proteins, satellite RNA, antisense RNAs, and ribozymes. The virus-coat protein—mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, when the transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV. The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa, etc. The viruses that have been used include Alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), Potato virus X (PVX), potato virus Y (PVY) etc. Resistance against Fungal and Bacterial infections: As a defense strategy against the invading pathogens (fungi and bacteria), the plants accumulate low molecular weight proteins which are collectively known as pathogenesisrelated (PR) proteins. Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani. Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed. Delayed fruit ripening: The gas hormone, ethylene, regulates the ripening of fruits therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene, forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripened by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.

Concepts and Scope of Plant Biotechnology

10.9

The most common example is the ‘Flavr Savr’ transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisense mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly, strategies have been developed to block the ethylene biosynthesis, thereby, reducing the fruit ripening. E.g., transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening. The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis. Male Sterility: The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA–hydrolyzing enzyme, that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce, etc. with male sterility have been developed. Nutritional quality: Transgenic crops with improved nutritional quality have already been produced by introducing genes involved in the metabolism of vitamins, minerals and amino acids.

• A transgenic Arabidopsis thaliana that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant has been developed. The biochemical machinery to produce a compound close in structure to alpha-tocopherol is present in A. thaliana. A gene that can finally produce alpha-tocopherol is also present, but is not expressed. This dormant gene was activated by inserting a regulatory gene from a bacterium which resulted in an efficient production of vitamin E.



• Glycinin is a lysine-rich protein of soybean and the gene encoding glycinin has been introduced into rice and successfully expressed. The transgenic rice plants produced glycinin with high contents of lysine. • Using genetic engineering, Prof. Potrykus and Dr. Peter Beyer have developed rice, which is enriched in pro-vitamin A, by introducing



10.10



Environmental Biotechnology

three genes involved in the biosynthetic pathway for carotenoid, the precursor for vitamin A. The aim was to help millions of people who suffer from night blindness due to Vitamin A deficiency, especially, whose staple diet is rice. The presence of beta-carotene in the rice gives a characteristic yellow/orange colour, hence this pro-vitamin A enriched rice is named as Golden Rice. • The genetic engineering is also being used to improve the taste of food e.g., a protein ‘monellin’ isolated from an African plant (Dioscorephyllum cumminsii) is about 100,000 sweeter than sucrose on molar basis. Monellin gene has been introduced into tomato and lettuce plants to improve their taste.

10.3  TRANSGENIC PLANTS FOR THE PRODUCTION OF VIRAL ANTIGENS A viral Antigen is an antigen with multiple antigenicities that is protein in nature, strain-specific, and closely associated with the virus particle. A viral antigen is a protein, encoded by the viral genome. A viral protein is an antigen specified by the viral genome that can be detected by a specific immunological response. Viruses are infectious pathogens that cause serious diseases & major threats for global public health, such as influenza, hepatitis, & AIDS. Virus is a sub-micrometer particle that has DNA or RNA packed in a shell called capsid. Viral antigens protrude from the capsid and often fulfill important function in docking to the host cell, fusion, and injection of viral DNA/RNA. Antibodybased immune responses form a first layer of protection of the host from viral infection; however, in many cases a vigorous cellular immune response mediated by T-cells and NK-cells is required for effective viral clearance. When cellular immunity is unable to clear the virus, the infection can become chronic, and serum antibodies to the viral pathogen are used as first indicator for the diagnosis of the disease. ELISAs provide a valuable tool in the detection and diagnosis of virus infection. The ability to produce recombinant viral proteins will ensure that future ELISAs are safe, specific and rapid. Even when a virus cannot be cultured, provided gene sequence is available, it is possible to rapidly respond to emerging viruses and new viral strains of existing pathogens. Recombinant viral antigens contain part of viral sequence, meaning that, the recombinant antigen contains a region which can be recognized by different antibodies produced by different individuals. This reduces the risk of false negatives which can occur with synthetic peptides, which contain only a small portion of the entire protein. If an individual infected with a viral antigen makes antibodies to a part of the protein not included in the synthetic peptides, a false negative results. Recombinant viral protein usually contains a fusion protein/partner which produces superior attachment to assay surfaces such as wells. For this

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10.11

reason, smaller amounts of recombinant protein will produce the same results as larger amounts of infused protein. The choice of fusion partner prevents false positives, allowing superior adhesion without incorrect results. Recombinant Viral proteins are expressed in bacteria, yeast, mammalian cells, and viruses. E. coli cells were first to be used for this purpose but the expressed proteins were not glycosylated, which was a major drawback since many of the immunogenic proteins of viruses such as the envelope glycoproteins, were glycosylated. Nevertheless, in many instances, it was demonstrated that the non-glycosylated protein backbone was just as immunogenic. The obvious advantage of recombinant viral antigens is that they are available in unlimited quantities and the production and quality control processes is simple. Advantages of using recombinant viral antigens:

• Production and quality control is simple.



• No nucleic acids or other viral or external proteins, therefore less toxic.



• Safer in cases where viruses are oncogenic or establish a persistent infection.



• Feasible even if virus cannot be cultivated Disadvantages:



• May be less immunogenic than conventional inactivated whole-virus vaccines.



• Requires adjuvant.



• Fails to elicit CMI. Facts about Viral Antigens:



• A Viral Protein Mimics its Way into cells.



• Viral Protein Helps Infected T Cells Stick To Uninfected Cells.



• The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Trans-activation Pathway.



• Viral Protein is an effective preventative against ear infection.



• HIV-1 Viral Protein R Induces Apoptosis via a Direct Effect on the Mitochondrial Permeability Transition Pore.



• The Level of Viral Antigen, presented by Hepatocytes, Influences CD8 T-Cell Function.



• Antigen-presenting cells from calves persistently infected with bovine viral diarrhea virus, a member of the Flaviviridae, are not compromised in their ability to present viral antigen.



• There is a difference in the distribution and spread of a viral antigen, development of lesions and correlation between presence of viral antigen and lesions.

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Environmental Biotechnology



• The absence of viral antigens on the surface of equine herpesvirus1-infected peripheral blood mononuclear cells is a strategy to avoid complement-mediated lysis.



• Viral Protein Influences Key Cell-signaling Pathway.



• A viral protein produced by cancer-causing virus influences a key signaling pathway in the immune cells that the virus infects. This stimulates the cells to divide, helping the virus spread through the body.



• Protection by recombinant viral proteins against a respiratory virulent avian metapneumovirus has been achieved.



• Viral O-acetylesterases are found in Influenza C viruses and Coronaviruses. Viral O-acetylesterases remove cellular receptors from the surface of target cells which destroys the receptor. Recombinant viral O-acetylesterases, derived from Sf9 insect cells as chimeric proteins fused to eGFP, specifically hydrolyze 9-O-acetylated sialic acids while that of Sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids.

To date, many plant species have been used for vaccine production. Early studies used tobacco and potato but now tomato, banana, corn, lupine, lettuce and others are being used for this purpose. The choice of the plant species (and tissue, in which the protein accumulates) is important and is usually determined through, how the vaccine is to be applied in the future. For example, an edible, palatable plant is necessary if the vaccine is planned for raw consumption. This limitation is overcome in non-edible plants by vaccine antigen extraction and purification. Antigen extraction is often performed when using tobacco, a plant that offers considerable experimental advantages such as ease of transformation and extensive genomic sequence knowledge. Heat treatment is feasible only if there is no deleterious effect on antigen stability. Recently, a “cooked” GM corn snack that accumulates the E. coli heatlabile enterotoxin has been proposed. In the case of vaccines for animal use, the plant should preferentially be selected among those consumed as normal component of the animals’ diet. The production of a vaccine in plants depends upon the availability of a DNA sequence coding for a protective antigen and on the construction of an expression “cassette” suitable for plant transformation. Stable plant transformation currently offers two options: insertion of the foreign gene into the nuclear genome or into the chloroplast genome. Transient plant transformation has also been used for plant expression of vaccine antigens through integration of the gene of interest into a plant virus and subsequent

Concepts and Scope of Plant Biotechnology

10.13

infection of susceptible plants. Plants producing two or more antigens may also be obtained through transformation with multiple gene constructs or through sexual crossing. The strategies for plant expression cassette construction and plant transformation depend on the desired goal.

10.3.1  Edible Vaccines Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The approach is to isolate genes encoding antigenic proteins from the pathogens and then expressing them in plants. Such transgenic plants or their tissues producing antigens can be eaten for vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops like banana and tomato are useful for immunization of humans, since banana and tomato fruits can be eaten raw. Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses, e.g. rabies virus, herpes virus. In 1990, the first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco at 0.02% of total leaf protein level was published in the form of a patent application under the International Patent Cooperation Treaty (Mason and Arntzen,1995). The first clinical trials in humans, using a plant derived vaccine were conducted in 1997 and were met with limited success. This involved the ingestion of transgenic potatoes with a toxin of E.coli causing diarrhea. The process of making of edible vaccines involves the incorporation of a plasmid carrying the antigen gene and an antibiotic-resistance gene, into the bacterial cells, e.g. Agrobacterium tumefaciens. The small pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. The surviving cells with altered genes multiply and form a callus. This callus is allowed to grow and subsequently transferred to soil to form a complete plant. In about a few weeks, the plants bear potatoes with antigen vaccines. The bacteria E.coli, V. cholerae cause acute watery diarrhea by colonizing the small intestine and by producing toxins. Chloera toxin (CT) is very similar to E.coli toxin. The CT has two subunits, A and B. Attempt was made to produce edible vaccine by expressing heatlabile enterotoxin (CT-B) in tobacco and potato. Another strategy adopted to produce a plant-based vaccine, is to infect the plants with recombinant virus carrying the desired antigen that is fused to viral coat protein. The infected plants are reported to produce the desired fusion protein in large amounts in a short duration. The technique involves, either placing the gene downstream a subgenomic promoter, or fusing the gene with capsid protein, that coats the virus.

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Environmental Biotechnology

Technical and social benefits envisaged in plant-derived edible vaccines No.

Benefit

Characteristics

1.

Oral delivery

The plant cell wall, consisting essentially of cellulose and sugars, provides protection in the stomach and gradual release of the antigen in the gut.

2.

Use as raw food or dry powder

The vaccinogenic plant tissue may be used as raw food, dried or, alternatively, proteins may be partially or fully purified and administered in capsules as dry powder.

3.

No need for “cold chain”

The vaccinogenic plant parts or plant extracts can be stored and shipped at room temperature.

4.

Mucosal and serum immune response

Plant-derived vaccines are primarily designed to trigger the mucosal immune system (IgA), thus preventing pathogen entry at mucosal surfaces; they also elicit serum and, possibly, cytotoxic responses.

5.

Cost efficiency

Production cost will be reduced 100–1000 times as compared with that of traditional vaccines.

6.

Optimised expression system

Plants may be engineered to accumulate the antigenin – convenient intracellular compartments (endoplasmic reticulum, chloroplast)

7.

Ease of genetic manipulation

Procedures essentially rely on established molecular and genetic manipulation protocols; these are already available in developing countries.

8.

Ease of production and scale-up

GM-plants can be stored as seeds. Unlimited vaccine quantity can be produced from these in limited time; production and management is suitable for developing countries.

9.

Safer than conventional vaccines

Lack of contamination pathogens.

10.

Ideal for ace bio-A. weapons

Safety and cost-efficiency propose, plants plant-derived vaccines, as an ideal tool to face bio-terrorism.

11.

Ideal for veterinary use

Cost-affordable, Ready for use as food additive.

with

mammalian

Concepts and Scope of Plant Biotechnology

10.15

Advantages of edible vaccines: The edible vaccines produced in transgenic plants will solve the storage problems, will ensure easy delivery system by feeding and will have low cost as compared to the recombinant vaccines produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera and hepatitis B, by feeding them banana, tomato, and vaccinating animals against important diseases, will be an interesting development. Safety and public acceptance: Plant-derived vaccines are certified free from animal pathogen contaminants. Furthermore, plant DNA is not known to interact with the animal DNA and plant viral recombinants do not invade mammalian cells. Further safety of plant-derived vaccines is obtained through following the same regulations established for traditional vaccines. Nevertheless, the present concern over the use of GM-plants is now affecting research in this important field, especially in Europe. One of the fears is that GM-pollen may outcross with sexually compatible plants (related crops or weeds) and affect biodiversity. In order to address this alarm, several pollen-containment approaches have been developed. These are essentially based on the exploitation of different forms of male sterility (suicide genes, infertility barriers, apomixis). An alternative way of solving the problem is engineering vaccines into the cpDNA, which is not transmitted to the sexual progeny through the pollen grains. An additional safety feature would be the recognition of GM-plants that produce vaccines by the addition of genes encoding coloured plant pigments. It is important to recognize that plants that produce vaccines are medicinal plants and should be grown, processed and regulated as pharmaceutical products. It is thought that pharmaceutical crops will be able to be grown on relatively small extensions of land, preferably contained within greenhouses, using controlled environmental conditions. In the majority of earlier papers, level of antigen accumulation in the plant organ was in the order of 0.1–0.4% of total soluble protein, while the more recent developments on cpDNA integration promises to increase this value to 30% or more. At the latter value, land requirements for industrial plant-derived vaccine-production will be in the order of a few thousand square meters. This will definitely enable vaccine-producing plants to be set apart from fieldgrown crop plants and offer added safety when engineered plant viruses are used for transient antigen expression. A further point of public concern in GMplants is the presence of antibiotic-resistance genes (used as selective marker in most transgenic plants). Approaches have now been developed to generate GM-plants (with both nuclear or cpDNA integration) that do not carry these genes.

10.4 BIOTECHNOLOGY IN AGRICULTURE—MERITS AND DEMERITS Biotechnology is the application of scientific techniques to modify and improve plants, animals, and microorganisms to enhance their value. Agricultural biotechnology is the area of biotechnology involving applications to agriculture.

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Environmental Biotechnology

Agricultural biotechnology has been practiced for a long time, as people have sought to improve agriculturally important organisms by selection and breeding. An example of traditional agricultural biotechnology is the development of disease-resistant wheat varieties by cross-breeding different wheat types, until the desired disease resistance is present in a resulting new variety. In the 1970s, advances in the field of molecular biology provided scientists with the ability to manipulate DNA—the chemical building blocks that specify the characteristics of living organisms—at the molecular level. This technology is called Genetic engineering. It also allows transfer of DNA between more distantly related organisms than was possible with traditional breeding techniques. Today, this technology has reached a stage where scientists can take one or more specific genes from nearly any organism, including plants, animals, bacteria, or viruses, and introduce those genes into another organism. An organism that has been transformed using genetic engineering techniques is referred to as a transgenic organism, or a genetically engineered organism. Many other terms are in popular use to describe these aspects of today’s biotechnology. The term “genetically modified organism” or “GMO” is widely used, although genetic modification has been around for hundreds, if not thousands–of years, since deliberate crosses of one variety or breed with another results in offspring that is genetically modified compared to the parents. Similarly, foods derived from transgenic plants have been called “GMO foods,” “GMPs” (genetically modified products), and “biotech foods.” While some refer to foods developed from genetic engineering technology as “biotechnology-enhanced foods,” others call them “frankenfoods.” For the reasons discussed later in this publication, controversy affects various issues related to the growing of genetically engineered organisms and their use as foods and feeds.

Genetic engineering Vs traditional biotechnology In traditional breeding, crosses are made in a relatively uncontrolled manner. The breeder chooses the parents to cross; but at the genetic level, the results are unpredictable. DNA from the parents recombines randomly, and desirable traits such as pest resistance are bundled with undesirable traits, such as lower yield or poor quality. Traditional breeding programs are time-consuming and labor-intensive. A great deal of effort is required to separate undesirable from desirable traits, and this is not always economically practical. For example, plants must be backcrossed again and again over many growing seasons to breed out undesirable characteristics produced by random mixing of genomes. Current genetic engineering techniques allow segments of DNA, that code genes for a specific characteristic, to be selected and individually recombined in the new organism. Once the code of the gene that determines the desirable

Concepts and Scope of Plant Biotechnology

10.17

trait is identified, it can be selected and transferred. Similarly, genes that code for unwanted traits can be removed. Through this technology, changes in a desirable variety may be achieved more rapidly than with traditional breeding techniques. The presence of the desired gene controlling the trait can be tested for at any stage of growth, such as in small seedlings in a greenhouse tray. The precision and versatility of today’s biotechnology enable improvements in food quality and production to take place more rapidly than when using traditional breeding.

10.4.1 Transgenic Crops in the U.S. Market Although genetically engineered organisms in agriculture have been available for only 10 years, their commercial use has expanded rapidly. Recent estimates are that, more than 60–70 percent of food products on store shelves may contain at least a small quantity of crops produced with these new techniques. Major crop plants produced by genetic engineering techniques have been so welcomed by farmers, that, currently a third of the corn and about threequarters of the soybean and cotton grown in the USA are varieties developed through genetic engineering (see: http://usda.mannlib.cornell.edu/reports/ nassr/field/pcp-bbp/pspl0302.pdf). Twelve transgenic crops (corn, tomato, soybean, cotton, potato, rapeseed [canola], squash, beets, papaya, rice, flax, and chicory) have been approved for commercial production in the USA. The most widely grown are “Bt” corn and cotton and glyphosate-resistant soybeans. Bt corn and cotton have had DNA from a naturally-occurring insecticidal organism, Bacillus thuringiensis, incorporated into their genome; it kills some of the most serious insect pests of these crops (European and southwestern corn borers, and cotton budworms and bollworms) after they feed on the plant, while beneficial insects are left unaffected. Glyphosate-resistant soybeans are unharmed by the broad-spectrum herbicide glyphosate, a characteristic that allows farmers to kill yield-reducing weeds in soybean fields without harming the crop.

Benefits of genetic engineering in agriculture Everything in life has its benefits and risks, and genetic engineering is no exception. Much has been said about potential risks of genetic engineering technology, but so far there is little evidence from scientific studies that these risks are real. Transgenic organisms can offer a range of benefits above and beyond those that emerged from innovations in traditional agricultural biotechnology. Following are a few examples of benefits resulting from applying currently available genetic engineering techniques to agricultural biotechnology. Increased crop productivity: Biotechnology has helped to increase crop productivity by introducing such qualities as, disease resistance and increased drought tolerance to the crops. Now, researchers can select genes for disease resistance from other species and transfer them to important crops. For

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Environmental Biotechnology

example, researchers from the University of Hawaii and Cornell University developed two varieties of papaya, resistant to papaya ring spot virus, by transferring one of the virus’ genes to papaya to create resistance in the plants. Seeds of the two varieties, named ‘SunUp’ and ‘Rainbow’, have been distributed under licensing agreements to papaya growers since 1998. Further examples come from dry climates, where crops must use water as efficiently as possible. Genes from naturally drought-resistant plants can be used to increase drought tolerance in many crop varieties. Enhanced crop protection: Farmers use crop-protection technologies because they provide cost-effective solutions to pest problems which, if left uncontrolled, would severely lower yields. As mentioned above, crops such as corn, cotton, and potato have been successfully transformed through genetic engineering to make a protein that kills certain insects when they feed on the plants. The protein is from the soil bacterium Bacillus thuringiensis, which has been used for decades as the active ingredient of some “natural” insecticides. In some cases, an effective transgenic crop-protection technology can control pests better and more cheaply than existing technologies. For example, with Bt engineered into a corn crop, the entire crop is resistant to certain pests, not just the part of the plant to which Bt insecticide has been applied. In these cases, yields increase as the new technology provides more effective control. In other cases, a new technology is adopted because it is less expensive than a current technology with equivalent control. There are cases in which new technology is not adopted because, for one reason or another, it is not competitive with the existing technology. For example, organic farmers apply Bt as an insecticide to control insect pests in their crops, yet, they may consider transgenic Bt crops to be unacceptable. Improvements in food processing: The first food product resulting from genetic engineering technology to receive regulatory approval, in 1990, was chymosin, an enzyme produced by genetically engineered bacteria. It replaces calf rennet in cheese-making and is now used in 60 per cent of all cheese manufactured. Its benefits include increased purity, a reliable supply, a 50 percent cost reduction, and high cheese yield efficiency. Improved nutritional value: Genetic engineering has allowed new options for improving the nutritional value, flavor, and texture of foods. Transgenic crops in development include soybeans with higher protein content, potatoes with more nutritionally available starch and an improved amino acid content, beans with more essential amino acids, and rice with the ability to produce beta-carotene, a precursor of vitamin A, to help prevent blindness in people who have nutritionally inadequate diets. Better flavor: Flavor can be altered by enhancing the activity of plant enzymes that transform aroma precursors into flavoring compounds. Transgenic peppers and melons with improved flavor are currently in field trials.

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10.19

Fresher produce: Genetic engineering can result in improved keepingproperties to make transport of fresh produce easier, giving consumers access to nutritionally-valuable whole foods and preventing decay, damage, and loss of nutrients. Transgenic tomatoes with delayed softening can be vine-ripened and still be shipped without bruising. Research is underway to make similar modifications to broccoli, celery, carrots, melons, and raspberry. The shelflife of some processed foods such as peanuts has also been improved by using ingredients that have had their fatty acid profile modified. Environmental benefits: When genetic engineering results in reduced pesticide dependence, we have less pesticide residues on foods, we reduce pesticide leaching into groundwater, and we minimize farm worker exposure to hazardous products. With Bt cotton’s resistance to three major pests, the transgenic variety now represents half of the cotton crop and has thereby reduced total world insecticide use by 15 per cent! Also, according to the U.S. Food and Drug Administration (FDA), “increases in adoption of herbicidetolerant soybeans were associated with small increases in yields and variable profits but significant decreases in herbicide use”. Benefits for developing countries: Genetic engineering technologies can help to improve health conditions in less developed countries. Researchers from the Swiss Federal Institute of Technology’s Institute for Plant Sciences inserted genes from a daffodil and a bacterium into rice plants to produce “golden rice,” which has sufficient beta-carotene to meet total vitamin A requirements in developing countries with rice-based diets. This crop has potential to significantly improve vitamin uptake in poverty-stricken areas where vitamin supplements are costly and difficult to distribute and vitamin A deficiency leads to blindness in children.

10.4.2 Possible Risks Associated with using Transgenic Crops in Agriculture Some consumers and environmentalists feel that inadequate effort has been made to understand the dangers in the use of transgenic crops, including their potential long-term impacts. Some consumer-advocate and environmental groups have demanded the abandonment of genetic engineering research and development. Many individuals, when confronted with conflicting and confusing statements about the effect of genetic engineering on our environment and food supply, experience a “dread fear” that inspires great anxiety. This fear can be aroused by only a minimal amount of information or, in some cases, misinformation. With people thus concerned for their health and the well-being of our planetary ecology, the issues related to their concerns need to be addressed. These issues and fears can be divided into three groups: health, environmental, and social.

10.4.3 Health-related Issues Allergens and toxins: People with food allergies have an unusual immune reaction when they are exposed to specific proteins, called allergens, in food.

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Environmental Biotechnology

About 2 per cent of people across all age groups have a food allergy of some sort. The majority of foods do not cause any allergy in the majority of people. Food-allergic people usually react only to one or a few allergens in one or two specific foods. A major safety concern raised with regard to genetic engineering technology is the risk of introducing allergens and toxins into, otherwise safe, foods. The Food and Drug Administration (FDA) checks to ensure that the levels of naturally-occurring allergens in foods made from transgenic organisms have not significantly increased above the natural range found in conventional foods. Transgenic technology is also being used to remove the allergens from peanuts, one of most serious causes of food allergy. Antibiotic resistance: Antibiotic resistance genes are used to identify and trace a trait of interest that has been introduced into plant cells. This technique ensures that a gene transfer during the course of genetic modification was successful. Use of these markers has raised concerns that new antibioticresistant strains of bacteria will emerge. The rise of diseases that are resistant to treatment with common antibiotics is a serious medical concern of some opponents of genetic engineering technology. The potential risk of transfer from plants to bacteria is substantially less than the risk of normal transfer between bacteria, or between us and the bacteria that naturally occur within our alimentary tracts. Nevertheless, to be on the safe side, FDA has advised food developers to avoid using marker genes that encode resistance to clinically important antibiotics.

10.4.4 Environmental and Ecological Issues Potential gene escape and super weeds: There is a belief among some opponents of genetic engineering technology that transgenic crops might cross-pollinate with related weeds, possibly resulting in “super weeds” that become more difficult to control. One concern is that pollen transfer from glyphosate-resistant crops to related weeds can confer resistance to glyphosate. While the chance of this happening, although extremely small, is not inconceivable; resistance to a specific herbicide does not mean that the plant is resistant to other herbicides, so, affected weeds could still be controlled with other products. Some people are worried that genetic engineering could conceivably improve a plant’s ability to “escape” into the wild and produce ecological imbalances or disasters. Most crop plants have significant limitations in their growth and seed dispersal habits that prevent them from surviving long without constant nurture by humans, and they are thus, unlikely to thrive in the wild as weeds. Impacts on “non-target” species: Some environmentalists maintain that once transgenic crops have been released into the environment, they could have unforeseen and undesirable effects. Although transgenic crops are rigorously tested before being made commercially available, not every potential impact

Concepts and Scope of Plant Biotechnology

10.21

can be foreseen. Bt corn, for instance, produces a very specific pesticide intended to kill only pests that feed on the corn. In 1999, however, researchers at Cornell University found that pollen from Bt corn could kill caterpillars of the harmless Monarch butterfly. When they fed Monarch caterpillars milkweed, dusted with Bt corn pollen in the laboratory, half of the larvae died. But follow-up field studies showed that, under real-life conditions, Monarch butterfly caterpillars are highly unlikely to come into contact with pollen from Bt corn that has drifted onto milkweed leaves—or, to eat enough of it to harm them. Insecticide resistance: Another concern related to the potential impact of agricultural biotechnology on the environment involves the question of whether insect pests could develop resistance to crop-protection features of transgenic crops. There is fear that large-scale adoption of Bt crops will result in rapid buildup of resistance in pest populations. Insects possess a remarkable capacity to adapt to selective pressures, but to date, despite widespread planting of Bt crops, no Bt tolerance in targeted insect pests has been detected. Loss of biodiversity: Many environmentalists, including farmers, are very concerned about the loss of biodiversity in our natural environment. Increased adoption of conventionallybred crops raised similar concerns in the past century, which led to extensive efforts to collect and store seeds of as many varieties as possible of all major crops. These “heritage” collections in the USA, and elsewhere, are maintained and used by plant breeders. Modern biotechnology has dramatically increased our knowledge of how genes express themselves and highlighted the importance of preserving genetic material, and agricultural biotechnologists also want to make sure that we maintain the pool of genetic diversity of crop plants needed for the future. While transgenic crops help ensure a reliable supply of basic foodstuffs, U.S. markets for specialty-crop varieties and locally-grown produce, appear to be expanding rather than diminishing. Thus, the use of genetically modified crops is unlikely to negatively impact biodiversity.

10.4.5 Social Issues Labeling: Some consumer groups argue that foods derived from genetically engineered crops should carry a special label. In the USA, these foods currently must be labeled only if they are nutritionally different from a conventional food. “Terminator” technology: Most farmers in the USA and elsewhere buy fresh seeds each season, particularly of such crops as corn, green peppers, and tomatoes. Anyone growing hybrid varieties must buy new seeds annually, because seeds from last year’s hybrids grown on the farm will not produce plants identical to the parent. For the same reason—to avoid random genetic diversity due to open pollination—farmers do not plant mango, avocado, or

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Environmental Biotechnology

macadamia from seed; instead, they clone individual plants of known quality through techniques such as grafting. In developing countries, many farmers who are not growing hybrids save harvested seeds for replanting the next year’s crop. A technology has been developed that might be used to prevent purchasers of transgenic crop seeds from saving and replanting them. Such “terminator” seeds are genetically engineered, along with other improvements more acceptable to farmers, to produce plants with seeds that have poor germination. This forces farmers, who otherwise save seed, to purchase it, if they wish to use these improved commercial varieties. And, in the USA, the crops engineered with various characters are sold alongside non-transgenic alternatives for which, growers also, typically, purchase seeds annually. Despite these mitigating circumstances, this is a serious issue among organic-growers and in developing countries, where the practice of saving seeds is the norm for farmers who are not growing hybrid crops. Inclusion of “terminator” genes means that these farmers cannot take advantage of improvements brought about by genetic engineering without being brought into the economic cycle that profits the seed companies. Without profit incentive, however, these companies are unlikely to invest in improving crops. This issue is analogous to that faced by pharmaceutical companies developing new medications against human diseases. Clearly, it is a difficult and divisive social issue.

Safety regulations in India A major contribution of Department of Biotechnology, Government of India has been to set up, in collaboration with other government departments, a framework for the evaluation and eventual clearance of transgenic crops for field cultivation. The release of transgenic crops is governed by the Indian Environment Protection Act (EPA) – 1986 which came into force from May, 1986. The Act provides a framework for the protection and improvement of environment. Later, the rules and regulations for the manufacture, use, import, export and storage of hazardous microorganisms, genetically engineered organisms or cells were notified under EPA, 1986 on 5th December, 1989. A mechanism based on interaction between committees and different departments of Government of India has been set up. Such materials will have to meet with the approval of the following Committees: Institutional Biosafety Committee (IBSC), Review Committee on Genetic Manipulation (RCGM) and Genetic Engineering Approval Committee (GEAC). Under the EPA 1986, the GEAC examines from the viewpoint of environmental safety and issues clearance or the release of genetically engineered organisms/transgenic crops and products into the environment. The GEAC also grants permits to conduct experimental and large-scale field trials which are beyond the limit of 20 acres. In case of transgenic crops, applicants are also required to seek clearance from the Ministry of Agriculture.

Concepts and Scope of Plant Biotechnology

10.23

Responsible scientists, farmers, food manufacturers, and policy makers recognize that the use of transgenic organisms should be considered very carefully to ensure that they pose no environmental and health risks or at least no more than the use of current crops and practices. Modern biotechnology represents unique applications of science that can be used for the betterment of society through development of crops with improved nutritional quality, resistance to pests and diseases, and reduced cost of production. Biotechnology, in the form of genetic engineering, is a facet of science that has the potential to provide important benefits if used carefully and ethically. Society should be provided with a balanced view of the fundamentals of biotechnology and genetic engineering, the processes used in developing transgenic organisms, the types of genetic material used, and the benefits and risks of the new technology.

CHAPTER

11

Animal Biotechnology

An important aspect of any biotechnological processes is the culture of animal cells in artificial media. These animal cells in culture are used in recombinant DNA technology, genetic manipulations and in a variety of industrial processes. Now-a-days it has become possible to use the cell and tissue culture in the areas of research which have a potential for economic value and commercialization. The animal cell cultures are being extensively used in production of vaccines, monoclonal antibodies, pharmaceutical drugs, cancer research, genetic manipulations etc. Animal cells, e.g. egg cells, are used for multiplication of superior livestock using a variety of techniques like cloning of superior embryonic cells, transformation of cultured cells, leading to the production of transgenic animals. The animal cells are also used, in vitro fertilization and transfer of embryos to surrogate mothers. Hence, the establishment and maintenance of a proper animal culture is the first step towards using them as tools for biotechnology.

11.1  HISTORY OF ANIMAL CELL CULTURE

• It was Jolly, who (1903) showed for the first time that the cells can survive and divide in vitro. Ross Harrison (1907) was able to show the development of nerve fibers from frog embryo tissue, cultured in a blood clot. Later, Alexis Carriel (1912) used tissue and embryo extracts as cultural media to keep the fragments of chick embryo heart alive.



• In the late 1940s, Enders, Weller and Robbins grew Poliomyelitis virus in culture which paved way for testing many chemicals and antibiotics that affect multiplication of virus in living host cells. The significance of animal cell culture was increased when viruses were used to produce vaccines on animal cell cultures in late 1940s.



• For about 50 years, mainly tissue explants rather than cells, were used for culture techniques; although, later, after 1950s, mainly dispersed

11.2







Environmental Biotechnology

cells in culture were utilized. In 1966, Alec Issacs discovered Interferon by infecting cells in tissue culture with viruses. He took filtrates from virus-infected cells and grew fresh cells in the filtered medium. When the virus was reintroduced in the medium, the cells did not get infected. He proposed that cells infected with the virus secreted a molecule which coated onto uninfected cells and interfered with the viral entry. This molecule was called “Interferon”. • Chinese Hamster Ovary (CHO) cell lines were developed during 1980s. Recombinant erythropoietin was produced on CHO cell lines by AMGEN (U.S.A.). It is used to prevent anaemia in patients with kidney failure who require dialysis. After this discovery, the Food and Drug Administration (U.S.A) granted the approval for manufacturing erythropoietin on CHO cell lines. In 1982, Thilly and co-workers used the conventional conditions of medium, serum, and O2 with suitable beads as carriers and grew certain mammalian cell lines to densities as high as 5x106 cells/ml. • A lot of progress has been also made in the area of stem cell technology which will have their use in the possible replacement of damaged and dead cells. In 1996, Wilmut and co-workers successfully produced a transgenic sheep named Dolly through nuclear transfer technique. Thereafter, many such animals (like sheep, goat, pigs, fishes, birds, etc.) were produced. • For animals, if the explant maintains its structure and function in culture, it is called as an ‘organotypic culture’. If the cells in culture re-associate to create a three dimensional structure irrespective of the tissue from which it was derived, it is described as a ‘histotypic culture’.

11.2  ANIMAL CELL CULTURE Salient Features of Animal cell culture



• Animal cells can grow in simple glass or plastic containers in nutritive media but they grow only to limited generations. • Animal cells exhibit contact inhibition. In culture the cancer cells apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape, they loose contact inhibition and pile over each other. • There is a difference in the in vitro and in vivo growth pattern of cells. For example, — there is an absence of cell-cell interaction and cell-matrix interaction, — there is a lack of three-dimensional architectural appearance, and — there is changed hormonal and nutritional environment. The way of adherence to glass or plastic container in which they grow, cell proliferation and shape of cell, results in alterations.

Animal Biotechnology

11.3



— The maintenance of growth of cells under laboratory conditions in suitable culture medium is known as primary cell culture.



— Cells are dissociated from tissues by mechanical means and by enzymatic digestion using proteolytic enzymes.



— Cells can grow as adherent cells (anchorage-dependent) or as suspension cultures (anchorage-independent).



— The primary culture is subcultured in fresh media to establish secondary cultures.



— The various types of cell lines are categorized into two types as Finite cell line and Continuous cell line. Finite cell lines are those cell lines which have a limited life span and grow through a limited number of cell generations. The cells normally divide 20 to 100 times (i.e., is 20–100 population doublings) before extinction. Cell lines transformed under in vitro conditions, give rise to continuous cell lines. The continuous cell lines are transformed, immortal and tumorigenic.



— The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protects the cells from chemical, physical, and mechanical stresses.



— Nutrient media is the mixture of inorganic salts and other nutrients capable of sustaining cell survival in vitro.



— Serum is essential for animal cell culture and contains growth factors which promote cell proliferation. It is obtained as exuded liquid from blood undergoing coagulation and filtered using Millipore filters.



— Cryopreservation is storing of cells at very low temperature (–180° C to –196° C), using liquid nitrogen. DMSO is a cryopreservative molecule which prevents damage to cells.



— In order to maintain the aseptic conditions in a cell culture, a LAF hood is used. Based on the nature of cells and organism, the tissue culture hoods are grouped into three types: Class I, Class II, and Class III.



— CO2 incubators are used and designed to mimic the environmental conditions of the living cells.



— An inverted microscope is used for visualizing cell cultures in situ.



— For most animal cell cultures, low-speed centrifuges are needed.



— Neuronal cells constitute the nervous system. In culture, the neuronal cells cannot divide and grow.



— The cells that form connective tissue (skin) is called fibroblast. The fibroblast can divide and grow in culture upto some generations after which they die. All normal animal cells are mortal.

11.4

Environmental Biotechnology



— Organ culture: The culture of native tissue that retains most of the in vivo histological features is regarded as organ culture. — Histotypic culture: The culturing of the cells for their re-aggregation to form a tissue-like structure represents histotypic culture. — Organotypic culture: This culture technique involves the recombination of different cell types to form a more-defined tissue or an organ. There are certain terms that are associated with the cell lines. These are as follows: (i) Split ratio: The divisor of the dilution ratio of a cell culture at subculture. (ii) Passage number: It is the number of times that the culture has been cultured. (iii) Generation number: It refers to the number of doublings that a cell population has undergone. In fact, these parameters help us to distinguish the cancer cells in culture from the normal cells because the cancer cells in culture, change shape (more rounded), loose contact inhibition, pile on each other due to overgrowth and have uncontrolled growth.

11.3  REQUIREMENTS FOR ANIMAL CELL CULTURE Among the essential requirements for animal cell culture are, special incubators to maintain the levels of oxygen, carbon dioxide, temperature, humidity as present in the animal’s body, the synthetic media with vitamins, amino acids, and fetal calf serum. Following parameters are essential for successful animal cell culture: (a) Temperature: In most of the mammalian cell cultures, the temperature is maintained at 37°C in the incubators, as the body temperature of Homo sapiens is 37°C. (b) Culture media: The culture media is prepared in such a way that it provides— (1) The optimum conditions of factors like pH, osmotic pressure, etc. (2) It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally, the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions. Glucose is added as energy source— it’s concentration varying, depending on the requirement. Phenol Red is added as a pH indicator of the medium. There are two types of media used for culture of animal cells and tissues—the natural media and the synthesized media. (3) Natural Media: The natural media are the natural sources of nutrient sufficient for growth and proliferation of animal cells and

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tissues. The Natural Media used to promote cell growth fall in three categories. I. Coagulant, such as plasma clots. It is now commercially available in the form of liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma can also be prepared in the laboratory by taking out blood from male fowl and adding heparin to prevent blood coagulation. II. Biological fluids such as serum. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium, etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum is obtained from human adult blood, placental-cord blood, horse blood, calf blood. The other forms of biological fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes, etc. III. Tissue extracts, for example, Embryo extracts: Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow, etc., are also used for culture of animal cells.

11.3.1  Synthetic Media Syntheic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors, etc. Different types of synthetic media can be prepared for a variety of cells and tissues to be cultured. Synthetic media are of two types—Serum containing media (media containing serum) and serum-free media (media with out serum). Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12, etc. Advantages of serum in culture medium are: (i) serum binds and neutralizes toxins, (ii) serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins, (iii) it contains the protease inhibitors, (iv) it increases the buffering capacity, (v) it provides trace elements. Disadvantages of serum in culture medium are: (i) it is not chemically defined and therefore it’s composition varies a lot, (ii) it is sometimes source of contamination by viruses, mycoplasma, prions, etc. (iii) it increases the difficulties and cost of downstream processing, (iv) it is the most expensive component of the culture medium.

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(4) pH—Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures. The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonateCO2 system as its major component. (5) Osmolality: A change in osmolality can affect cell growth and function. Salt, glucose and amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm.

11.4  CELL-BASED THERAPY The animal cell culture techniques are used in replacing the damaged and dead cells with normal and healthy cells using the stem cell technology. This therapy is called Cell-Based therapy which involves the use of stem cell technology involving the replacement of damaged and dead cells with normal and healthy cells. This is used to treat blood cancer, and other neurodegenerative diseases, etc.

11.5  APPLICATIONS OF ANIMAL CELL CULTURE The animal cell cultures are used for a diverse range of research and development. These areas are: (a) Production of antiviral vaccines, which requires the standardization of cell lines for the multiplication and assay of viruses. (b) Cancer research, which requires the study of uncontrolled cell division in cultures. (c) Cell fusion techniques. (d) Genetic manipulation, which is easy to carry out in cells or organ cultures. (e) Production of monoclonal antibodies requires cell lines in culture. (f) Production of pharmaceutical drugs, using cell lines. (g) Chromosome analysis of cells derived from womb. (h) Study of the effects of toxins and pollutants using cell lines. (i) Use of artificial skin. (ii) Study the functions of the nerve cells.

11.5.1  Somatic Cell Fusion One of the applications of animal cell culture is the production of hybrid cells by the fusion of different cell types. These hybrid cells are used for the following purposes:

(i) study of the control of gene expression and differentiation,

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Animal Biotechnology

(ii) study of the problem of ‘malignancy’, (iii) viral application, (iv) gene-mapping, (v) production of hybridomas for antibody production. In 1960s, in France, for the first time, hybrid cells were successfully produced from mixed cultures of two different cell lines of mouse. Cells growing in culture are induced by some of the viruses such as ‘Sendai virus’ to fuse and form hybrids. This virus induces two different cells first, to form heterokaryons. During mitosis, chromosomes of heterokaryon move towards the two poles, and later on, fuse to form hybrids. It is important to remove the surface carbohydrates to bring about cell fusion. Some other chemicals like polyethylene glycol also induce somatic cell fusion. Many commercial proteins have been produced by animal cell culture and their medical application is being evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.

Production of T-PA

11.5.2  Blood Factor VIII Haemophilia A is a blood disorder which is a sex-linked genetic disease in humans. The patients suffering from Haemophilia A lack factor VIII, which plays an important role in the clotting of blood. This factor VIII is secreted by a gene present on X-chromosome but this gene undergoes mutations in people suffering from Haemophilia. Current therapy for this disease is the transfusion of blood factor VIII into patients. Using rDNA technology, factor VIII has been produced from mammalian cell culture, e.g., Hamster kidney cell.

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11.5.3  Erythropoietin (EPO) The EPO is a glycoprotein consisting of 165 amino acids and is formed in the foetal liver and kidneys of the adults. It causes proliferation and differentiation of progenitor cells into erythrocytes (erythroblasts) in the bone marrow. Erythropoietin is hormone-like in nature and is released by the kidney under, hypoxic or anoxic conditions caused by anaemia. Amgen Inc. holds US patent for preparation of, eErythropoietin, by recombinant method using Chinese Hamster Ovary cell lines. Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic conditions caused by anaemia. r-HUEPO- recombinant human erythropoitein has been effectively used to treat anemia associated with AIDS, renal failure etc.

11.5.4 The production of Monoclonal Antibodies using Hybridoma Technology Antibodies are proteins synthesized in blood against antigens and are collected from the blood serum. The antibodies, which are heterogenous and nonspecific in action are called polyclonal antibodies. If a specific lymphocyte, after isolation and culture in vitro becomes capable of producing a single type of antibody bearing specificity against specific antigen, it is known as monoclonal antibody. The monoclonal antibodies are used in the diagnosis of diseases because of the presence of desired immunity. However, these antibody-secreting cells cannot be maintained in culture. It was observed that the myeloma cells (bone marrow tumour cells, due to cancer) grow indefinitely and also produce immunoglobulins which are, in fact, monoclonal antibodies. In 1974, George Kohler and Milstein isolated clones of cells from the fusion of two parental cell lines – lymphocytes from spleen of mice immunized with red blood cells from sheep and myeloma cells. These cells were maintained in vitro and produced antibodies. The hybrid cells maintained the character of lymphocytes to secrete the antibodies, and of myeloma cells to multiply in culture. These hybrid cell lines are called “Hybridoma” and are capable of producing unlimited supply of antibodies. Hybridoma are obtained by using an antibody, producing lymphocytes cell and a single myeloma cell. Monoclonal antibodies bind very specifically to an epitope (specific domains) on an antigen and, by using them, it is possible to detect the presence of specific antigens. The monoclonal antibodies are used for the treatment of patients with malignant leukaemia cells, B cell lymphomas and allograft rejection after transplantation. CD3 is an antigen present on the surface of mature T-cells lymphocytes. If T-cell population is depleted or controlled, the transplanted organ will not be rejected. An antibody that acts against CD3 surface antigen of T-cells is called OKT3, i.e., anti-CD3 Moab. OKT3 is a monoclonal antibody which has been licensed for clinical use for the treatment of acute renal allograft rejection. OKT3 removes antigen-bearing cells from circulation, thereby, helps in accepting the graft.

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Steps Involved in the production of monoclonal antibodies

When monoclonal antibodies are used as enzymes using the technique of enzyme engineering, then they are called abzymes. Using animal cell cultures, it is also possible to produce Polyclonal Antibodies. Polyclonal antisera are derived from many cells; therefore, contains heterogeneous antibodies that are specific for several epitopes or an antigen.

11.6 SCALE-UP OF ANIMAL CELL CULTURE Modifying a laboratory procedure, so that it can be used on an industrial scale is called scaling up. Laboratory procedures are normally scaled up via intermediate models of increasing size. The larger the plant, the greater the running costs, as skilled people are required to monitor and maintain the machinery. The first pre-requisite for any large scale cell culture system and its scaling up is the establishment of a cell bank. Master cell banks (MCB) are first established and they are used to develop Master Working Cell Banks (MWCB). The MWCB should be sufficient to feed the production system at a particular scale for the predicted life of the product. The cell stability is an important criteria, so MWCB needs to be repeatedly sub-cultured and each generation should be checked for changes. A close attention should be paid to the volume of cultured cells as the volume should be large enough to produce a product in amounts which is economically viable. The volume is maintained by: • increasing the culture volume,

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• by increasing the concentration of cells in a reactor by continuous perfusion of fresh medium, so that the cells keep on increasing in number without the dilution of the medium.

A fully automated bioreactor maintains the physicochemical and biological factors to optimum level and maintains the cells in suspension medium. The most suitable bioreactor used is a compact-loop bioreactor consisting of marine impellers. The animal cells unlike bacterial cells, grow very slowly. The main carbon and energy sources are glucose and glutamine. Lactate and ammonia are their metabolic products that affect growth and productivity of cells. So, the on-line monitoring of glucose, glutamate, and ammonia is carried out by on line flow injection analysis (FIA) using gas chromatography (GC), high performance liquid chromatography (HPLC), etc. In batch cultures, mainly Roller Bottles with Micro Carrier Beads (for adherent cells) and spinner flasks (for suspension cultures) are used in Scaleup of animal cell culture process.

11.6.1  Roller Bottles The Roller bottles provide total curved surface area of the micro carrier beads for growth. The continuous rotation of the bottles in the CO2 incubators helps to provide medium to the entire cell monolayer in culture. The roller bottles are well attached inside a specialized CO2 incubators. The attachments rotate the bottles along the long axis which helps to expose the entire cell monolayer to the medium during the one full rotation. This system has the following advantages over the static monolayer culture:

(a) it provides increase in the surface area,



(b) provides constant gentle agitation of the medium,



(c) provides increased ratio of surface area of medium to its volume, which allows gas exchange at an increased rate through the thin film of the medium over the cells. Typically, a surface area of 750-1500 cm2 with 200-500 ml medium will yield 1–2 × 108 cells.

The roller bottle cell culture

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11.6.2  Micro Carrier Beads Micro carrier beads are small spherical particles with diameter 90-300 micrometers, made up of dextran or glass. Micro Carrier beads, increase the number of adherent cells per flask. These dextran or glass-based beads come in a range of densities and sizes. The cells grow at a very high density which rapidly exhausts the medium, and therefore, the medium has to be replaced for the optimum cell growth. At the recommended concentration when the microcarriers are suspended, they provide 0.24 m2 area for every 100 ml of culture flask.

11.6.3  Spinner cultures The spinner flask, was originally developed to provide the gentle stirring of micro-carriers, but are now used for scaling up the production of suspension cells. The flat surface glass flask is fitted with a Teflon paddle that continuously turns and agitates the medium. This stirring of the medium improves gas exchange in the cells in culture. The spinner flask used at commercial scale consists of one or more side arms for taking out samples and decantation, as well.

11.7  TYPES OF CELL CULTURES Primary cell culture: The maintenance of growth of cells dissociated from the parental tissue (such as kidney, liver) using the mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers, is called Primary Cell Culture. The primary cell culture could be of two types depending upon the kind of cells in culture. (a) Anchorage Dependent/Adherent cells: Cells shown to require attachment for growth are set to be Anchorage Dependent cells. The Adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue. They grow adhering to the cell culture. (b) Suspension Culture/Anchorage Independent cells: Cells which do not require attachment for growth or, do not attach to the surface of the culture vessels, are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes. Secondary cell cultures :When a primary culture is sub-cultured, it becomes known as secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. Subculturing—Subculturing or splitting cells is required to periodically provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media, washing the plate, disassociating the adhered cells, usually enzymatically. Such cultures may be called secondary cultures.

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Cell Line: A Cell Line or Cell Strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types:

(a) Finite Cell Lines: The cell lines which have a limited life span and go through a limited number of cell generations (usually, 20–80 population doublings) are known as finite cell lines. These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24–96 hours.



(b) Continuous Cell Lines: Cell lines transformed under laboratory conditions or, in vitro culture conditions, give rise to continuous cell lines. The cell lines show the property of ploidy (aneuplacdy or heteroploidy), absence of contact inhibition and anchorage dependence. They grow in monolayer or suspension form. The growth rate is rapid and doubling time is 12–24 hours.



(c) Monolayer cultures: When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.



(d) Suspension cultures: Majority of continuous cell lines grow as monolayers. Some of the cells which are non-adhesive, e.g. cells of leukemia or, certain cells which can be mechanically kept in suspension, can be propagated in suspension. There are certain advantages in propagation of cells by suspension culture method. These advantages are:



(a) The process of propagation is much faster,



(b) The frequent replacement of the medium is not required,



(c) Suspension cultures have a short-lag period,



(d) treatment with trypsin is not required,



(e) a homogenous suspension of cells is obtained,



(f) the maintenance of suspension cultures is easy and bulk production of the cells is easily achieved,



(g) scale-up is also very convenient. The cell lines are known by:



(a) A code, e.g., NHB for Normal Human Brain.



(b) A cell line number—This is applicable when several cell lines are derived from the same cell culture source, e.g. NHB1, NHB2.



(c) Number of population doublings the cell line has already undergone e.g., NHB2/2 means two doublings.

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Salient features of cell culture with volution of a cell line

11.8  CHARACTERIZATION OF CELL LINES The cell lines are characterized by their—(a) growth rate and (b) karyotyping. (a) Growth Rate: A growth curve of a particular cell line is established taking into consideration the population doubling time, a lag time, and a saturation density of a particular cell line. A growth curve consist of:

(1) Lag Phase: The time the cell population takes to recover from such sub culture, attach to the culture vessel and spread.



(2) Log Phase: In this phase, the cell number begins to increase exponentially.



(3) Plateau Phase: During this phase, the growth rate slows or stops due to exhaustion of growth medium or confluency.

(b) Karyotyping: Karyotyping is important as it determines the species of origin and determine the extent of gross chromosomal changes in the line. The cell lines with abnormal karyotype are also used if they continue to perform normal function. Karyotype is affected by the growth conditions used, the way in which the cells are subcultured, and whether or not the cells are frozen. (c) There are certain terms that are associated with the cell lines.

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These are as follows: (i) Split ratio: The divisor of the dilution ratio of a cell culture, at subculture. (ii) Passage number: It is the number of times that the culture has been cultured. (iii) Generation number: It refers to the number of doublings that a cell population has undergone. Some Animal Cell Lines and the Products obtained from them

Cell line

Product

Human tumor

Angiogenic factor

Human leucocytes

Interferon

Mouse fibroblasts

Interferon

Human Kidney

Urokinase

Transformed human kidney Single chain urokinase-type plasminogen cell line, TCL-598 activator (scu-PA) Human kidney cell (293)

Human protein (HPC)

Dog kidney

Canine distemper vaccine

Cow kidney

Foot and Mouth Disease (FMD) vaccine

Chick embryo fluid

Vaccines for influenza, measles and pumps

Duck embryo fluid

Vaccines for rabies and rubella

Chinese Hamster (CHO) cells

Ovary 1. Tissue-type plasminogen activator (t-PA) 2. b-and gamma interferons 3. Factor VIII

11.9  STEM CELL TECHNOLOGY Stem cells retain the capacity to self-renew as well as to produce progeny with a restricted mitotic potential and restricted range of distinct types of differentiated cells, they give rise to. The formation of blood cells, also called haematopoiesis, is the classical example of concept of stem cells. Indirect assay methods were developed to identify the haematopoietic stem cells. The process of haematopoeis occurs in the spleen and bone marrow in mouse. In human beings, about 100,000 haematopoietic stem cells produce one billion RBCs, one billion platelets, one million T-cells, one million B-cells per kg body weight per day. Several methods have been developed to study haematopoiesis and stem cells:

(a) Repopulation assay: Edmens Snell’s group created mice which were genetically identical by mating of sibling mice after 21 generations. Two groups of mice were lethally X- irradiated to destroy their blood cell-forming capacity. One of this group was injected with marrow

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cells from the femur bone of a normal and healthy albino mice. It was observed that this group survived, whereas the mice in the other group died. The spleen of mice which survived had the colonies of the bone marrow cells just like bacterial colonies on a petri plate. This came to be known as colony-forming units of spleen (CFU-S) and the technique is known as repopulation assay. (b) The in vitro clonal assay: In this assay, the stem cells proliferate to form colonies of differentiated cells on semi-solid media. This assay helps in identifying growth factors required for the formation of blood cells from the primitive stem cells. One of the first commercialized biotechnology product – erythropoietin, was assayed by this procedure. (c) Long-term marrow culture: In this method, the marrow cells from femur bone were grown under in vitro conditions on plastic surfaces. These techniques were helpful in bone marrow transplantation and treatment of blood cancer by releasing immature blood cells into the blood stream. (d) Embryonic stem cell culture: Embryonic stem cells are cell lines derived from the inner cell mass of fertilized mouse embryo without the use of immortalizing or transforming agents. The Inner Cell Mass (ICM) are the cells that are maintained in tissue culture in the presence of irradiated fibroblast cells. These cells are often used in creating chimeric mice. In 1998, J.A. Thomson developed the method to multiply the human embryonic stem cells. Human ICM can also be now derived either by IVF or from germ cell precursors and cultured on a petri plate. The differentiation of these cells into lineage restricted (neuronal and glial) cells can be accomplished by altering the media in which the cells grow.

Scheme of obtaining chimeras

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Environmental Biotechnology

(e) The ICM cells could be used to create chimeric mice. In chimeric mice, it was possible to take ES cells from a black mouse and implant it into the embryo of an albino mouse (white). The progeny, so developed, had skin colour of black and white (a chimera).

11.9.1  Genetic Engineering of Animal Cells and their Applications The mammalian cells are genetically modified by introducing the genes needed for specific purposes such as production of specific proteins or to improve the characteristics of a cell line. The methods used to introduce the foreign genes/ DNA into mammalian cells are: Electroporation, Lipofection, Microinjection and/or fusion of mammalian cells with bacteria or viruses. After the integration of the foreign DNA into the mammalian cells, the transfected/transformed cells are selected by using suitable markers. Some of such markers in use are: Viral thymidine kinase, Bacterial dihydrofolate reductase, Bacterial neomycin phosphotransferase. It has been possible to overproduce several proteins in mammalian cells through genetic manipulations, e.g. tissue plasminogen activator, erythropoietin, interleukin-2, interferon-beta, clotting factors VIII and IX, tumor necrosis factors. The recombinant mammalian cells are also conveniently used for the production of monoclonal antibodies.

11.9.2  Manipulation of Gene Expression in Eukaryotes The eukaryotic organisms have the capability to bring about the posttranslational modifications such as glycosylation, phosphorylation, proteolytic cleavage, etc., which ultimately help in the production of stable and biologicallyactive proteins. Due to these reasons, the use of eukaryotic expression system is preferred, however, it is difficult to conduct experiments with eukaryotic cells. The introduction of a foreign DNA into animal cells is called transfection. The insert, DNA in the eukaryotic cells may be associated with vector or integrated into the host chromosomal DNA. Among the various hosts used for the expression of cloned genes, the common yeast Saccharomyces cerevisiae is the most extensively used. Besides this, the cultured insect cells are in use for expressing cloned DNAs. Baculoviruses exclusively infect insect cells. The DNA of these viruses encode for several products and their productivity in cells is very high to the extent of more than 10,000 times compared to mammalian cells. The Baculoviruses not only carry a large number of foreign genes but can also express and process the products formed. By using baculovirus as an expression vector system, a good number of mammalian and viral proteins have been synthesized. The most commonly used baculovirus is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). It grows on the insect cell lines and produces high levels of polyhedrin or a recombinant protein. The mammalian cell expression vectors are used for the production of specific recombinant proteins and to study the function and regulation of

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mammalian genes. However, large-scale production of recombinant proteins with engineered mammalian cells is costly. The mammalian vector contains a eukaryotic origin of replication from an animal virus such as Simian virus 40 (SV 40) and a prokaryotic origin of replication. It has a multiple cloning site and a selectable marker gene, both of which remain under the control of eukaryotic promoter and polyadenylation sequences. These sequences are obtained from either animal viruses (SV40, herpes simplex virus) or mammalian genes (growth hormone, metallothionein). The promoter sequences facilitate the transcription of cloned genes (at the multiple cloning site) and the selectable marker genes. On the other hand, the polyadenylation sequences terminate the transcription.

11.9.3  Collection and purification process of Recombinant proteins As the recombinant proteins start accumulating in the host cells, it becomes important to collect and purify them. This is a tricky process since and many times, the recombinant protein is a foreign body for the host cells the enzyme machinery of the host cell becomes activated to degrade the outside protein. One of the strategies adopted is the use of bacterial strains, deficient in proteases or, alternatively, the recombinant proteins are fused with the nativehost proteins. The fusion proteins are resistant to protease activity. Sometimes, the foreign proteins accumulate as aggregates in the host organism which minimizes the protease degradation. The best way out is to quickly export and secrete out the recombinant proteins into the surrounding medium. The recovery and the purification of foreign proteins is easier from the exported proteins. The efforts have been made to develop methods to increase the export of recombinant proteins. Some of the species of the bacterium, Bacillus subtilis, normally secrete large quantities of extracellular proteins. A short DNA sequence, called signal sequence from such species, is introduced into other B. subtilis. These bacteria produce recombinant DNA tagged with signal peptide, which promotes export and secretion. This signal peptide is removed after the purification of foreign protein. The techniques used for the purification of recombinant proteins from the mixture of secreted proteins are affinity-tagging, immunoaffinity purification, etc.

11.9.4  Organ culture and Histotypic cultures The cell-cell interaction leads to a multistep events in in vivo situations. For example, hormone stimulation of fibroblasts is responsible for the release of surfactant by the lung alveolar cells. Androgen-binding to stomal cells stimulates the prostrate epithelium. In other words, hormones, nutritional factors and xenobiotics exert stimulating effects on the cells to function in a coordinated manner. Xenobiotics broadly refers to the unnatural, foreign, and synthetic chemicals such as pesticides, herbicides, refrigents, solvents and

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other organic compounds. It is impossible to study these cellular interactions that occur in the in vivo system with isolated cells or, cells in culture. This has lead to the attempts to develop organ and histotypic culture with the aim of creating in vitro models comparable to the in vivo system. The three types of such cultures are:

(a) Organ culture: In this type of culture, the whole organs or small fragments of the organs, with their special and intrinsic properties intact, are used in culture.



(b) Histotypic culture: The cell lines grown in three dimensional matrix to high density represent histotypic cultures.



(c) Organotypic cultures: A component of an organ is created by using cells from different lineages in proper ratio and spatial relationship under laboratory conditions.

11.9.5  Organ culture In the organ culture, the cells are integrated as a single unit which helps to retain the cell to cell interactions found in the native tissues or organs. Due to the preservation of structural integrity of the original tissue, the associated cells continue to exchange signals through cell adhesion or communications. Due to the lack of a vascular system in the organ culture, the nutrient supply and gas exchange of the cells become limited. In order to overcome this problem, the organ cultures are placed at the interface between the liquid and gaseous phases. Sometimes, the cells are exposed to high O2 concentration which may also lead to oxygen-induced toxicity. Due to the inadequate supply of the nutrients and oxygen, some degree of necrosis at the central part of the organ may occur. In general, the organ cultures do not grow except some amount of proliferation that may occur on the outer cell layers.

11.9.6  Techniques and Procedure for organ culture In order to optimize the nutrient and gas exchanges, the tissues are kept at gas-limited interface using the support material which ranges from semi-solid gel of agar, clotted plasma, micropore filter, lens paper, or strips of Perspex or plexiglass. The organ cultures can also be grown on top of a stainless steel grid. Another popular choice for growing organ cultures is the filter-well inserts. Filter-well inserts with different materials like ceramic, collagen, nitrocellulose, are now commercially available. Filter well inserts have been successfully used to develop functionally-integrated thyroid epithelium, stratified epidermis, intestinal epithelium, and renal epithelium. The procedure for organ cultures has the following steps:

(a) The organ tissue is collected after the dissection.



(b) The size of the tissue is reduced to less than 1mm in thickness.



(c) The tissue is placed on a gas medium interface support.

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(d) Incubation in a CO2 incubator.

(e) M199 or CMRL 1066 medium is used and changed frequently. (f) The techniques of histology, autoradiography, and immunochemistry are used to study the organ cultures.

11.9.7  The advantages of organ culture The organ cultures can be used to study the behavior of an integrated tissue in the laboratory. It provides an opportunity to understand the biochemical and molecular functions of an organ/tissue.

11.9.8  Limitations of organ culture It is a difficult and expensive technique. The variations are high with low reproducibility. For each experiment, a new or fresh organ is needed, as organ cultures are not propagated.

11.9.9  Histotypic cultures Using histotypic culture, it is possible to use dispersed monolayers to regenerate tissuelike structures. It is the growth and propagation of cell lines in three-dimensional matrix to high cell density that contributes to this. The techniques used in histotypic cultures are:

(a) Gel and sponge technique: In this method, the gel (collagen) or sponges (gelatin) are used which provides the matrix for the morphogenesis and cell growth. The cells penetrate these gels and sponges, while growing.



(b) Hollow fibers technique: In this method, hollow fibers are used which helps in more efficient nutrient and gas exchange. In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures. The cells get attached to capillary fibers and increase in cell density to form tissue like structures.



(c) Spheroids: The re-association of dissociated cultured cells leads to the formation of cluster of cells called spheroids. It is similar to the reassembling of embryonic cells into specialized structures. The principle followed in spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue-like architecture. However, there is a limitation of diffusion of nutrients and gases in these cultures.



(d) Multicellular tumour spheroids: These are used as an in vitro proliferating models for studies on tumor cells. The multicellular tumor spheroids have a three-dimensional structure which helps in performing experimental studies related to drug therapy, penetration of drugs besides using them for studying regulation of cell proliferation,

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immune response, cell death, and invasion and gene therapy. A size bigger than 500 mm leads to the development of necrosis at the centre of the MCTS. The monolayer of cells or aggregated tumour is treated with trypsin to obtain a single cell suspension. The cell suspension is inoculated into the medium in magnetic stirrer flasks or roller tubes. After 3–5 days, aggregates of cells representing spheroids are formed. Spheroid growth is quantified by measuring their diameters regularly. The spheroids are used for many purposes. They are used as models for a vascular tumour growth. They are used to study gene expression in a three-dimensional configuration of cells. They are also used to study the effect of cytotoxic drugs, antibodies, radionucleotides, and the spread of certain diseases like, rheumatoid arthritis.

11.9.10  Organotypic cultures These cultures are used to develop certain tissues or tissue models, for example, skin equivalents have been created by culturing dermis, epidermis and intervening layer of collagen, simultaneously. Similarly, models have been developed for prostrate, breast, etc. Organotypic culture involves the combination of cells in a specific ratio to create a component of an organ.

11.10  CELL AND TISSUE ENGINEERING Tissue engineering refers to the application of the principles of engineering to cell culture for the construction of functional anatomical units—tissues/ organs. The aim of tissue engineering is nothing but to supply the various body parts for the repair or replacement of damaged tissues or organs. It is now possible to grow skin cells, blood cells, cardiac cells, etc. by using the ability of stem cells to proliferate and differentiate. During the last decade, the tissue culture work in animals demonstrated that virtually any human tissue or organ can be grown in culture. This became possible only after it became known that the ability of cultured cells to undergo differentiation can be restored. ‘Skin’ was the first organ to be cultured in artificial media and could be successfully used for transplantation following serious skin burns. For past few years, some of the biotech companies like, ATS (Advanced Tissue Science, USA), Biosurface Technology (BTI, Cambridge) and Organogenesis, are developing artificial skins to the stage of clinical trials. In the field of tissue replacement, focus of attention is the Artificial cartilage. As it is not vascularized, it is not rejected due to immunogenic response. This will have lots of implications in the treatment of sports related injuries and diseases like, arthritis.

11.10.1  Design and Engineering of Tissues The design and tissue engineering should essentially cause minimal discomfort to the patient. The damaged tissues should be easily fixed with the desired

Animal Biotechnology

11.21

functions, quickly restored. Another important factor controlling the designing of tissue culture is the source of donor cells. The cells from the patient himself, is always preferred as it considerably reduces the immunological complications. However, under certain situations, allogeneic cells (cells taken from a person other than the patient), are also used. The other important factors are – the support material, it’s degradation products, cell adhesion characteristics, etc. It was demonstrated in 1975 that human keratinocytes could be grown in the laboratory in a form suitable for grafting. A continuous sheet of epithelial cells can be grown now; however, there is still difficult to grow TE skin with the dermal layer with all the blood capillaries, nerves, sweat glands, and other accessory organs. Some of the implantable skin substitutes which are tissue engineering skin constructs with a limited shelf life of about 5 days are: (a) Integra TM—A bioartificial material composed of collagenglycosaminoglycan and is mainly used to carry the seeded cells. (b) Dermagraft TM—This is composed of polyglycolic acid polymer-mesh seeded with human dermal fibroblasts from neonatal foreskins. (c) Apligraf TM—It is constructed by seeding human dermal fibroblasts into collagen gel with the placement of a layer of human keratinocytes on the upper surface. These tissue-constructs integrate into the surrounding normal tissue and form a good skin cover with minimum immunological complications. The urothelial cells and smooth muscle cells from bladder are now being cultured and attempts are on to construct TE urothelium. Some progress has also been made in the repair of injured peripheral nerves using tissueengineered peripheral nerve implants. The regeneration of the injured nerve occurs from the proximal stump to rejoin at distal stump. The regeneration process requires substances like (a) Conduct material: The conduct material is composed of collagenglycosaminoglycans, PLGA (poly lactic-co-glycolicacid), hyaluronan and fibronectin and forms the outer layer. (b) Filling material: The filling material contains collagen, fibrin, fibronectin and agarose. This supports the neural cells for regeneration. And, (c) Additives: A large number of other factors are also added, e.g. growth factors, neurotrophic factors such as fibroblast growth factor (FGF), nerve growth factor (NGF).   The other important applications of tissue engineering are in gene therapy, pseudo-organs and as model cell systems for developing new therapeutic approaches to human diseases. The attempts are on to create tissue models in the form of artificial organs using tissue engineering. The artificial liver is being created using hepatocytes cultured as spheroids and held suspended in

11.22

Environmental Biotechnology

artificial support system such as porous gelatin sponges, agarose or collagen. Some progress has been made in the area of creating the artificial pancreas using spheroids of insulin-secreting cells which have been developed from mouse insulinoma beta cells. Three-dimensional brain cell cultures have been used for the study of neural myelination, neuronal regeneration, and neurotoxicity of lead. The aggregated brain cells are also being used to study Alzheimer’s disease and Parkinson’s disease. Thyroid cell spheroids are being used to study cell adhesion, motility, and thyroid follicle biogenesis. Table depicting the technological goals and areas of research in tissue engineering Growth of cells in three-dimensional systems Delivery systems for protein therapeutics Cell cultivation methods for culturing ‘recalcitrant cells’ Expression of transgenic proteins in transplantable cells To develop vehicles for delivering transplantable cells Development of markers for tracking transplanted cells Avoiding immunogenicity in transplantable cells Development of in vivo and ex vivo biosensors for monitoring cell behaviour during tissue production Downstream Processing: Downstream processing or down-streaming is the extraction and purification of the desired end products of fermentation processes. Such products might include cells, solvents or solutes. Various processes are available for the separation of cells from the fermentation broth in which they are grown, including flocculation, filtration, centrifugation, sedimentation or flotation. The procedure adopted depends on whether it is the cells, or the solution surrounding them, that contains the desired endproducts. Bioethics in Animal Genetic Engineering: There are some serious issues related to genetic modification of animals using animal genetic engineering techniques. One is not sure of the consequences of these genetic modifications and the further interaction with the environment. Proper clinical trials are also necessary before one can use it for commercial purposes. In the recent past, people have raised objections on some of the methods used, e.g., the transfer of a human genes into food animals, use of organisms containing human genes as animal feed. Some religious groups have expressed their concern about the transfer of genes from animals whose flesh is forbidden for use as food into the animals that they normally eat. Transfer of animal genes into food plants may be objectionable to the vegetarians. Besides this, there are several other aspects of this issue that have to be sorted out.

Animal Biotechnology

11.23



(a) What will be the consequences, if a modified animal will breed with other domestic or wild animals, thereby, transferring the introduced genes to these populations?



(b) What are the health risks to human on consumption of genetically modified animals and their products?



(c) With the production of disease-resistant animals, what will be the effect on ecology?



(d) There is also widespread concern about the risks of human recipients getting infected with animal viral diseases after a xenotransplantation., which might infect the population at large.



(e) There are also concerns about the risk that drug resistance gene markers used in genetic engineering procedures might inadvertently be transferred and expressed.

The need of the hour is to formulate clear guidelines which should be followed while using genetic engineering techniques in bio-medical research e.g., products from transgenic organisms should be clearly marked to give choice to people who follow dietary restrictions due to religious beliefs. In fact, all the ethical and moral issues raised by some aspects of biotechnology should be addressed by open discussion and dialogue.

CHAPTER

12

Biotechnology of Aquaculture

Aquaculture, also known as aquafarming, is the farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants. Aquaculture involves cultivating freshwater and saltwater populations under controlled conditions, and can be contrasted with commercial fishing, which is the harvesting of wild fish. Mariculture refers to aquaculture practiced in marine environments.

• According to the Food and Agriculture Organization (FAO), aquaculture “is understood to mean the farming of aquatic organisms including fish, molluscs, crustaceans and aquatic plants. Farming implies some form of intervention in the rearing process to enhance production, such as regular stocking, feeding, protection from predators, etc. Farming also implies individual or corporate ownership of the stock being cultivated.

Biotechnology provides powerful tools for the sustainable development of aquaculture, fisheries, as well as the food industry. Increased public demand for seafood and decreasing natural marine habitats have encouraged scientists to study ways that biotechnology can increase the production of marine food products, and making aquaculture as a growing field of animal research.

• Biotechnology allows scientists to identify and combine traits in fish and shellfish to increase productivity and improve quality.



• Scientists are investigating genes that will increase production of natural fish growth factors as well as the natural defense compounds, marine organisms use, to fight microbial infections.



• Modern biotechnology is already making important contributions and poses significant challenges to aquaculture and fisheries development.



• It perceives that modern biotechnologies should be used as adjuncts to, and not as substitutes for, conventional technologies in solving problems, and that their application should be need-driven rather than technology-driven.

12.2

Environmental Biotechnology



• The use of modern biotechnology to enhance production of aquatic species holds great potential not only to meet demand but also to improve aquaculture.



• Genetic modification and biotechnology also holds tremendous potential to improve the quality and quantity of fish reared in aquaculture.



• There is a growing demand for aquaculture; biotechnology can help to meet this demand. As with all biotech-enhanced foods, aquaculture will be strictly regulated before approved for market.



• Biotech aquaculture also offers environmental benefits. When appropriately integrated with other technologies for the production of food, agricultural products and services, biotechnology can be of significant assistance in meeting the needs of an expanding and increasingly urbanized population.



• Successful development and application of biotechnology are possible only when a broad research and knowledge base in the biology, variation, breeding, agronomy, physiology, pathology, biochemistry and genetics of the manipulated organism exists.



• Benefits offered by the new technologies cannot be fulfilled without a continued commitment to basic research.



• Biotechnological programmes must be fully integrated into a research background and cannot be taken out of context if they are to succeed.

Biotechnology in fish breeding: Gonadotropin releasing hormone (GnRH) is now the best available biotechnological tool for the induced breeding of fish. GnRH is the key regulator and central initiator of reproductive cascade in all vertebrates. It is a decapeptide  and was first isolated from pig and ship hypothalami with the ability to induce pituitary release of luteinising  hormone (LH) and follicle stimulating hormone (FSH). Since then, only one form of GnRH has been identified in most placental mammals including, human beings, as the sole neuropeptide causing the release of LH and FSH. However, in non-mammalian species (except guinea pig) twelve GnRH variants have now been structurally elucidated, among them seven or eight different forms have been isolated from fish species. Depending on the structural variant and their biological activities, number of chemical analogues have seen prepared and, one of them is salmon GnRH analogue, profusely used now in fish breeding and marked commercially throughout the world. The induced breeding of fish is now successfully achieved by development of GnRH technology.   Transgenesis: Transgenesis or transgenics may be defined as the introduction of exogenous gene/DNA into host genome resulting in its stable maintenance, transmission and expression. The technology offers an excellent opportunity for modifying or improving the genetic traits of commercially important fishers, molluscs and crustaceans for aquaculture. The idea of

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12.3

producing transgenic animals became popular when Palmitter and his group in 1982, first produced transgenic mouse by introducing metallothionein human growth hormone fusion gene (mT-hGH) into mouse egg, resulting in dramatic increase in growth. This triggered a series of attempts on gene transfer in economically important animals, including fish. The first transgenic fish was produced by Zhu and his group in 1985 in China, who claimed the transient expression n putative transgenics, although they gave no molecular evidence for the integration of the transgene. The technique has now seen successfully applied to a number of fish species. Dramatic growth enhancement has been shown using this technique, especially in salmonids. Some studies have revealed enhancement of growth in adult salmon to an average of 3 Ð, 5 times the size of non-Ð transgenic controls, with some individuals, especially during the first few months of growth, reaching as much as 10 Ð, 30 times the size of the controls. The introduction of transgenic technique has simultaneously put more emphasis on the need for production of sterile progeny in order to minimize the risk of transgenic stocks mixing in the wild populations. The technical development has expanded the possibilities for producing either sterile fish or those whose reproductive activity can be specifically turned on or off using inducible promoters. This would clearly be of considerable value allowing both optimal growth and controlled reproduction of the transgenic stocks while ensuring that any escaped fish would be unable to breed. An increased resistance of fish to cold temperatures has been another subject of research in fish transgenics for the past several. Cold water temperatures pose a considerable stressor to many fish and few are able to survive water temperatures much below 0–1°C. This is often a major problem in aquaculture in cold climates. Interestingly, some marine teleosts have high levels (10 Ð 25 mg/ml) of serum antifreeze proteins (AFP) or glycoproteins (AFGP) which effectively reduce the freezing temperature by preventing ice-crystal growth. The isolation, characterization and regulation of these antifreeze proteins, particularly, of the inter-flounder Pleuronectas americanus, has been the subject of research for a considerable period in Canada. Consequently, the gene encoding the liver AFP from winter flounder was successfully introduced into the genome of Atlantic salmon where it became integrated into the germ line and then passed onto the off Ð spring F3, where it was expressed specifically in the liver. The introduction of AFPs to gold fish also increased their cold tolerance, to temperatures at which all the control fish died. Similarly, injection or oral administration of AFP to juvenile milkfish or tilapia led to an increase in resistance to a 26 to 13°C. drop in temperature. The development of stocks harboring this gene would be a major benefit in commercial aquaculture in countries where winter temperatures often border the physiological limits of these species. The most promising tool for the future of transgenic fish production is  undoubtedly in the development of the embryonic stem cell (ESC) technology. There cells are undifferentiated and remain totipotent, so, they

12.4

Environmental Biotechnology

can be manipulated in vitro, and subsequently reintroduced into early embryos where they can contribute to the germ line of the host. This would facilitate the genes to be stably introduced or deleted. Although significant progress has been made in several laboratories around the world, there are numerous problems to be resolved before the successful commercialization of the transgenic brood stock for aquaculture. To realize the full potential of the transgenic fish technology in aquaculture, several important scientific break through are required. These include:

(i) more efficient technologies for mass gene transfer,



(ii) targeted gene transfer technologies such as embryonic stem cell gene transfer,

(iii) suitable promoters to direct the expression of transgenes at optimal levels during the desired developmental stages, (iv) identified genes of desirable traits for aquaculture and other applications,

(v) information on the physiological, nutritional, immunological and environmental factors that maximize the performance of the transgenics,

(vi) safety and environmental impacts on transgenic fish. Chromosome Engineering: Chromosome sex manipulation techniques to induce polyploidy (triploidy and tetraploidy) and uniparental chromosome inheritance (gynogenesis and androgenesis) have been applied extensively in cultured fish species. These techniques are important in the improvement of fish breeding as they provide a rapid approach for gonadal sterilization, sex control improvement of hybrid viability and clonation. Most vertebrates are diploid, meaning that, they possess two complete chromosome sets in their somatic cells. Polyploidy individuals possess one or more additional chromosome sets, bringing the total to three in triploids, four in tetraploids and so on. Induced triploidy is widely accepted as the most effective method for producing sterile fish for aquaculture and fisheries management. The methods used to induce triploids and other types of chromosome set manipulations in fishes and the applications of these biotechnologies to aquaculture and fisheries management are well described by many scientists. Tetraploid breeding lines are of potential benefit to aquaculture, by providing a convenient way to produce large numbers of sterile triploid fish through simple interploidy crosses between tetraploids   and diploids. Although tetraploidy has been induced in many finfish species, the viability of tetraploids was low in most instances. In teleosts, technique for inducing sterility, include exogenous hormone treatment and triploidy induction. The use of hormone treatments, however, could be limited by governmental regulation and a lack of consumer acceptance of hormone-treated fish products. Triploidy can be induced by exposing eggs

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12.5

to physical or chemical treatment shortly after fertilization to inhibit extrusion of the second polar body triploid; fish are expected to be sterile because of the failure of homologous chromosomes to synapse correctly during the first meiotic division. Methods of triploidy induction included exposing fertilized eggs to temperature shock (hot or cold), hydrostatic pressure shock or chemicals such an colchicines, cytochalasin-B or nitrous oxide. Triploid can also be produced by crossing teraploids and diploids. Tetraploid induction involves fertilizing eggs with normal sperm and exposing the diploid zygote for physical or chemical treatment to suppress the first mitotic division. Gynogenesis is the process of animal development with exclusive maternal inheritance. The production of gynogenetic individuals is of particular interest to fish breeders because a high level of inbreeding can be induced in single generation. Gynogenesis may also be used to produce all Ð female populations in species with female homogamety and to reveal the sex determination mechanisms in fish. It is convenient to use all female gynogenetic progenies (instead of normal bisexual progenies) for sex inversion experiments. Methodologies combining use of induced gynogenesis with hormonal sex inversion have been developed for several aquaculture species. Androgenesis is the process which have commercial application in aquaculture. It can also be used in generating homozygous lines of fish and in the recovery of lost genotypes from the crypreserved sperms. Androgenetic individuals have been produced in a few species of cyprinids, cichlids and salmonids. Biotechnology and fish health management:  Disease problem area is a major constraint for development of aquaculture. Biotechnological tools such as molecular diagnostic methods, use of vaccines and immunostimulants are gaining popularity for improving the disease resistance in fish and shelfish species world over for viral diseases, avoidance of the pathogen is very important. In this context, there is a need to rapid method for detection of the pathogen. Biotechnological tools such as gene probes and polymerase chain reaction (PCR) are showing great potential in this area. Gene probes and PCRbased diagnostic methods have been developed for a number of pathogens affecting fish and shrimp. In case of finfish aquaculture, number of vaccine against bacteria and viruses have been developed. Some of these have been conventional vaccines consisting of killed microorganism but new generation of vaccine consisting of protein subunit vaccine, genetically engineered organism, and DNA vaccine, are currently under development. In the vertebrate system, immunization against disease is a common strategy. However, the immune system of shrimp is rather poorly developed; biotechnological tools are helpful for development of molecule, which can stimulate this immune system of shrimp. Recent studies have shown that the non-specific defense system can be stimulated using microbial product such as lipopolysacharides, peptidoglycans or glucans. Among the immunostimulants known to be effective in fish, glucan and levamisole enhance phagocytic activities and specific antibody responses.

12.6

Environmental Biotechnology

Cryopreservation of gametes or gene banking:   Cryopreservation is a technique, which involve long-term preservation and storage of biological material at a very low temperature, usually at -196°C, the temperature of liquid nitrogen. It is based on the principle that very low temperature tranquilize or immobilize the physiological and biochemical activities of cell, thereby, making it possible to keep them viable for very long period. The technology of cryopreservation of fish spermatozoa (milt)  has been adopted for animal husbandary . The first success in preserving fish sperm at low temperature was reported by Blaxter (1953) who fertilized Herring (Clupea herengus) eggs with frozen, thawed semen. The spermatozoa of almost all cultivable fish species has now been cryopreserved. Cryopreservation overcomes problems of male maturing before female, allows selective breeding and stock improvement and enables conservation. One of the emerging requirements for that can be used by breeders for evolving new strains. Most of the plant varieties that have been produced are based on the gene bank collections. Aquatic gene bank, however, suffers from the fact that, at present, it is possible to cryopreserve only the male gametes of finfishes and there in no viable technique for finfish eggs and embryos..

Conclusion Biotechnological research and development are growing at a very fast rate. Biotechnology has assumed greatest importance in recent years in the development of fisheries, agriculture and human health. The science of biotechnology has endowed us with new tools and tremendous power to create novel genes and genotypes of plants, animals and fish. The application of biotechnology in the fisheries sector is a relatively recent practice. Nevertheless, it is a promising area to enhance fish production. The increased application of biotechnological tools can certainly revolutionize our fish farming besides its role in biodiversity conservation.  

12.1  PRODUCTION OF TRANSGENIC FISH By using different transgenic techniques, researchers are seeking to improve the genetic traits of the fish used in aquaculture. Researchers are trying to develop fish which are: larger and grow faster, more efficient in converting their feed into muscle, resistant to diseases, tolerant of low-oxygen levels in the water, and tolerant to freezing temperatures.

• For example, some species of fish make a protein which allows them to survive in the Arctic. This “antifreeze” gene has been transplanted into other species of fish so, they also can survive in very cold waters.

Growing fish, that are longer and heavier, is the goal of researchers, who are experimenting with applying various types of growth hormone to fish. One method of doing this is to dip the fish in a solution which contains the hormone. However, there are some problems with this technique. First, it

Biotechnology of Aquaculture

12.7

may be difficult to produce large quantities of purified growth hormone, the method is labor-intensive, and it’s difficult to determine whether the fish are getting the right amount of growth hormone. Therefore, researchers want to develop new strains of transgenic fish which naturally produce just the right amount of growth hormone to speed their growth. Such fish would be more cost-effective since, they would produce higher levels of growth hormone on their own, and they would pass this trait to their offspring. There has been some success in this area. For example, a researcher at the University of Connecticut has developed a tilapia fish that grows twice as fast and up to five times as large as wild strains. The scientist introduced an extra copy of the growth hormone gene into fish embryos at a very early stage, resulting in the unique growth characteristics. There are two main techniques which researchers use to transfer genetic material in fish. • One is called microinjection, in which the genetic material is injected into newly-fertilized fish eggs. However, this method is time-consuming, so researchers may prefer to use electroporation. • Electroporation involves transferring the genetic material, or DNA, into fish embryos through the use of an electrical current. • Besides these two techniques, Retroviral vectors, containing the envelope protein of vesicular stomatitis virus, have been developed, and used to produce transgenic fish. Microinjection: Gene transfer research with fish began in the mid 1980s utilizing microinjection. Zhu and his group, in 1980, published the first report of transgenes microinjected into the fertilized eggs of goldfish. In almost all fish gene transfer research, the foreign gene was microinjected into the cytoplasm of one-to-four cell embryos, as pronuclei are extremely difficult to visualize in live one-cell fish embryos. Microinjection is a tedious and slow procedure and can result in high egg mortality. After the initial development of microinjection, new techniques such as electroporation, retroviral integration, liposomal-reverse-phase-evaporation, sperm-mediated transfer and high velocity micro-projectile bombardment were developed that sometimes can more efficiently produce large quantities of transgenic individuals in a shorter time period. Electroporation: It involves placing the eggs in a buffer solution containing DNA and applying short electrical pulses to theoretically create a transient openings of the cell membrane, allowing the transfer of genetic material from solution into the cell. The efficiency of the electroporation is affected by a variety of factors including voltage, number of pulses and frequency of pulses. The first successful gene transfer utilizing electroporation produced integration rates and survival similar to that for microinjection. It is proved experimentally that electroporation can be more efficient than microinjection with integration rates sometimes as high as 30–100%. Hatching rates are higher

12.8

Environmental Biotechnology

for electroporated embryos than for microinjected channel catfish embryos, and post-fertilization electroporation treatments had higher hatching rates than electroporation of sperm and then eggs, prior to fertilization. Efficiency of gene transfer is determined by several factors including: hatching percentage, gene integration frequency, the number of eggs which can be manipulated in a given amount of time and the quantity of effort required to manipulate the embryos. In this regard, electroporation is a powerful technique for mass production of transgenic fish. Retroviral vectors: Retroviral vectors containing the envelope protein of vesicular stomatitis virus have been developed, and used to produce transgenic fish. Integration rates may be increased because of active infection. Unfortunately, these vectors are prone to unstable expression or even complete silencing of transgene expression. Sarmasik and his group (2001) successfully utilized retroviral constructs to produce transgenic crayfish and topminnows, Poeciliposis lucida. The pantropic retroviral vectors were derived from the Hepatitis B virus and the Vesicular stomatitis virus, a pathogen similar to hoof and mouth disease which infects mammals, insects and possibly, plants. The vector sticks to most cell membranes of any species. Transgenic crayfish and topminnows were produced by injecting immature gonads with a solution of the vector, about one month before the normal age of first reproduction. Matured injected individuals were mated with normal individuals, and produced 50% transgenic offspring. Integration, expression and transmission of the pantropic retroviral-reporter transgene were observed, for at least three generations. This is a very good gene transfer technique for live-bearers and fish, in general, but, introduction of viral sequences into food fish may not be accepted by the public. Use of transposases to enhance integration rates may be a more viable option than retroviral vectors for oviparous aquatic organisms, but does not solve the problem of live-bearers. Theoretically, inactivation of a gene can be accomplished by knockout of the gene by replacing the original gene with a mutated copy of the gene, or by disruption of gene expression using the antisense approach or the ribozyme technology. Although, the later two approaches are currently feasible, the knockout approach is the ultimate method for gene inactivation because it will eliminate the gene products completely. Another technique for post-transcriptional gene silencing is utilization of RNA antisense constructs. Both double-stranded RNA and antisense RNA were effective in disrupting the expression of GFP in transgenic zebra fish. Various antisense technologies appear feasible. Regardless of the method of transfer, the foreign DNA introduced to the developing embryo, it appears to initially replicate and amplify rapidly in the cytoplasm of the developing embryo, and then disappears, as development proceeds. Integration at the one-cell stage has never been observed, thus the delayed integration causes mosaicism, and, not all tissues contain the transgene and, not all cells within

Biotechnology of Aquaculture

12.9

the transgenic tissues, harbor the transgene. Copy numbers can range from one to several thousand at a single locus, and, in contrast to the head-to-tail organization observed in the mouse system, in some but not all cases, the DNA can also be found organized in all possible concatemeric forms, suggesting random end-to-end ligation of the injected DNA prior to integration. Transgenes can integrate at single or multiple chromosomal locations for individual transgenic fish. For salmonids, the frequency of transgene transmission from founder animals averages about 15%, suggesting that integration of the foreign DNA occurs on average at the two-to-four cell stage of development. Transmission of transgenes to F2, or later progeny, occurs at Mendelian frequencies, indicating that the DNA is stably integrated into the host genome and passes normally through the germ line.

12.2  PEARL OYSTER CULTURE The pearl oyster industry, traditionally, relied upon spat collection in the field to supply its needs for farm requirements. As the industry has expanded, the need for a more predictable source of the spat has emerged. Likewise, the thought of controlling some desirable characteristics of the pearl oysters has resulted in the setup of hatchery and nursery facilities. The hatchery and nursery will give the farmer a predictable supply of spat and will allow greater manipulation of genetic traits. This is the road to proper animal husbandry. Every hatchery and nursery has site-specific conditions which require fine-tuning of the initiating protocol used at the facility. This development of the specific protocol for a hatchery will take place as the start-up begins and, as spat are produced. In the end, the protocol for the given hatchery will be the most efficient possible for that hatchery. Good observation and thoroughness are necessary to fine-tune the initiating protocol. The hatchery/nursery staff must exhibit these qualities in their work. It is the intent of the consultant that the start-up and training stage of this new hatchery and nursery will be greatly facilitated by the information contained in a manual which is prepared for each hatchery. Broodstock: Central to larval culture is, the availability of good quality broodstock. Broodstock Pinctada margaritifera may be obtained from shell that have settled on collectors set in the lagoons of atolls or, in some cases, from wildstock shell. Freshly-collected shell, which are potential broodstock, should be assessed for DVM (Dorso-Ventral Measurement) size and shape of the shell. If the shell looks suitable for broodstock-use, the shell should be drilled in the appropriate place and hung on chaplets from a long line or they may be placed into 8–pocket panel nets and suspended from a long line. They should remain on the long line at least 4–6 months before they may be used during a spawning for the hatchery. This allows time for the shell to acclimate to the lagoon conditions, and for gametogenesis to proceed toward ripe gametes. Another very suitable source of broodstock are seeded shell which

12.10

Environmental Biotechnology

are already hanging on long lines. Although the farmer must be careful not to have excessive handling of these seeded shell, it is normal in most places that cleaning of these shell is carried out on a regular basis to reduce fouling organisms growing on the outside of the shell. Cleaning of these shells can be combined with their use as broodstock for a planned spawning in the hatchery. Large quantities of good quality eggs can be obtained by this method because shells which have been hanging on chaplets and long lines generally are in good condition and release gametes readily upon handling. The numbers of shell being cleaned are generally large, so there is no problem with obtaining sufficient eggs for stocking into the hatching tanks. Spawning: Other than the thermal method of spawning stimulation, chemical methods can be used to induce spawning. These include different concentrations [1.532, 3.064, or 6.128 millimolars] of hydrogen peroxide, either in normal seawater or alkaline seawater (pH 9.1). The pH media can be prepared using Tris buffer or Sodium hydroxide pellets. The Pearl Oyster Farming and Pearl Culture Manual in India [Central Marine Fisheries Research Institute at Tuticorin, India, published, February 1981] stated that, when inducing spawning chemically, “A pH value of 9.0 in the case of Tris buffer and 9.5 in NaOH, gives 78.6% and 68.4% of spawning, respectively.” Further, they say, “Injection of 0.2 ml of N/10 ammonium hydroxide solution into the adductor muscle of the pearl oyster results in 48% spawning.” It should be noted here that serotonin-induced spawning such as used with giant clams (1–4 ml of 2 millimolar serotonin solution) may also be a possibility with pearl oysters. However, information from the James Cook University Blacklip Pearl Oyster Project indicates that serotonin is not so effective with pearl oysters as it is with giant clams or other bivalves. Although, initial spawnings may utilize relatively large numbers of brood stock that will not be identified as the parents, eventually, the hatchery will be used for crosses between broodstock shell with desirable traits. In this case, the broodstock should be kept on tagged chaplets or 8–pocket panel nets so that the parents can be identified. DVM measurements would also be recorded at each spawning date that the shells are used as broodstock. Do pearl oysters which produce good quality, round pearls, at the first harvest, possess a genetic trait for round pearls? This question could be tested by using these shell for a special spawning after they have been re-seeded a second time and had 4–6 months rest in the lagoon. The growth, survival, and general development of their offspring would be carefully recorded up, until the time, they were old enough for their first seeding. The result will then come out in the first harvest. There are other characteristics of the shell shape, the nacre, etc., which are likewise important for genetic manipulation. Larval Rearing in the Hatchery: A most important factor in larval rearing success is cleanliness. It is essential that egg and sperm collection materials have been chlorine-cleaned and are stored dry for use during a spawning and during the larval cycle.

Biotechnology of Aquaculture

12.11

The following details on the development of embryos and larvae are taken from the Pearl Oyster Farming and Pearl Oyster Culture Manual:

12.2.1  Early Development and Larval Rearing Cleavage: The first cell division is seen 45 minutes after fertilization resulting in the formation of a micromere and a macromere. The polar body is placed at the cleavage furrow. During the second cleavage the micromere divides into two and the macromere divides unequally into a micromere and macromere. The stage with three micromeres and a macromere is called Trefoil stage. The macromere does not take part in further divisions. Micromeres divide repeatedly, thus becoming smaller and smaller and passing through 8-cell, and so on, until the morula stage. Each micromere develops a small cilium which helps in the movement of the embryo. Blastula: The embryo is ball-like with transparent cells and a blastocoel. The embryos lift themselves in the water column and congregate at the surface. The floating embryos are siphoned out to clean containers and the residues at the bottom, containing broken tissues, undeveloped embryos, unfertilized eggs, sperm, etc., are discarded. Reorientation of cells starts and the blastocoel and blastopore are formed. The blastula stage is reached 5 hours after fertilization. Gastrula: Gastrulation takes place by epiboly. The cells convolute and differentiate into different dermal layers. The archenteron is formed. The embryo is bean-shaped as there is convolution of cells. The gastrula exhibits negative phototropism. The stage is reached in 7 hours. Trochophore larva: The minute cilia present in the gastrula stage disappear and the pre-oral and post-oral tufts of cilia develop, thus marking antero-posterior differentiation of the embryo. A single apical flagellum is developed at the anterior side. The anterior portion of the larva is broader while the posterior end is tapering like an inverted triangle. The movement of the larva is affected by the propulsive movement of the flagellum. The dorsal ectodermal cells secrete the embryonic shell, known as the prodissoconch I. Veliger: A definite ‘D’ shape is obtained by the secretion of the prodissoconch I having a hinge line, mantle and rearrangement of the pre-oral tuft of cilia into a velum. The single flagellum, pre-oral and post-oral tufts of cilia disappear. The veliger larva [of Pinctada fucata] measures 67.5 um along the antero-posterior axis and 52.5 um along the dorso-ventral axis. This stage is reached in 20 hours. A Quick Reference to Feeding Schedules for Larvae and Spat are prepared in the manual. This useful reference should be placed on the Algal Lab wall and used daily during the larval feeding. Another part of the protocol shown in the manual are, days of larval life shown against the columns of Stocking Density (of larvae), Algal Feed, Flushing requirements, Draindown requirements,

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Microscope checks on size of larvae, % feeding, and Spat collectors. This table will allow a quick visual check over the larval cycle by technicians to see what is required. Technician Duty Schedule: Along with the above tables which help the technicians to keep up with the protocol, there need to be forms for Weekly Duty Schedules for, 1.) the Hatchery Phase, 2.) the Nursery Stage, and 3.) the Algal Production. These schedules list the most important duties that the technicians need to do over a weekly period. Some duties are required daily, whilst others are required less regularly. Land Nursery Culture of Spat: The manual discusses settling materials to use for late stage larvae and the potential positive effects of conditioning the collector materials. Spat can be left on their collectors until they are large enough to be safely removed and placed into trays with the appropriate size mesh to retain the spat. The minimum protocol required in the raceways is discussed in the manual. Algal Culture: The usual food of bivalve larvae such as blacklip pearl oysters is unicellular algae ranging from 2–10 microns (um). Generally, it is wise to be careful in feeding new veliger larvae with too much unicellular algae as the gut may only just be in the process of completion and the possibility exists of the gut becoming plugged up with algal cells that cannot be completely digested. The protocol on the feeding density over days of the larval cycle are shown in the manual. Monospecific cultures: Whether a hatchery is located in a temperate or tropical area, the monospecific unicellular algal cultures are required for larval rearing needs. A considerable amount of time is needed to set up and maintain these cultures. The trained technicians handling the algae cultures must keep careful attention to detail and hygiene. The manual shows the steps involved in starting with stock cultures of unicellular algae (= microalgae) to large mass cultures of 60–250-L. The f/2 medium is one of the standard microalgal culture mediums in use around the world. This will be the medium to be used at hatchery for cultures from 50 ml flasks to the 250-L cylinder cultures, but where the budget is restricted, Aquasearch has it’s own cheap medium which works nearly as well, as the f/2 medium. The stocks must be cared for because all the cultures come from the stocks. It should be noted here that sodium metasilicate is only added to the media which will be used to grow diatoms in. Diatoms have an outside shell (like a jewelry box that fits neatly into top and bottom) made of silicate, so, this material becomes limiting in dense cultures. Mass microalgal cultures that will be grown in the outdoor algal culture area will grow well with other culture media that are tested for mass culture. These media are cheaper than f/2 and quite suitable for the large volumes of algae being grown to feed spat in the land nursery raceways.

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12.3  SHRIMP FARMING Shrimp are decapod crustaceans, that can be found in both fresh and salt water. Shrimp are swimming crustaceans living close to the bottom of the water bodies. With firm and translucent flesh and their delicious taste, shrimp are one of the most popular seafood, just next to fish. Low in saturated fats and calories, but high in essential fats and other nutrients, shrimp are a healthy food, with innumerable health benefits. So let’s take a brief look at shrimp nutritional value and benefits. A shrimp farm is an aquaculture business for the cultivation of marine shrimp or prawns for human consumption. Commercial shrimp farming began in the 1970s, and production grew steeply, particularly to match the market demands of the United States, Japan and Western Europe. The total global production of farmed shrimp reached more than 1.6 million tonnes in 2003, representing a value of nearly 9 billion U.S. dollars. About 75% of farmed shrimp is produced in Asia, in particular, in China and Thailand. The other 25% is produced mainly in Latin America, where Brazil, Ecuador, and Mexico are the largest producers. The largest exporting nation is Thailand. Shrimp farming has changed from traditional, small-scale businesses in Southeast Asia into a global industry. Technological advances have led to growing shrimp at ever higher densities, and broodstock is shipped worldwide. Virtually, all farmed shrimp are of the family Penaeidae, and just two species – Penaeus vannamei (Pacific white shrimp) and Penaeus monodon (giant tiger prawn) – account for roughly 80% of all farmed shrimp. These industrial monocultures are very susceptible to diseases, which have caused several regional wipe-outs of farm shrimp populations. Increasing ecological problems, repeated disease outbreaks, and pressure and criticism from both NGOs and consumer countries, led to changes in the industry in the late 1990s and, generally stronger regulation by governments. In 1999, a program aimed at developing and promoting more sustainable farming practices was initiated, including governmental bodies, industry representatives, and environmental organizations. Farming methods: When shrimp farming emerged to satisfy demand that had surpassed the wild fisheries’ capacity, the subsistence farming methods of old were rapidly replaced by the more productive practices required to serve a global market. Industrial farming, at first, followed traditional methods, with so-called “extensive” farms, compensating for low density with increased pond sizes; instead of ponds of just a few hectares, ponds of sizes up to 100 hectares (1.0 km2) were used and huge areas of mangroves were cleared in some areas. Technological advances made more intensive practices possible that increased yield per area, helping reduce pressure to convert more land. Semi-intensive and intensive farms appeared, where the shrimp were reared on artificial feeds and ponds were actively managed. Although many extensive farms remain, new farms typically are of the semi-intensive kind.

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Until the mid-1980s, most farms were stocked with young wild animals, called ‘postlarvae’, typically, caught locally. Post larvae fishing became an important economic sector in many countries. To counteract the depletion of fishing grounds and to ensure a steady supply of young shrimp, the industry started breeding shrimp in hatcheries.

Life cycle

• Shrimp mature and breed only in a marine habitat. The females lay 100,000 to 500,000 eggs, which hatch after some 24 hours into tiny nauplii.



• These nauplii feed on yolk reserves within their bodies, and then metamorphose into zoeae.



• Shrimp in this second larval stage, feed in the wild on algae, and after a few days, morph again into myses.



• The myses look akin to tiny shrimp, and feed on algae and zooplankton.



• After another three to four days, they metamorphose a final time into postlarvae—young shrimp, that have adult characteristics.



• The whole process takes about 12 days from hatching.

In the wild, post larvae then migrate into estuaries, which are rich in nutrients and low in salinity. They migrate back into open waters when they mature Supply chain: In shrimp farming, this life cycle occurs under controlled conditions. The reasons to do so include, more intensive farming, improved size control resulting in more uniformly-sized shrimp, and better predatorcontrol, but also the ability to accelerate growth and maturation by controlling the climate (especially, in farms in the temperate zones, using greenhouses). There are three different stages:

• Hatcheries breed shrimp and produce nauplii or even post larvae, which they sell to farms. Large shrimp farms maintain their own hatcheries and sell nauplii or post larvae to smaller farms in the region.



• Nurseries grow post larvae and accustom them to the marine conditions in the grow-out ponds.



• In the grow-out ponds the shrimp are grown from juveniles to marketable size, which takes between three to six months.

Most farms produce one to two harvests a year; in tropical climates, even three are possible. Because of the need for salt water, shrimp farms are located on or near a coast. Inland shrimp farms have also been tried in some regions, but, the need to ship salt water and competition for land with agricultural users, led to problems. Hatcheries: Small-scale hatcheries are very common throughout Southeast Asia. Often run as family businesses and using a low-technology approach,

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they use small tanks (less than ten tons) and often low animal densities. They are susceptible to disease, but due to their small size, they can typically restart production quickly after disinfection. The survival rate is anywhere between zero and 90%, depending on a wide range of factors, including disease, the weather, and the experience of the operator.

• Green water hatcheries are medium-sized hatcheries, using large tanks with low animal densities. To feed the shrimp larvae, an algal bloom is induced in the tanks. The survival rate is about 40%.



• Galveston hatcheries (named after Galveston, Texas, where they were developed) are large-scale, industrial hatcheries using a closed and tightly-controlled environment. They breed the shrimp at high densities in large (15 – 30 t) tanks. Survival rates vary between 0% and 80%, but typically, achieve 50%.



• In hatcheries, the developing shrimp are fed on a diet of algae, and later also, brine shrimp nauplii, sometimes (especially in industrial hatcheries) augmented by artificial diets. The diet of later stages also includes fresh or freeze-dried animal protein, for example, krill. Nutrition and medication (such as antibiotics) fed to the brine shrimp nauplii are passed on to the shrimp that eat them.

Nurseries: Farmers transferring post larvae from the tanks on the truck to a grow-out pond Many farms have nurseries where the post-larval shrimp are grown into juveniles for another three weeks in separate ponds, tanks, or socalled raceways. A raceway is a rectangular, long, shallow tank through which water flows continuously.

• In a typical nursery, there are 150 to 200 animals per square metre. They are fed on a high-protein diet for at most three weeks before they are moved to the grow-out ponds. At that time, they weigh between one and two grams. The water salinity is adjusted gradually to that of the grow-out ponds.



• Farmers refer to post larvae as “PLs”, with the number of days suffixed (i.e., PL-1, PL-2, etc.). They are ready to be transferred to the growout ponds after their gills have branched, which occurs around PL13 to PL-17 (about 25 days after hatching). Nursing is not absolutely necessary, but is favored by many farms because it makes for better food utilization, improves the size uniformity, helps use the infrastructure better, and can be done in a controlled environment to increase the harvest. The main disadvantage of nurseries is that some of the postlarval shrimp die upon the transfer to the grow-out pond.



• Some farms do not use a nursery, but stock the post larvae directly in the grow-out ponds after having acclimated them to the appropriate temperature and salinity levels in an acclimation tank. Over the course of a few days, the water in these tanks is changed gradually to match

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that of the grow-out ponds. The animal density should not exceed 500/ liter for young post larvae and 50/liter for larger ones, such as PL-15. Feeding the shrimp: While extensive farms mainly rely on the natural productivity of the ponds, more intensively managed farms rely on artificial shrimp-feeds, either exclusively or as a supplement to the organisms that naturally occur in a pond. A food chain is established in the ponds, based on the growth of phytoplankton. Fertilizers and mineral conditioners are used to boost the growth of the phytoplankton to accelerate the growth of the shrimp. Waste from the artificial food pellets and shrimp excrement can lead to the eutrophication of the ponds. Artificial feeds come in the form of specially formulated, granulated pellets that disintegrate quickly. Up to 70% of such pellets are wasted, as they decay before the shrimp have eaten them. They are fed two to five times daily; the feeding can be done manually either from ashore or from boats, or using mechanized feeders distributed all over a pond. The feed conversion rate (FCR), i.e., the amount of food needed to produce a unit (e.g., one kilogram) of shrimp, is claimed by the industry to be around 1.2–2.0 in modern farms, but this is an optimum value that is not always attained, in practice. For a farm to be profitable, a feed conversion rate below 2.5 is necessary; in older farms or under suboptimal pond conditions, the ratio may easily rise to 4:1. Lower FCRs result in a higher profit for the farm. Farmed species: Although there are many species of shrimp and prawn, only a few of the larger ones are actually cultivated, all of which belong to the family of penaeids (family Penaeidae), and within it, to the genus Penaeus. Many species are unsuitable for farming: they are too small to be profitable, or simply stop growing, when crowded together, or are too susceptible to diseases. The two species dominating the market are:

• Pacific white shrimp (Litopenaeus vannamei, also called “whiteleg shrimp”) is the main species cultivated in western countries. Native to the Pacific coast from Mexico to Peru, it grows to a size of 23 cm. L. vannamei accounts for 95% of the production in Latin America. It is easy to breed in captivity.



• Giant tiger prawn (Penaeus monodon, also known as “black tiger shrimp”) occurs in the wild in the Indian Ocean and in the Pacific Ocean from Japan to Australia. The largest of all the cultivated shrimp, it can grow to a length of 36 cm and is farmed in Asia. Because of its susceptibility to white spot disease and the difficulty of breeding it in captivity, it is gradually being replaced by L. vannamei since 2001.

Together, these two species account for about 80% of the whole farmed shrimp production.

• Indian white shrimp (P. indicus) is a native of the coasts of the Indian Ocean and is widely bred in India, Iran and the Middle East and along the African shores.

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Shrimp marketing: For commercialization, shrimp are graded and marketed in different categories. From complete shrimp (known as “headon, shell-on” or HOSO) to peeled and deveined (P&D), any presentation is available in stores. The animals are graded by their size uniformity and then also, by their count per weight unit, with larger shrimp attaining higher prices.

12.3.1  Shrimp Nutritional Value Details Shrimp Nutrition Facts: Shrimp are quite popular as a low calorie food source of protein. About 85 g of shrimp provide just 84 calories. The same amount of shrimp contain approximately—

• 0.9 g fats, with 0.2 g saturated fats,



• 0.2 g monounsaturated fats and



• 0.4 g poly unsaturated fats.

Apart from these, 85 g shrimp contain about 17.8 g proteins, 166 mg cholesterol, 295 mg omega-3 fatty acids, 17.9 mg omega-6 fatty acids, 191 IU of vitamin A, 1.9 mg vitamin C, 1.3 mcg vitamin B12, 2.2 mg niacin, 3.4 mcg folate, 68.8 mg choline, 33.2 mg calcium, 2.6 mg iron, 28.9 mg magnesium, 155 mg potassium, 116 mg phosphorus, 1.3 mg zinc and 33.7 mg selenium. Shrimp Nutritional Benefits: A mere look at the shrimp nutrition facts can give you an idea about how nutritious this seafood is. The only thing that can confuse people is that shrimp are quite high in cholesterol. Though shrimp contains a high amount of cholesterol and increases the level of LDL cholesterol in the body, it can also raise the level of HDL cholesterol, which is considered as good for the health of the cardiovascular system. In fact, increase in HDL cholesterol is more than the increase in LDL cholesterol, when a shrimp diet is followed. Thus, eating shrimp can lower the ratio of LDL to HDL cholesterol, which can prove beneficial for the heart health. In addition to increasing the level of HDL cholesterol, shrimp can provide a number of health benefits, which are explained below:

• Shrimp are rich in omega-3 and 6 fatty acids, the two types of essential fatty acids that can boost heart and cardiovascular health. Eating foods rich in these fatty acids can significantly lower the risk for cardiovascular diseases, by reducing the level of cholesterol in the body, and preventing blood clotting.



• Omega-3 essential fatty acids can also slow down or prevent the development of diseases like rheumatoid arthritis, high blood pressure, colorectal cancer and cancerous tumors.



• Being a very important dietary source of selenium, shrimp can provide protection against degenerative diseases and cancer. Selenium is the trace mineral, which can prevent the proliferation of cancerous cells and neutralize the damaging effects of free radicals, which is associated with a number of diseases including, cancer.

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• Vitamin B12, found in shrimp, ensures proper formation and maturation of the blood cells. This vitamin is also essential for the functions of the brain. • Shrimp contain significant amount of vitamin D and the mineral calcium. Vitamin D is required by the body for the proper absorption of the mineral calcium, which is essential for the formation of strong bones and teeth. • Phosphorus, found in shrimp, can also contribute towards the development of strong bones and teeth. • Shrimp do not contain significant level of saturated fats, but contains unsaturated fatty acids, which again can help to improve the health of heart and the cardiovascular system. This seafood can also help to lower the level of triglycerides, and thereby, reduce the risk of heart disease and stroke. To sum up, shrimp is one of the highly nutritious seafood around. However, it is also common food allergens, and many individuals can experience severe allergic reactions due to their intake. An allergic reaction to shrimp can produce itching, hives, skin rash, breathing problems, wheezing and unusual swelling—especially of the face, tongue, throat and the lips. To avoid such severe reactions, it is generally advised not to take or consume shrimp in its pure form. But those who are not allergic to shrimp, can realize the shrimp nutritional value or health benefits, while enjoying the delectable taste of this seafood. Shrimp can be prepared in a number of ways, but the point to be kept in mind is that it should be cooked quickly, so as to preserve its taste and flavor.

12.4  GROWTH AND REPRODUCTION OF EDIBLE CRUSTACEANS Crustaceans (Crustacea) form a very large group of arthropods, usually treated as a subphylum, which includes such familiar animals as crabs, lobsters, crayfish, shrimp, krill and barnacles. The 67,000 described species range in size from Stygotantulus stocki at 0.1 mm (0.004 in), to the Japanese spider crab with a leg span of up to 12.5 ft (3.8 m) and a mass of 44 lb (20 kg). Like other arthropods, crustaceans have an exoskeleton, which they moult to grow. They are distinguished from other groups of arthropods, such as insects, myriapods and chelicerates, by the possession of biramous (two-parted) limbs, and by the nauplius form of the larvae. Most crustaceans are free-living aquatic animals, but some are terrestrial (e.g. woodlice), some are parasitic (e.g. Rhizocephala, fish lice, tongue worms) and some are sessile (e.g. barnacles). The group has an extensive fossil record, reaching back to the Cambrian, and includes living fossils such as Triops cancriformis, which has existed apparently unchanged since the Triassic period. More than 10 million tons of crustaceans are produced by fishery or farming for human consumption, the majority of it being shrimps and prawns.

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Krill and copepods are not as widely fished, but may be the animals with the greatest biomass on the planet, and form a vital part of the food chain. The scientific study of crustaceans is known as carcinology (alternatively, malacostracology, crustaceology or crustalogy), and a scientist who works in carcinology is a carcinologist. Consumption by humans: Many crustaceans are consumed by humans, and nearly 10,700,000 tons were produced in 2007; the vast majority of this output is of DECAPOD CRUSTACEANS: crabs, lobsters, shrimp, and prawns. Over 60% by weight of all crustaceans caught for consumption are shrimp and prawns, and nearly 80% are produced in Asia, with China alone producing nearly half the world’s total. Non-decapod crustaceans are not widely consumed, with only 118,000 tons of krill being caught, despite krill having one of the greatest biomasses on the planet Decapod Crustaceans (edible crustaceans): The decapods or Decapoda (literally “ten-footed”) are an order of crustaceans within the class Malacostraca, including many familiar groups, such as crayfish, crabs, lobsters, prawns and shrimp. Most decapods are scavengers. It is estimated that the order contains nearly 15,000 species in around 2,700 genera, with approximately 3,300 fossil species. Nearly half of these species are crabs, with the shrimp and Anomura (including hermit crabs, porcelain crabs, squat lobsters), making up the bulk of the remainder. Classification within the order Decapoda depends on the structure of the gills and legs, and the way in which the larvae develop, giving rise to two suborders:

• Dendrobranchiata and



• Pleocyemata.



• Dendrobranchiata consists of prawns, including many species colloquially referred to as “shrimp”, such as the “white shrimp”, Litopenaeus setiferus.



• Pleocyemata includes the remaining groups, including true shrimp. Those groups which usually walk rather than swim (Pleocyemata, excluding Stenopodidea and Caridea) form a clade called Reptantia.

Decapods are primarily marine animals and are most abundant in warm, shallow tropical waters, but they are exploited commercially throughout the world. Some shrimp, for example, live in the open ocean and possess light organs, or photophores, which are thought to aid in feeding, species recognition, or camouflage (by counter-illumination). Approximately, 10 per cent of known decapod species occur in freshwater or terrestrial habitats. Survival in freshwater depends upon an organism’s ability to keep its blood concentration at a level higher than the medium and to reduce the permeability of its body surface. Those decapods that have colonized terrestrial environments, such as some species of hermit and fiddler crab, have evolved mechanisms to

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protect against desiccation and overheating while regulating the internal concentrations of their body fluids. Vascularization of the gill surfaces has made respiration possible on land for some species of decapods. Terrestrial decapods must usually return to the sea to spawn, while most freshwater decapods spend their entire life cycle in fresh water, commonly hatching their young as miniature adults. Growth and reproduction: Decapods exist in a variety of relationships with other organisms. Members of some hermit crab species, for example, carry anemones or bryozoan colonies on the shell in a commensal relationship (one, in which, the colonies do not feed on the host tissue). The pea crab Pinnotheres ostreum, on the other hand, parasitically feeds on the American oyster, causing gill damage. Some shrimp have symbiotic relationships with fish; they remove parasites from the mouths and gills of the fish. Decapods are behaviorally complex. Hermit crabs seek out empty shells to use as a protective covering, selecting successively larger ones to accommodate their growth. They discriminate between available shells based on each shell’s size, species, weight, and degree of physical damage. The two basic types of locomotion are swimming and crawling, though the macruran decapods are able to move swiftly backward by flexing their abdomens. Burrowing is accomplished by beating the leaf like swimmerets, or pleopods, or by digging with the thoracic legs. There is generally a separation between the sexes, although there are some examples of simultaneous hermaphroditism (i.e., individuals with both male and female reproductive organs). In most groups, fertilization is external, although in some species it is internal. Variations in patterns of mating activity are believed to be linked to the molting cycle. Male decapods can copulate only when their exoskeleton is fully hardened, while some females are capable of copulation only after a molt, when their shells are soft. In most decapods, the fertilized eggs are carried cemented to the abdominal appendages until they are hatched. After hatching, they can be classified as one of four basic larval types, partly by their mode of locomotion: nauplius, protozoea, zoea, and postlarva. Most decapod crustacean larvae hatch in the zoea stage. Decapods have three distinct body regions, each made up of segments, or somites: the head, thorax, and abdomen. The head and the thorax are fused and are often referred to as the cephalothorax. A pair of appendages is attached to each somite. The first two pairs, the first and second antennae, consist of a segmented stalk and flagella, and serve such sensory functions as olfaction, touch, and balance. The remaining three head appendages are either the crushing and chewing mandibles or the flattened, multi-lobed food manipulators. The anterior thoracic appendages serve as mouthparts, while the posterior pairs are the walking legs, or pereiopods. The remaining appendages may be modified for swimming, sperm transfer, pinching claws, or even forming a tail fan with the telson.

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A head shield, or carapace, covers the cephalothorax and extends over the gills, which are attached to the body wall of the thorax. The heart is located to the rear of the carapace above the gut, which is basically a straight tube consisting of the stomodeum, or foregut, the mesenteron, or midgut, and the proctodeum, or hindgut. The primary excretory organ is a gland (the “green gland”) that opens at the base of the antennae. The central nervous system consists of a supraesophageal ganglion with lateral connections to a subesophageal ganglion. The eyes, which may be absent in some deep-sea species, are usually well-developed with a pigmented, multifaceted cornea. Reproduction: Crustaceans produce from eggs, which have been fertilized by sperm in much the same manner as other animals. The eggs are produced in the ovaries in the female and passed to the outside through oviducts. The sperms are produced in tubular testes in the male. After the eggs have been fertilized, they begin development and then hatch.

• When the eggs hatch, this can take several days to several weeks; depending on the species, the young larvae are detached. From this point on, they are on their own and must fee, grow, swim and survive. After a series of transformations, the larvae becomes a miniature adult.



• Crustaceans cannot grow as many other animals do because of their outer skeleton. Instead, they periodically shed the outer skeleton, grow rapidly for a short time, and then, form another hard skeleton. While this process is taking place, they hid in an isolated place.



• Another remarkable ability, the crustacean has is, to be able to break off or to drop their appendages. This is called autotomy. They have special breaking-off points near the body. If caught, they can quickly break-off this appendages to get away. A new appendage is more easily grown. Following table illustrates the nutritional value of edible crustaceans. Nutritional value per 100 g Energy

410 kJ (98 kcal)

Carbohydrates

0g

Sugars

0g

Dietary fiber

0g

Fat

0.59 g

saturated

0.107 g

monounsaturated

0.091 g

polyunsaturated

0.16 g

Protein

20.5 g

Thiamine (vit. B1)

0 mg (0%)

Riboflavin (vit. B2)

4 mg (333%)

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Niacin (vit. B3)

4 mg (27%)

Vitamin B6

4 mg (308%)

Pantothenic acid (B5)

2 mg (40%)

Folate (vit. B9)

2 mg (1%)

Vitamin C

0 mg (0%)

Calcium

6 mg (1%)

Iron

2 mg (15%)

Magnesium

8 mg (2%)

Phosphorus

15 mg (2%)

Potassium

0 mg (0%)

Zinc

15 mg (158%)

12.5  NEUROENDOCRINE PRINCIPLES INVOLVED IN THE REGULATION OF GROWTH, REPRODUCTION AND METABOLISM OF PRAWNS AND CRABS (EDIBLE CRUSTACEANS) Programming of reproductive events of a species is being regulated by interactions between various exogenous and endogenous factors. In the tropical brachyurans, reproduction occurs round the year due to the relatively stable environmental situations. Estuaries are characterized by fluctuating conditions of salinity and temperature to the extent that both are considered dominant ecological factors which may act either singly, or in concert, to modify programming the reproduction and growth and distribution of estuarine organisms. Significantly, many estuarine invertebrates require specific temperature and salinity conditions at different developmental stages. Many species use photoperiod as an external cue to initiate a series of physiological events like molting, reproduction and hatching. Stephens (1955) established photoperiod as a determinant factor in the induction of moulting in the crayfish Procambarus sp. Photoperiod and temperature influence ovarian development and spawning in the American lobster H.americanus. The neurosecretory system of the eyestalk consists of a group of peptidergic neurons clustered in the medulla terminalis of X-organ (XO) and their bulbous axonic terminals that constitute the sinus gland (SG), which is a neurohaemal organ that releases a number of peptide hormones. XO-SG complex is the neurendocrine system located within the eyestalk of crustaceans that produces hormonal factors that control physiological processes of gonads. Neurological hormones controlling moult and reproduction come under two categories, viz., inhibitory and stimulatory principles. Several neuropeptides, forming the so-called crustacean hyperglycemic hormone family, have been isolated from the Xorgan–SG complex: the moult-inhibiting hormone (MIH) involved in moulting, the vitellogenesis (or gonad)-inhibiting hormone (VIH/GIH) involved in reproduction, the mandibular organ-inhibiting hormone (MOIH) involved in reproduction and development, and the crustacean hyperglycemic

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hormone (CHH) involved in the regulation of hemolymph glucose level, are examples. Regulation of growth: The physiological process of growth is hormonally co-ordinated and regulated. The rigid exoskeleton in crustaceans and insects require periodic molts to accomplish growth and metamorphosis and this is harmoniously regulated by a cascade of neuropeptides. Stimulatory principles: In crustaceans, the ecdysial glands known as the Y-organs are responsible for ecdysteroid synthesis. Y-organ is located in the anterior branchial chamber in all crustaceans; it appears as a compact mass in crabs and a less compact mass in crayfishes and lobsters. Moulting is stimulated directly by the presence of moulting hormone, ecdysteroids (a group of polyhydroxylated ketosteroids) circulating in the hemolymph. The synthesized ecdysone is released into the hemolymph and converted into active 20-hydroxyecdysone (20-HE) by peripheral tissues and promotes the physiological changes associated with moulting. In Cancer antennarius, additional ecdysteroids are released by the Y-organs, 3-dehydroxyecdysone and 25-deoxyecdysone, a precursor for active ponasterone A. Ponasterone A is the main serum ecdysone present during premoult in Carcinus maenas and the land crab, Gecarcinus lateralis. In the fiddler crab, Uca pugilator and other crustaceans, several ecdysteroids (e.g. 25-deoxyecdysone, ponasterone A, and ecdysone, 20E) circulate in the hemolymph. Changes in these ecdysteroid titers and ratios during the molt cycle are temporally correlated with major physiological events involved in moulting and regeneration of lost limbs. The haemolymph ecdysteroid titres are known to vary with stages in the moulting cycle, with high levels during premoult and low levels during postmoult and intermoult. However, no significant change in haemolymph ecdysteroid titres was observed in relation to oocyte development in Carcinus maenas. The Y-organ in the cephalothorax of crustaceans and the integument of ticks has been reported to be the sources of secreted ecdysteroids in adults, as in earlier stages, but the tissue source is not known for adults in many arthropod groups. In arthropods, the ecdysteroid hormones regulate growth, differentiation, and reproduction by influencing gene expression. Ecdysteroids enhance the formation of ecdysteroid receptor–ultraspiracle protein (EcR–USP) heterodimers which regulate gene transcription. The close relationships between Y-organ activities and hemolymph ecdysteroid levels suggest that the Y-organ activity directly affects the hemolymph ecdysteroid levels in M. rosenbergii.

12.5.1  Inhibitory Principles Moult-inhibiting hormone (MIH): The neurosecretory system of the eyestalk consists of a group of peptidergic neurons clustered in the medulla terminalis of X-organ and their bulbous axonic terminals that constitute the sinus gland, which is a neurohaemal organ that releases a number of peptide hormones including the complex of the eyestalks. Crustacean moult-inhibiting hormone (MIH), a polypeptide secreted by the X-organ sinus gland complex of the

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eyestalks, regulates molting by inhibiting ecdysteroid synthesis of Y-organs. The peptidic nature of MIH has been established for many brachyuran and macruran species. Aguilar and his group (1996) isolated the MIH from the SG of the Mexican crayfish, Procambarus bouvieri and compared its sequences with other four known peptides from Homarus americanus, Carcinus maenas, Callinectes sapidus and Penaeus vannamei and found that the length varied between 72–78 residues and molecular mass between 8 and 9 KDa. All had six cysteines that form three disulfide bonds. Yang and his group (1996) isolated and sequenced a peptide with MIH activity from the sinus gland of the kuruma prawn, P. japonicus. In the crabs Carcinus maenas, Cancer pagurus, and Gecarcinus lateralis, the MIH gene encodes a 113-amino acid prohormone (proMIH) composed of a 35-residue signal peptide and a 78-amino acid mature peptide. The mature peptide had the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine, and one aspargine in identical positions in the highly conserved sequence, characteristic of other crustacean MIHs. Scientists revealed that the MIH genes of Cancer feriatus and C. pagurus consist of three exons and two introns, and span approximately 4.3 kb. The hormone-binding domain of the MIH receptor is likely to be highly conserved, and therefore, the MIH from one crab species is capable of inhibiting ecdysteroid production by Y-organs in other. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using yeast, Pichia pastoris expression system. Regulation of reproduction: Crustacean reproduction is under the control of stimulatory and inhibitory principles. High titres of MIH and GSH and low titres of GIH and MH always result in reproduction. While GSH acts as a direct acceleratory principle, MIH acts indirectly as gonad acceleratory principle by inhibiting the production of MH by Yorgan. Reproduction is under endogenous regulation by nervous, endocrine and/or neurendocrine systems. Regulation of crustacean reproduction is now thought to be based on a multi-hormonal system in which factors of different chemical nature and origin may play a direct role in gonad development, sexual differentiation and mating behavior. Maturation of the gonads in crustaceans is regulated by two antagonistically acting neurological hormones; the gonad inhibiting hormone (GIH) from the sinus gland and the gonad stimulating hormone (GSH) found in the brain and thoracic ganglia. Decapod females depend mainly on neurological endocrine centers outside the eyestalk for the gonad stimulatory principles and that from the eyestalk for gonad inhibitory principles. Crustacean mandibular organs are suggested to play a role in regulating reproduction in crustacean females. Mandibular organ that control growth and reproduction is known to be controlled by mandibular organ inhibiting hormone (MOIH), originating from the eyestalk. Recent developments in crustacean endocrinology have given an insight into the importance of ecdysteroids in female reproduction.

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Eyestalk neuropeptides such as MIH is found to have an inhibitory control on ecdysteroid synthesis by the Y-organ. Mandibular organ is also suggested to have a stimulatory role for Y organ.

12.5.2  Stimulatory principles Gonad stimulating hormone (GSH): The hormonal factors that have a stimulatory effect on reproduction in crustaceans include, the vitellogenesis stimulating ovarian hormone, methyl farnesoate, a JH analogue, and the ecdysteroids. The existence of a female hormone of ovarian origin in the Decapoda, as in the amphipods and isopods, has been reported to stimulate development of secondary sexual characters. However, its role in stimulating oogenesis has not been proved. Vitellogenesis Stimulating Ovarian Hormone (VSOH): A possible existence of an ovarian hormone or VSOH from the ovary and/or the follicle cells surrounding the ovary was strongly considered by Charniaux-Cotton (1960). However, the investigators who proposed the existence of VSOH were not able to substantiate it. It has been suggested that estradiol-17b secreted from ovarian follicle cells induces vitellogenin synthesis in the ovary as VSOH in the Penaeid shrimp, P. vannamei. Mandibular organ: In decapods, a glandular tissue is located at the base of the tendon associated with the posterior abductor muscle of the mandibles, the mandibular organ (MO). Mandibular organ produces methyl farnesoate (MF), a juvenile hormone-related compound which is involved in crustacean reproduction and development. Ultra-structural studies reveal the occurrence of cells with extensive SER and abundant mitochondria. Initial studies by several authors suggested that the mandibular organ might be involved in regulating reproduction, and moulting. Current evidence indicates that the MO is negatively regulated by peptides present in the eyestalk, mandibular organ inhibiting factor (MOIH). Methyl farnesoate was identified as a secretory product of MO. The mandibular organ when implanted into immature females, stimulated ovarian growth accompanied by morphological and ultra-structural signs of active vitellogenesis.

12.5.3  Inhibitory principles Gonad inhibiting hormone (GIH): GIH is secreted by the neurosecretory cells of the XO in the eyestalks and is transported intra-axonally through the XO-SG tract to the sinus gland (eyestalk). GIH is suggested to suppress the ovarian development by blocking vitellogenin synthesis by hepatopancreas and/or vitellogenin uptake by oocytes. GIH repress vitellogenin synthesis in adipose tissue or vitellogenin uptake by oocytes. XO-SG extract when injected to ablated animals suppressed ovarian development. It has also been suggested that GIH inhibits only the secondary phase of the maturation of

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oocytes and probably, has no effect on the primary growth. The primary phase has been suggested to be under the control of MH. Mandibular organ inhibiting hormone (MOIH): The methyl farnesoate (MF) secretion, by mandibular organ is suggested to be negatively controlled by the mandibular organ inhibiting hormone produced by the eyestalk. MOIH has been isolated and found to consist of 72–76 peptides and a MW of 8.0–8.5 Kd. MOIH peptides resemble that of the GIH. Removal of the eyestalk removes MOIH inhibition and results in the hypertrophy of MO with increased secretion of MF. Injection of sinus gland extracts decreases secretion of methyl farnesoate by the mandibular organ. The amino acid sequences of these MOIH peptides are similar to peptides in the crustacean hyperglycemic hormone (CHH) family of neuropeptides. In addition, there appears a compound in the eyestalk that lowers hemolymph levels of methyl farnesoate in vivo, but does not directly affect the mandibular organ in vitro. The inhibition of methyl farnesoate synthesis by eyestalk peptides involves the inhibition of farnesoic acid O-methyl transferase, the last enzyme in the methyl farnesoate biosynthetic pathway. The activity of this enzyme is affected by cyclic nucleotides, suggesting that these compounds may be involved in the signal transduction pathway mediating the effects of MOIH. G-protein in the crustacean mandibular organ participate in the signal transduction from the eyestalk neuropeptides (MOIH) to the enzyme farnesoic acid O-methyl transferase responsible for methyl farnesoate synthesis. Farnesoic acid O-methyl transferase (FAMeT), directly or indirectly (through MF), modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurological secretory cells of the X-organ-sinus gland . Interaction between growth and reproduction: Growth and reproduction are two related processes among crustaceans. These processes are regulated by two related endocrine axes that are governed by inhibitory neurological hormones secreted from the X-organ-sinus gland complex (XO-SG) in the eyestalk. Hormones involved in the two processes are related. MH is needed for the prepubertal development. In adults, alternatively high levels of MIH and GSH and low levels of MH and GIH bring about seasonal changes in the moulting and reproduction. In brachyuran crabs and lobsters, these are two antagonistc events wherein the animal enters into either moult or reproduction at a particular period. Surgical extirpation of the eyestalks can induce both moulting and vitellogenesis. The reproductive programming of a species or population is the outcome of the interaction between various exogenous and endogenous factors. In the tropical brachyurans, reproduction occurs round the year due to the relatively stable environmental situations. Much research has been conducted on the normal growth and reproductive cycles of several crustaceans, simply because of their high reproductive potential which enable successful culture for food purposes and the high nutritional

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values. The general pattern of growth and reproduction of a population varies even among the same species. Identification of gonad stimulating substances is greatly used for domestication of commercially important species that do not reproduce easily under captivity. Precocious development of gonad by way of unilateral and bilateral eyestalk extirpation in the prawn and shrimp species has been employed in aquaculture. The larvae produced by eyestalk ablation have been found to be of low quality than those of the intact ones and this warrants more research in this field. The methyl farnesoate produced by mandibular organ stimulates and enhances reproduction. Detailed study of mandibular organ and its role in stimulation of ovarian maturation in more species may provide better understanding. Environmental variables play an important role in growth and reproductive behavior. In estuarine species, the behavior patterns are affected by hormones, temperature, tidal conditions and photoperiod.

CHAPTER

13

Nutrient Film Culture Techniques

Nutrient Film culture Technique or NFT is a hydroponic technique wherein a very shallow stream of water containing all the dissolved nutrients required for plant growth is recirculated past the bare roots of plants in a watertight gully, also known as channels. In an ideal system, the depth of the recirculating stream should be very shallow, little more than a film of water, hence the name ‘nutrient film’.

• This ensures that the thick root mat, which develops in the bottom of the channel, has an upper surface, which, although moist, is in the air. Subsequent to this, an abundant supply of oxygen is provided to the roots of the plants. A properly designed NFT system is based on using the right channel slope, the right flow rate, and the right channel length.



• The main advantage of the NFT system over other forms of hydroponics is that the plant roots are exposed to adequate supplies of water, oxygen and nutrients. In all other forms of production, there is a conflict between the supply of these requirements, since excessive or deficient amounts of one results in an imbalance of one or both of the others. NFT, because of its design, provides a system wherein all three requirements for healthy plant growth can be met at the same time, provided that the simple concept of NFT is always remembered and practiced.



• The result of these advantages is that higher yields of high-quality produce are obtained over an extended period of cropping. A downside of NFT is that it has very little buffering against interruptions in the flow, e.g., power outages; but, overall, it is one of the most productive techniques.

Though it is a simple hydroponic system, for NFT to function efficiently and to get better results, one should need to carefully monitor the following:

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• Type of channels: There is a variety of channel available on the market, and to select an appropriate one you need to consider factors such as cost, availability on the market and requirements of your hydroponic plants. They are available with pre-punched holes and removable lids for placing plant seedlings and cleaning the system and the length should be around 10–15 m. Ensure, that it should be flat and not curved to maintain a considerable depth of liquid at the centre of a channel. It is also important to have correct channel slope to get constant flow of nutrients.



• Aeration: In NFT, since plant roots are submerged in water, it leads to oxygen deficiency, at times. To eliminate this problem, you need to install small compact air pumps or air stones to supply oxygen.



• Pathogens and Pest infestation: Moulds and pathogens are prone to grow in moist environment, so a regular check on the condition of your plants will help you to spot the early signs of pests and diseases. To prevent your plants from Pythium infection, it is better to add H2O2 to your solution.



• Nutrient check: One needs to make sure that one’s plants receive balanced nutrients comprising all the essential micro- and macronutrients that are available during the different growth phases of one’s hydroponic plants. Inadequate nutrient supply can affect the overall growth of plants. If your crops grow faster than normal, it can be a sign of nitrogen depletion in the nutrient solution.



• Pump maintenance: One can come across pump failures at some point of time as the submerged water pumps are continuously working. However, these pumps are small and inexpensive and can be easily replaced. You also need to clean your system on a regular basis to ensure smooth flow of nutrients, without which, the crops might suffer.

13.1  PLANT DISEASES Plant disease is the abnormal growth and development of a plant. Growth and development of the plant does not live up to the normal expectations. A diseased plant is incapable of carrying out its normal physiological functions to the best of its genetic potential. Many different living and non-living entities can have a negative effect on plants. A. Infectious Diseases (caused by biotic organisms): (a) Fungi

(b) Prokaryotes:

( ) Bacteria (ii) Mycoplasmas

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(c) Viruses and viroids (d) Nematodes (e) Parasitic Higher Plants B. Non-Infectious (caused by abiotic factors) (a) Temperature extremes (b) Moisture extremes (c) Light extremes (d) Nutrient extremes (e) Soil acidity or alkalinity (salt problems) (f) Pesticide toxicity (g) Air conditions: pollution, strong winds, etc. (h) Improper cultural practices 2. A disease episode requires the interaction of three components, which we call “The Disease Triangle”. A. The host must be susceptible to the disease, at the proper age and physiological state, for infection and development of disease to occur. Healthy, strong-growing, non-­stressed plants are less susceptible to disease than plants, under stress. B. The pathogen must be virulent (able to cause disease), not in a state of dormancy and must be present at a certain minimum population level. C. The environment must be conducive (favorable) for the development of disease: temperature, moisture, nutrients, wind, etc. must all favor the pathogen. D. The degree to which these three components interact, relates to the severity of the disease episode (e.g., if the host is highly susceptible, the pathogen, highly virulent and the environment, highly conducive, then, the disease will be very severe). E. Successful disease-control depends on the integrated use of available control methods.

Symptoms and Signs

1. Symptoms are the response of the plant to attack by a disease causing agent. A. Examples: leaf spots, wilting, stunting, chlorosis, necrosis, etc. 2. Signs are the visual presence of some structure formed by the pathogen on the host. A. Examples: mycelium, spores, fruiting bodies, bacterial ooze, etc.

Organisms Associated with Diseased Tissue

1. Primary organism: The organism is directly responsible for the disease.

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2. Secondary organism: The organism(s) is(are) taking advantage of weakened tissue. 3. Disease complexes and organism succession. A. Disease complex refers to the situation where the disease is caused by more than one organism. Disease complexes are especially common in turf. B. Organism succession refers to the fact that plants are colonized over time by many different organisms. For example, when plants are healthy, they are colonized by non­-pathogenic symbionts. When the plants become diseased, they are first colonized by primary pathogens, then by secondary organisms, and eventually, by other saprophytes. Saprophytic organisms are in association with healthy plants. The primary disease-causing agent is only operating by itself for a short period of time. Secondary organisms may be weakpathogens or pathogens. Weak pathogens are organisms that are not aggressive and (typically) do not cause disease by themselves. C. Disease complexes add to difficulty in disease management, as identification and control of one organism, may accelerate activity of another organism in the complex. D. Organism succession makes primary pathogen identification difficult. The diseased specimen must be examined relatively quickly after disease symptoms begin; otherwise, secondary pathogens or saprophytes are all that can be found. Accurate diagnosis of the causal agent is required for effective use of chemical control measures. Use of an inappropriate chemical will not only be ineffective against the disease agent, but can also lead to additional disease problems by killing beneficial microorganisms in the environment.

13.2  PHYTODIAGNOSTICS BASED ON IMMUNOLOGICAL AND MOLECULAR TECHNIQUES Accurate detection and identification of plant pathogens are fundamental to plant pathogen diagnostics, and thus, plant disease management. The lack of rapid, accurate and reliable means by which plant pathogens can be detected and identified has been one of the main limitations in plant disease management and has prompted the search for alternative diagnostic techniques. The advent of enzyme-linked Immunosorbent assay (ELISA) and polymerase chain reaction (PCR) has caused a shift towards the use of molecular approaches in modern plant pathogen diagnostics. Nowadays, many techniques have been developed for the detection and identification of plant pathogens, each requiring its own protocol, equipment, and expertise. In addition, some of these techniques permit reliable quantification of the target pathogen as well, and supply the information required to estimate potential risks regarding disease development, spread of the inoculum, and economic

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losses. The major challenge at the moment is the development of multiplex assays that allow accurate detection and quantification of multiple pathogens in a single assay. In this chapter, we discuss recent advances in molecular plant pathogen diagnostics that are likely to impact future plant disease-controlling and, preventing strategies. Immunological or serological techniques: A first development towards techniques for molecular pathogen detection was the advent of immunological/ serological or antibody-based detection methods almost 30 years ago. These techniques were originally developed to detect viruses, as those cannot be cultured in vitro. • Immunological or Serological techniques are based on the binding between diagnostic antibodies and specific antigenic determinants of the target pathogen.

• Several serological plant pathogen detection methods have been described, of which, the enzyme-linked immunosorbent assay (ELISA) is, by far, the most common technique.



• Although, different types of ELISA have been developed, all involve an enzyme-mediated color change reaction to detect and often, also quantify antibody binding as a measure for pathogen presence.



• Since its introduction in the late 1970s, ELISA assays have been routinely used for virus and bacteria detection because of their highthroughput capacity, the rapid, relatively cheap and simple nature, and the possibility to quantify the target pathogen.



• A major limitation for the development of serological methods is the labor-intensive procedure to obtain reliable assays, often due to the difficulty to generate selective antibodies.



• Although polyclonal antibodies, which recognize multiple epitopes of the pathogen, have been used successfully for detecting many viruses, they do not always have the desired degree of specificity and, importantly, the specificity may vary with each newly-produced batch.



• The accuracy of detection is often improved by using either monoclonal or recombinant antibodies. Both of these allow the selection of specific target epitopes to avoid “false positives”.



• However, developing antibodies with the required degree of specificity is difficult for complex organisms such as bacteria and fungi. In those cases, it is often hard to find reliable species-specific epitopes that are ubiquitously shared within a species but not with other species.

Therefore, most antibody-based assays currently available are for the detection of relatively unsophisticated organisms such as plant viruses, while those available for the detection of fungi and bacteria are less common.

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13.3  MOLECULAR OR NUCLEIC ACID-BASED TECHNIQUES Before the possibility to amplify nucleic acid sequences existed, the sensitivity of detection based on those sequences totally relied on the method to translate their presence into a detectable signal. Since the introduction of amplification methods for nucleic acids, in particular—the polymerase chain reaction (PCR), nucleic acid-based methods are increasingly developed for the detection and identification of plant pathogens. This trend is enhanced by the growing availability of pathogen sequence data in public databases like GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) and COGEME (http://www.cogeme. man.ac.uk/). A crucial step in the development of nucleic acid-based diagnostic assays is the selection of sequences that can be employed for pathogen identification. In general, there are two main approaches that can be used to select target sequences. The first, and most widespread strategy, involves the use of ubiquitously conserved genes. The second strategy involves the screening of random parts of the genome in order to find sequences, harboring the desired selectivity. Currently, the nuclear ribosomal DNA (rDNA) operon is the most commonly used target for bacteria as well as for fungi for a number of reasons. First, it has been found that this gene provides a powerful means for analyzing phylogenetic relationships over a wide range of taxonomic levels including, for example, genus, species, and even below. Apart from this potential, the large amount of ribosomal sequences in public databases allows to determine genomic regions that can be used to design selective primers or probes. This is facilitated even more by the structural nature of this type of gene since it contains alternating regions with high and low degrees of conservation. This allows to design primers on sequences that are conserved between species which span variable domains that can be used for species identification. In addition, the multiple copies of the gene present in each cell permit a very sensitive detection. Although rDNA is the main target of many nucleic acid-based analyses, other targets for detecting fungi include b-tubuline, actin, elongation factor 1 alpha, and mating-type genes. However, if these genes do not display the desired degree of selectivity, other regions of the genome need to be assessed. The screening of arbitrary regions in the genome to find sequences with the required selectivity can be achieved by several techniques, including RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) technology. Diagnostic markers identified with these approaches can be sequenced and are used to design specific SCAR (sequence characterized amplified region) primers. Nevertheless, as these sequences can be derived from anywhere in the genome, there often is few sequence data available for comparison to multiple other organisms. Therefore, extensive screening is required to validate the specificity of the marker.

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Two specific problems can hamper detection of plant pathogens based on nucleic acid-based techniques because they complicate the identification of reliable markers. • First, misclassification of strains is a regularly-occurring phenomenon in fungal taxonomy. Historically, taxonomists have grouped closelyrelated fungi in a single genus or species largely based on similarities of structural and morphological characteristics. However, especially large fungal genera and genera containing asexual fungal species are known to often contain unrelated species. As a result, taxonomic relationships are not always reflected by the evolutional relationships that are often revealed using nucleic acid-based techniques. Therefore, finding selective sequences shared by all members of a species is complicated for certain species.

• A second problem for molecular detection of certain plant pathogens is the existence of fungal species that harbor pathogenic as well as nonpathogenic or, even beneficial strains. This is a known phenomenon for complex species such as Fusarium oxysporum and Rhizoctonia solani. In those cases, target sequences should preferably be directly associated with virulence traits which severely limits the number of sequences.

Nucleic acid-based techniques can be divided into DNA- and RNA-based technologies DNA-based techniques: DNA is a highly attractive target for the detection of plant pathogens in biological samples because it is easier to handle and more resistant to degradation than RNA. With improved extraction methods (34) and commercially available extraction kits, highly purified DNA can rather easily be obtained from complex environmental samples.

13.3.1 Polymerase Chain Reaction (PCR) Using PCR, millions of copies of specific DNA sequences may be rapidly synthesized in a thermocyclic process that consists of repetitive cycles of DNA denaturation, primer annealing, and extension using a thermostable DNA polymerase. If a DNA sequence unique to a particular organism is determined, specific PCR primers or probes can be designed that enable determination of the presence or absence of that sequence, and thus, of the specific organism. The presence of amplified DNA is traditionally detected by gel electrophoresis, but alternative detection formats including, colorimetric and fluorimetric assays, do exist. PCR-based detection methods are very sensitive and can detect minute quantities of pathogen DNA—even the amount derived from a single fungal spore. To improve specificity, but sometimes also sensitivity, PCR products may also be detected using a probe. Other approaches to increase sensitivity and specificity include the use of immunocapture PCR (IC-PCR) or nested PCR. IC-PCR utilizes antibodies to isolate the pathogen

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from a sample prior to PCR amplification, and has mainly been used to detect plant pathogenic viruses. Nested PCR involves two consecutive PCR reactions, the second one using primers that share a sequence within the target DNA fragment that is amplified in the first reaction. As a result, a specific reaction products that are generated in the first PCR reaction should not be amplified in the second reaction. Many reports describe specific applications of PCR technology in plant pathology. In addition, increasingly, companies providing diagnostic services are using PCR to routinely detect and identify plant pathogens. Quantification of the amount of pathogen DNA, supplying the information required for disease management decisions and for monitoring the effects of these decisions has also been pursued using PCR-based methods. Although, it is relatively easy to quantify the amount of amplicon generated, it is more difficult to relate this quantity to the initial amount of target DNA present in a sample. This is caused by the typical non-linear kinetics of template amplification. Nevertheless, in theory, the exponential nature of PCR allows the initial amount of DNA to be calculated from the amount of product at any time point in the reaction. In practice, however, as the reaction proceeds, reagents become limiting and a plateau level is reached, where the amount of product is no longer proportional to the original amount of template. Target DNA can be quantified using competitive PCR, which is based on the co-amplification of target DNA and a competitor DNA, both with the same primer pair. The amount of target DNA is subsequently determined on agarose gel by comparing the relative amounts of target and competitor PCR product. This method has been used to successfully quantify, for instance, Verticillium wilt pathogens.

13.3.2 Real-time PCR Especially with respect to quantification purposes, real-time PCR is a powerful development. This technology differs from conventional PCR by monitoring products on-line while they accumulate at each reaction cycle in a closed tube format, without the need of post-reaction processing such as gel electrophoresis. As a consequence, real-time PCR is generally faster than conventional PCR, enabling high throughput analyses. In addition, the risk of post-PCR carry-over contamination of amplicons is eliminated. Real-time PCR allows accurate template quantification during the exponential phase of the reaction, before reaction components become limiting. The initial amount of target DNA can be related to a threshold cycle, defined as the cycle number at which fluorescence increases above the background level. Target DNA is quantified using a calibration curve that relates threshold cycles to a specific amount of template DNA. Typically, DNA amplification is monitored each cycle based on the emission of fluorescence. Amplicons can be detected using several chemistries, which can be divided into amplicon non-specific or amplicon specific methods, using DNA-binding

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Main chemistries for amplicon detection in real-time PCR application (A) As a DNA-intercalating dye such as SYBR Green® (S) binds to dsDNA, fluorescence is recorded. (B) Taqman® probes, (C) Molecular Beacons® as well as (D) Scorpion® primers use a strategy to extinguish fluorescence at certain conditions using a reporter fluorophore (R) and a fluorogenic quencher (Q). Upon physical separation of both molecules fluorescence is emitted. (E) The use of FRET probes involves the hybridization of two labeled oligonucleotides in close proximity. When both probes bind to the target fragment, energy is transferred from the donor (d) to the acceptor (a) molecule resulting in fluorescence.

dyes and sequence-specific probes, respectively. The use of DNA-intercalating dyes such as SYBR Green® is a more straightforward and less expensive approach compared to using probes, but it is also less specific since the dye binds to all double-stranded DNA (dsDNA) present in the sample. In addition, the interpretation of results can be disturbed by formation of primer-dimers or a specific reaction products. It is therefore, crucial to use highly specific primers and to determine optimal reaction conditions. In addition, melt curve analysis, at the end of the PCR reaction, allows evaluating the accuracy of the amplification reaction. In contrast to amplicon non-specific chemistries, probe-based assays often offer the advantages of increased specificity, certainly in combination with specific primers, and reducing signals due to mispriming or primer-dimer formation. Most applications, to date, have used TaqMan® probes. These probes are single-stranded, short oligonucleotides which are labeled with a fluorophore and a fluorogenic quencher. Because of the close proximity of both groups, the fluorescent signal is quenched. During the annealing phase of each PCR cycle, the probe hybridizes to a specific region within the target- amplified fragment. The probe is degraded by 5’ exonuclease activity when the DNA polymerase extends the primer. Consequently, the fluorophore and the quencher are released independently, resulting in a fluorescent signal. Variants of this quenching chemistry include, hairpin-shaped Molecular Beacons® and Scorpion® primers. Whereas, the loop portion of these molecules contains the probe sequence, the stem, which is formed by complementary sequences added to both ends of the probe, holds a fluorophore and a quencher in close proximity. In addition, Scorpion primers couple the stem-loop-based probe to a PCR primer. Specific binding of the probe to its target opens the structure, producing a fluorescent

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signal. A completely different detection chemistry comprises the use of fluorescent resonance energy transfer (FRET) probes. With this technology, two oligonucleotide probes are designed such that they hybridize in very close proximity to the amplified fragment. Whereas one of the probes contains a donor fluorophore at its 3’ end, the other probe is labeled at its 5’ end with an acceptor fluorophore. When both probes properly hybridize to the target fragment, the energy excited by the donor is transferred to the acceptor resulting in a fluorescent signal. Closely related microbial species often, only differ in a single or a few bases of ubiquitously conserved genes, for instance, the rDNA. The high degree of specificity of real-time PCR technology allows, independent of the detection chemistry, the detection of single-nucleotide polymorphisms (SNPs), meaning that specificity is determined by a single base pair. Therefore, this technology offers many opportunities in plant pathogen diagnostics. In recent years, real-time PCR assays have been developed for accurate detection and/or quantification of specific plant pathogens as well as for monitoring pathogen infections. Although, not yet used routinely in phytodiagnostics, real-time PCR has much potential for future applications. Ligase Chain Reaction (LCR): The ligase chain reaction (LCR) uses two complementary pairs of oligonucleotides that hybridize in close proximity on the target fragment. Only when the oligonucleotides correctly hybridize to the target sequence, the remaining nick between the oligonucleotides is ligated by a DNA ligase and, a fragment equating to the total sequence of both oligonucleotides, is generated. Similar as in a PCR reaction, the products of one reaction serve as templates for subsequent cycles, resulting in an

General principle of the Ligase Chain Reaction (LCR) Two complementary pairs of adjacent oligonucleotides (pla and plb, p2a, and p2v) bind to the target sequence. Only if the oligonucleotides bind in close proximity, DNA ligase seals the nicks and the cycle can be repeated.

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exponential amplification of the desired fragment. To further enhance sensitivity and, sometimes, also specificity, LCR can also be used following a PCR pre-amplification. Detection of LCR products can be performed by polyacrylamide gel electrophoresis. With this technology, SNPs can easily be differentiated. Although LRC is regularly applied in human disease detection, it has rarely been reported for detection of plant pathogens.

13.3.3 Rolling Circle Amplification (RCA) Originally, padlock probes were developed as a new approach for molecular analysis of complex DNA samples, including analysis of alleles and point mutations in the human genome. A padlock probe consists of a singlestranded linear oligonucleotide of about 70–100 nt in length with a targetcomplementary region at both ends and a linker segment, in between. The 5’ and 3’ end regions are designed to hybridize next to each other on a target strand. When properly hybridized to the target sequence, the molecule can be circularized upon ligation. Because of the need for precise base pairing at the junction where ligation should take place and the simultaneous hybridization of two different fragments, padlock probes ensure high specificity. For sensitive pathogen detection, however, signal amplification is a prerequisite. One approach for the amplification of padlock probes is a PCR reaction using primers that hybridize to sequences within the spacer region of the probe. Another method to amplify padlock probes is rolling circle amplification (RCA), analogous to replication mechanisms of several viruses with circular genomes. Two types of RCA have been described: linear and hyper-ranched RCA. In the first procedure, a primer hybridized at some point on the circular DNA is extended continuously using a DNA polymerase that lacks exonuclease activity. As a result, a long linear fragment composed of many tandem repeats of the complement to the circularized molecule is generated. In addition, hyperbranched (or cascade) RCA uses a second primer that binds to each generated RCA repeat. During elongation, the Exonuclease-deficient DNA polymerase displaces the polymerized strand in front of it. Next, the displaced strands, which are tandem, repeat with identical sequences to the original padlock probe, serve again as template for the first primer, resulting in a cascade of DNA amplification. As for conventional PCR, detection of amplified products can be achieved using gel electrophoresis or labeled probes enabling real-time monitoring of the amplification process. However, although RCA is considered to be one of the most sensitive amplification methods, the procedure is fairly complicated and relatively expensive. Therefore, it is important to realize what level of sensitivity is required for a method to be used for plant pathogen diagnostics. The most sensitive technique will probably not be required when assessing whether measures have to be taken in a certain crop to prevent yield losses. Often, such a decision requires a threshold level to be crossed which can be detected by many less sensitive techniques. In contrast, sensitivity is very important when it comes to zero tolerance of quarantine diseases.

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General principle of hyperbranched rolling Circle Amplification (RCA) The 5′ and 3′ ends of a linear padlock probe are designed to hybridize next to each other on a target strand. When properly hybridized, the molecule is circularized by ligation. Synthesis of the complementary strand of the circularized padlock probe is initiated by primer pl. As a strand of linear tandem repeats is generated, a second primer (p2) hybridizes to each newly generated repeat. During elongation, the exomclease-deficient DNA polymerase displaces the polymerized strand in front of it which, in turn, serves as template for the first primer.

All DNA-based methods have in common that they might detect DNA from dead or non-active organisms as well. Therefore, detection of non-viable propagules cannot be ruled out. However, DNA from dead cells in soils should be degraded fairly rapid due to the high microbial activity, suggesting that interference by DNA from dead cells might be negligible. Nevertheless, the rate of DNA degradation depends on soil type and moisture content. As DNA degradation occurs slower in desiccated soils, accurate diagnosis of samples from dry fields may be biased by detection of dead organisms. However, since long-lasting soil desiccation generally does not occur in horticultural or agricultural practice, this should not be a major concern. To exclude detection of dead organisms, a culturing step on or in a suitable medium, or even in plants, prior to PCR amplification, could be included. This technique is referred to as BIO-PCR. Because, only active propagules will be able to grow, this technique enables selection of viable organisms. In addition, PCR sensitivity is increased by the culturing step. However, disadvantages are its labor-intensive and time-consuming nature, and the inability to detect nonculturable organisms. As an alternative, attempts are made to use DNA-binding dyes such as ethidium monoazide (EMA) to distinguish viable from non-viable organisms. Since the membranes of dead cells quickly disintegrate, EMA is able to selectively enter dead cells where it covalently binds to dsDNA upon lightexposure. EMA-bound DNA is blocked for PCR amplification, thus enabling the selective amplification of targets from living organisms. Another alternative

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to prevent detection of dead organisms is the use of RNA as target molecule. Since RNA is less stable than DNA, RNA will be degraded much faster in dead organisms. In addition, the stability of DNA can be the cause of persisting contaminations in diagnosis laboratories where large numbers of samples, which often contain the same pathogen, are processed. In addition, messenger RNA (mRNA) is only produced in metabolically-active cells, making mRNA attractive to selectively detect living microorganisms. However, extraction of RNA from environmental samples should be a careful procedure. RNA-based techniques: Whereas DNA-based detection techniques are increasingly being used to detect and identify pathogenic fungi, bacteria as well as nematodes, RNA-based techniques are mainly used to detect plant viruses since most of them have RNA genomes. However, since mRNA may more accurately reflect metabolicallyactive pathogen material, these techniques can also be used to selectively detect viable pathogen propagules. Reverse Transcriptase PCR (RT-PCR): Since PCR can only amplify doublestranded templates such as DNA, RNA should be converted to DNA (called complementary DNA or cDNA) prior to use in a PCR-based assay. Typically, such reverse transcriptase PCR (RT-PCR) consists of an annealing step for one primer and an extension step to synthesize the complementary or second strand, followed by a (real-time) PCR reaction. In plant pathology, RT-PCR is a common strategy to detect plant viruses . Nucleic Acid Sequence-Based Amplification (NASBA), Transcription Mediated Amplification (TMA), or Self-Sustained Sequence Replication (3SR): Nucleic acid sequence-based amplification (NASBA), also known as transcription mediated amplification (TMA) or self-sustained sequence replication (3SR), has been used for the direct amplification of RNA. In contrast to conventional PCR, amplification is carried out in an isothermal process (avoiding the need for a thermocycler) using three different enzymes, including a reverse transcriptase, RNase H, and T7 RNA polymerase. Initially, a primer containing an RNA polymerase promoter sequence at its 5’ end and a targetspecific sequence at its 3’ end is extended by reverse transcription to produce a cDNA strand. The resulting hybrid is a substrate for RNase H, which degrades the original RNA strand. Subsequently, a second DNA strand is produced from a primer designed to bind to the 3’ end of the cDNA, resulting in a dsDNA molecule that contains the sequence information of the original RNA and the promoter sequence of the T7 RNA polymerase. In a next step, T7 RNA polymerase initiates DNA transcription leading to the production of a large number of antisense RNA molecules. Each antisense RNA molecule is used to generate new dsDNA molecules based on the same principle, and initiates a new round of replication. The amplification products can be visualized using a specific labeled probe which hybridizes to the RNA amplicons. In addition, amplicons can be

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Environmental Biotechnology

monitored in real-time using a specific detection probe such as a Molecular Beacon®. This procedure is referred to as AmpliDet RNA and combines the advantages of both NASBA and real-time PCR.

General principle of Nucleic Acid Sequence-Based Amplification (NASBA) Upon binding of primer pl that is tailed with a T7 RNA polymerase promoter, reverse transcriptase (RT) generates a cDNA strand. The result hybrid is a substrate for , which degrades the original RNA strand. Subsequently, reverse transcriptase generates a complementary strand to the first cDNA strand using a second primer (p2), resulting in double-stranded DNA (dsDNAW) with a T7 RNA polymerase promoter. This is a template for T7 RNA polymerase (T7 pol) that transcribes a large number of antisense RNA molecules (asRNA) which, in turn, are converted into dsDNA for a next amplification cycle.

13.4 ANTAGONISTIC FUNGI Fungal antagonists are those fungus species, which act against the harmful microorganisms and their products. Trichoderma spp.: Trichoderma viride, T. harzianum and T. virens are most extensively used fungal antagonists. They are mass-produced using fermentation technology. They are used for the management of soil-borne pathogens by seed treatment and soil application. Aspergillus niger: Aspergillus niger, an ubiquitous fungus is present in all types of soil with no specific moisture and pH requirements. It is formulated as wettable powder. It controls a number of devastating soil-borne pathogens, e.g. Fusarium oxysporum, Macrophomina phaseolina, Pythium

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aphanidermatum, Rhizoctonia solani, and Sclerotinia sclerotiorum, belonging to different classes of fungi by a single application under different agro-climatic conditions in different crops Myrothecium verrucaria: It is a deuteromycete fungus, which produces cuticle degrading enzymes as well as mycolytic enzymes. It is formulated either in the form of conidia or unicellular yeast like cells. It is used to control root-infecting fungus, Sclerotium rolfsii on groundnut. Trichoderma spp. has been widely used as antagonistic fungal agents against several pests as well as plant growth enhancers. Faster metabolic rates, antimicrobial metabolites, and physiological conformation are key factors which chiefly contribute to antagonism of these fungi. Mycoparasitism, spatial and nutrient competition, antibiosis by enzymes and secondary metabolites, and induction of plant defence system are typical biocontrol actions of these fungi. On the other hand, Trichoderma spp. have also been used in a wide range of commercial enzyme productions, namely, cellulases, hemicellulases, proteases, and 1,3-glucanase. Information on the classification of the genus, Trichoderma, mechanisms of antagonism and role in plant growth promotion has been well documented. Trichoderma viride is an antagonistic fungus which prevents the crops from diseases such as Root rots, Wilts, brown rot, damping off, Charcoal rot and other soilborne diseases in crops. It is suitable for Sugarcane, Pulses, Oilseeds, Cotton, Vegetables, Banana, Coconut, Oil palm, Chilies, Lime, Coffee & Tea, Areca nut & Rubber, Flower crops and Spices.

Mode of disease control

• Trichoderma fungus is well known for disease and nematode control of crop plants.



• Trichoderma controls diseases by the production of several lytic enzymes and antibiotics-controlling disease-causing microbes.



• Trichoderma controls nematode infestation by feeding on infective nematodes.



• These fungi compete with other disease-causing microbes for nutrients and space.



• These fungi increase the rate of plant growth and development, by developing more robust roots. These deep roots cause crops, such as corn, and ornamental plants, to become more resistant to drought.



• Trichoderma also solubilizes phosphates and micronutrients.



• Trichoderma harzianum produces enzymes such as protease which controls Botrytis cinerea.



• Trichoderma are also helpful in solubilization and sequestration of inorganic nutrients.

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• Induces defense responses in crop plants (Induced resistance)



• Inactivation of the pathogen’s enzymes

13.5 ANTAGONISTIC BACTERIA Bacterial antagonists are those bacterial species, which act against the harmful microorganisms and their products. The best example for antagonistic bacteria is Pseudomonas fluorescens. Pseudomonas fluorescens: Antagonistic Pseudomonas fluorescens is a bacteria with high antibiosis potentia. It enters the plant vascular system, and reaches the various parts of the plant system and acts as systemic bio-control agent against various fungal and bacterial diseases such as Pythium spp., Phytophtora spp., Rhizoctonia solani, Fusarium spp., Botrytis cinerea, Sclerotium spp., Sclerotinia sp. and Ustilogo spp. It is suitable for plants like Tomatoes, Chilly, Cut flowers, Orchards, Vineyards Ornamentals, Potato, Cucumbers and Eggplant.

Mode of disease control Pseudomonas produces secondary metabolites, many extracellular hydrolytic enzymes, which suppress plant disease.

• Antibiotics such as pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol inhibit phytopathogen growth. Diseases from Rhizoctonia solani and Pythium ultimum that affect cotton plants are inhibited by this strain.



• Pseudomonas fluorescens produces hydrogen cyanide and the siderophores pyocheline and pyoverdine, which it uses to outcompete with many pathogenic bacteria and suppress pathogens in the rhizosphere.



• Pseudomonas fluorescens produce exopolysaccharides which are used for protection against bacteriophages or dehydration as well as for defense against the host immune system.



• Pseudomonas fluorescens possess viscosin which is a peptidolipid that enhances antivirality.

13.6 ANTIFEEDANTS The possibility of using non-toxic deterrents and repellents as crop protectants is intuitively attractive. The concept of using insect antifeedants (=feeding deterrents) gained strength in the 1970s and 1980s with the demonstration of the potent feeding deterrent effect of azadirachtin and neem seed extracts to a large number of pest species.

• Indeed, considerable literature, scientific and otherwise, touts neem as a successful demonstration of the antifeedant concept.

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• In reality, it is the physiological actions of azadirachtin that appear most reliably linked to field efficacy of neem insecticides; although purely behavioral effects cannot be ruled out, there is hardly any irrefutable evidence or documentation of field efficacy based on the antifeedant effects of neem alone.



• As an academic exercise, the discovery and demonstration of plant natural products as insect antifeedants has been unquestionably successful. In addition to the neem triterpenoids, extensive work has been performed on clerodane diterpenes from the Lamiaceae and sesquiterpene lactones from the Asteraceae.



• On the other hand, not a single crop protection product, based unequivocally on feeding or oviposition deterrence, has been commercialized.





Two main problems face the use of antifeedants in agriculture. • The first is interspecific variation in response—even closely related species can differ dramatically in behavioral responses to a substance— limiting the range of pests affected by a particular antifeedant. Some substances that deter feeding by one pest can even serve as attractants or stimulants for other pests. • The second is the behavioral plasticity in insects—pests can rapidly habituate to feeding deterrents, rendering them ineffective in a matter of hours. This has been recently demonstrated not only for pure substances like azadirachtin, but also for complex mixtures (plant extracts). Whereas a highly mobile (flying) insect may leave a plant upon first encountering an antifeedant, a less mobile one (larva) may remain on the plant long enough for the deterrent response to wane. Such behavioral changes are important in light of the observation that some plant substances are initially feeding deterrents but lack toxicity, if ingested. Azadirachtin is clearly an exception to this rule, as ingestion leads to deleterious physiological consequences, but many other compounds or extracts with demonstrated antifeedant effects lack toxicity when administered topically or via injection.

13.7 INSECTICIDAL ACTIVITIES OF THE COMPOUNDS OF BOTANICS (BOTANICAL INSECTICDES) Botanical insecticides are naturally-occurring secondary metabolites synthesised by plants species, which act on the insect growth and survival. Natural pesticidal products are available as an alternative to synthetic chemical formulations, but they are not necessarily less toxic to humans. Azadirachtin: Azadirachtin is just one of more than 70 limonoids produced by the neem tree. It is a powerful insect antifeedent and growth regulators. It is structurally similar to insect hormones called ‘ecdysones,’ which control

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the process of metamorphosis as the insects pass from larva to pupa to adult stage. Azadirachtin occurs in all parts of the neem tree, but is concentrated in the kernel. Azadirachtin is used to control whiteflies, aphids, thrips, fungus gnats, caterpillars, beetles, mushroom flies, mealybugs, leafminers, gypsy moths and others.

13.8 PREDATORS An entomophagous species that generally consumes more than one prey individual to complete its development is called a predator. Cryptolaemus montrouzieri: C. montrouzieri is a voracious feeder of mealybugs in both larval and adult stage. It is small (about 3–4 mm), dark brown lady beetle with orange head, larvae have woolly appendages of wax, which makes them resemble mealy bugs. It is mass produced on the mealy bugs, Ferrisia virgata or Planococcus sp. and multiplied on sprouted potatoes or pumpkin fruits. C. montrouzieri larvae and adults attack citrus and closely related mealy bugs and some soft scales. Adults predators are released @ 5-10 beetles/vine/tree. Chrysoperla carnea: They are cosmopolitan in distribution. Adult chrysopids are medium-sized (7–15 mm), yellowish green to grey in colour with red, yellow or brown markings. Most of the adults depend upon pollen, nectar and honeydew and the larval stages are predatory in nature. These are mass-produced using UV sterilized eggs of Corcyra cephalonica. The first instar larvae are released in the field @ 1.25 lakh/ha to control sucking pests and eggs and first instar larvae of lepidopteran insects. Weed Feeder: Those organisms, which feed on the weeds depleting or eliminating them, are called Weed Feeder. Zygogramma bicolorata: This is Chrysomelid beetle used as a biocontrol agent for the control of the carrot weed, Parthenium hysterophorus. Larvae and adult beetles feed on the leaves of this weed. The life cycle of this beetle is completed in 20–43 days. The insect is capable of remaining under soil in diapause from November to June. Once rain starts, diapause is broken and the beetles come out of soil and the new cycle is started

CHAPTER

14

Biotechnology: Industrial Sustainability

uman activities – industrialization, urbanization, agriculture, fishing and H aquaculture, forestry and silviculture as well as petroleum and mineral extraction – have profound impacts on the world’s environment, as well as, on the quality of life. As a result, there is a growing appreciation that nationally, regionally and globally, the management and utilization of natural resources need to be improved and that the amounts of waste and pollution generated by human activity need to be reduced on a large scale. This will require a reduction and, if possible, elimination of unsustainable patterns of production and consumption. As a result, emphasis is growing on industrial sustainability because this is increasingly recognized as a key means of bringing about such reduction of environmental impacts and improving quality of life.

14.1  INDUSTRIAL SUSTAINABILITY The World Commission on Environment and Development has provided insight on sustainable patterns of production and consumption through its description of sustainable development: “Sustainable Development: Strategies and actions that have the objective of meeting the needs and aspirations of the present without compromising the ability to meet those of the future”. This definition of sustainable development can be adapted to provide a conceptual definition of industrial sustainability: “Industry is sustainable when it produces goods and services in such a manner as to meet the needs and aspirations of the present without compromising the ability of future generations to meet their own needs”. A closer look shows that industry is sustainable when it is:

• Economically viable (uses natural, financial and human capital to create value, wealth and profits).

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• environmentally compatible (uses cleaner, more eco-efficient products and processes to prevent pollution, depletion of natural resources as well as loss of biodiversity and wildlife habitat). • Socially responsible (behaves in an ethical manner and manages the various impacts of its production through initiatives such as Responsible Care). This “triple bottom line” for industry is captured in a quote from the Shell Report 2000: “Excellent environmental performance is meaningless if no wealth is created. Wealth in a destroyed environment is equally senseless. No matter how wealthy, a society fundamentally lacking in social equity cannot be sustained.” Moving Toward More Sustainable Industries: Developing sustainable industries implies constantly assessing and improving industrial performance. The aim is to uncouple economic growth from environmental degradation so that industry will be more profitable and, simultaneously, environmental quality will also improve. Economic growth provides jobs and income, goods and services and opportunities to improve the standard of living for an increasing world population. Environmental protection recognizes the intrinsic value of nature and living things. It also recognizes the potential of organisms living in ecosystems to provide insights and the means for developing sustainable industrial products, processes and production systems. Sustainable industrial development can be achieved if the three requirements (economic, environmental and social) outlined above are applied to guide the pathway and shape the process by which industry and the economy grow. At a very basic level, sustainable industrial development means doing more with less – increasing eco-efficiency, that is, decreasing the level of pollution and at the same time, the amount of energy, material and other inputs required to produce a given product or service. A major way of accomplishing this is through cleaner production. Cleaner production involves a paradigm shift where innovation is used to develop: • processes and production systems which: — save costs and are more profitable because they are less wasteful of materials and energy (resulting in less emissions of greenhouse gases, persistent organic chemicals and other pollutants). — enable greater and more efficient utilization of renewable resources (energy, chemicals and materials), lessening our dependence on nonrenewable resources such as petroleum and reducing associated greenhouse gas emissions. • products which are: — Better performing, more durable and don’t persist after their useful life. — Less toxic, more easily recyclable and more biodegradable than their conventional counterparts.

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14.3

— Derived as much as possible from renewable resources and contribute minimally to net greenhouse gas emissions.

14.2 TECHNOLOGY, CLEANER PRODUCTION AND SUSTAINABILITY Technological innovation is a key means of achieving cleaner production and sustainable industrial growth. However, “cleaner” should not be confused with “sustainable”. Sustainable means, clean enough to meet the needs of the present without compromising the ability of future generations to meet their own needs. Making the distinction between “cleaner” and “sustainable” requires the tools to assess and compare the performance of different technologies used for industrial production. Companies that have to take decisions on implementing and improving production processes can develop these processes on the basis of best available technologies. Some sources of information already exist on best available technology, for example, the European Integrated Pollution Prevention and Control Bureau (http://eippcb.jrc.es) or the UNEP International Cleaner Production Information Clearinghouse (www.emcentre.com/unepweb/). Scientifically validated criteria and methods for evaluating the longterm sustainability of industrial production are still being developed (see, for example, the Web site of the Canadian National Round Table on the Environment and the Economy: www.nrtee-trnee.ca). Nevertheless, it is possible to estimate what is sustainable (“clean enough”) from an environmental perspective based on the present situation and some simple assumptions. This helps answer the question: “If one is to approach environmental sustainability while achieving sustained economic growth, what should be the environmental performance targets for technology at the R&D phase today compared to the performance of technology which is currently the industry standard?”

Eco-efficiency of the economy to keep the environmental footprint constant

To answer this question, it is necessary to determine what environmental performance will be required of technology in order to keep the environmental

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“footprint” (impact) of industrial production at a constant level. Experience has shown that environmental footprint of industry is directly proportional to the level of economic activity (that is, if production doubles, then the environmental footprint doubles), other things being equal. So, as production increases, so too, must the environmental performance, or “eco-efficiency”, of technology used if concomitant increases in the environmental footprint of industrial activities is to be avoided. What this means is that new technologies, which bring improvements in production, must also bring improvements in “eco-efficiency”. As is explained more fully in Fig. 14.1, the lag between the R&D phase of new technologies and the point at which these become industry standards, means that work at the R&D stage today needs to target quite significant improvements in environmental performance. If the present environmental impact of existing industrial production is not sustainable, then the environmental performance targets for new technology to help address this will have to be raised even higher. The following sections show that advances in technology, especially biotechnology, can help deliver improvements in environmental performance beyond the factor of 3–4 identified in the example in Fig. 14.1. The graph sets out a picture of a growing economy over time. The curve represents two functions. First, it represents the rising environmental footprint or impact from 4% economic growth without any changes in the environmental performance of the technology used. And second, the same curve delineates the factor of eco-efficiency gain required to deliver the same growth with no impact on the environmental footprint. So the graph in Fig. 14.1 shows that, in order to bring the environmental impact back to its original level: • technologies that are ready to be introduced into the market today (it takes an average of 25 years for these to become average industry practice), should have an environmental performance at least three times better than the current industry average (that is, emissions only 33%, of present). • technologies at the R&D stage today (it will take an average of 35 years for these to become average industry practice) should have an environmental performance at least four times better than the current industry average (that is, emissions only 25%, of present). The assumptions built into Fig. 14.1 include:

• environmental impacts are directly proportional to level of economic activity.



• economic growth (the rate of increase in production), for purposes of this analysis, is set at 4% per year. • improving the environmental performance (“eco-efficiency”) of production technology decreases the environmental footprint for a given level of economic activity.



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• in order to be adopted, a technology must provide a significant net positive value in terms of its economic and/or environmental performance.



• if newly developed technology is now beginning to be introduced into industry, it will take an average of 25 years for it to become the average performance of the industry as a whole.



• technologies at the R&D stage today will take an average of 10 years to develop to the “market-ready” stage, that is, where it is attractive for industry to begin adopting them.

14.3 LEARNING FROM NATURE: BIOMIMICRY AND BIOTECHNOLOGY It is difficult to achieve a four-fold improvement in environmental performance through incremental improvements in conventional production technologies. Improvements of this magnitude usually call for a paradigm shift. For a growing number of companies, the inspiration for such a paradigm shift is coming from the products and processes found in natural ecosystems and the organisms that live in them. Biomimicry is the name coined for this approach in which industrial production systems imitate nature. Industrial biotechnology is that set of technologies that come from adapting and modifying the biological organisms, processes, products, and systems found in nature for the purpose of producing goods and services. The organisms, processes, products and systems found in natural ecosystems have evolved over millions of years to become highly efficient. For example, all energy in natural ecosystems is renewable and is initially captured from sunlight through photosynthesis. Also, all bio-organic chemicals and materials are renewable, biodegradable and recycled. There is no such thing as “waste” – the by-products of one organism are the nutrients for another. Most, if not all, metabolic processes are catalyzed by enzymes and are highly specific and efficient. Biotechnology has evolved over the last 25–30 years into a set of powerful tools for developing and optimizing the efficiency of bioprocesses and the specific characteristics of bioproducts. This increase in efficiency and specificity has great potential for moving industry along the path to sustainability. Increased efficiency allows for greater use of renewable resources without leading to their depletion, degradation of the environment and a negative impact on quality of life. Biotechnology can become an important tool for decoupling economic growth from degradation of the environment and the quality of life. Biotechnology can also enable the design of processes and products whose performance cannot be achieved using conventional chemistry or petroleum as feedstock. Here are some examples of some of the industrial efficiency tools now coming from the application of biotechnology:

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Environmental Biotechnology

• Enzymes extracted from naturally-occurring micro-organisms, plants and animals can be used biologically to catalyze chemical reactions with high efficiency and specificity. Compared to conventional chemical processes, bio-catalytic processes usually consume less energy, produce less waste and use less organic solvents (that, then require treatment and disposal). • By imitating natural selection and evolution, the performance of naturally-occurring enzymes can be improved. Enzymes can rapidly be ‘evolved’ (this technique is called “molecular evolution”) through mutation or genetic engineering and selected using high-throughput screening to catalyze specific chemical reactions and to optimize their performance under certain conditions such as elevated temperature. • The metabolic pathways of micro-organisms can also be modified by genetic engineering. The aim is to turn each cell into a highly efficient “mini reactor” that produces in one step, and at high yield, what would take an organic chemist a number of steps with much lower yield (this technique is called “metabolic engineering”). • A further improvement on metabolic engineering involves engineering the enzymes in the optimal configuration onto the cell membrane and, when the cell is ruptured, the cell membrane becomes a bio-catalytic surface that provides the high efficiency of metabolic engineering without the energy penalty of keeping the organism alive. • Plant biomass can be processed and converted by fermentation and other processes into chemicals, fuels and materials that are renewable and result in no net emissions of greenhouse gases. Also, these biologically-derived products (“bio products”) are generally less toxic and less persistent than their petrochemical counterparts. • Groups of companies can mimic the co-operative action of organisms in natural ecosystems by clustering around the processing of a feedstock such as biomass, so that the by-product of one is the starting material for another. Also, energy, such as waste heat, can be used efficiently. This approach is called “industrial ecology”. • The ability to “evolve” bioprocesses and bio production systems allows for major improvements in both economic and environmental performance. This permits a manufacturing facility to increase its profitability and capacity while maintaining or, even reducing its environmental footprint.

14.4 BIO-SAFETY The micro-organisms used for industrial bio-processing or for production of industrial enzymes are selected to avoid use of pathogenic organisms. They are subject to stringent environmental regulations in Organization for Economic Co-operation and Development (OECD) countries. Occupational

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health regulations also impose rules on their handling in the workplace and, after they are used, they are inactivated by sterilization. The resulting organic material is usually composted. This breaks down the DNA and protein components and the compost can be used as fertilizer to maintain the level of organic material in the soil.

Examples of Case Studies The OECD Task Force on Biotechnology for Sustainable Industrial Development has recently published a report entitled “The Application of Biotechnology to Industrial Sustainability”. This report provides case studies of how companies in a wide range of industrial sectors have used biotechnology to reduce the cost and environmental impact of their production activities. Summaries of the case studies are provided below. Fine Chemicals: Given the cost of developing new bio-processes and bioproducts, it is not surprising that some of the first applications of industrial biotechnology appear in the pharmaceutical and fine chemicals segment of the chemical industry, where the value of the products can bear the cost of technology development. It has long been known that enzymes can catalyze certain chemical reactions with high efficiency and specificity. Since 1970, Tanabe Seiyaku (Japan) has used enzymes-derived from certain microorganisms to produce amino acids. Immobilizing the enzymes on a surface, so they could be used again and again, led to 40% cost savings. Improving this system of immobilization of the micro-organisms to optimize the performance of the enzymes yielded a further 15-fold increase in productivity (i.e., the ratio of product yield to starting material used), resulting in a major reduction of costs and waste. Enzymes usually function in an aqueous solution and this can reduce the requirement in equivalent conventional chemical processes for organic solvents, that will later need to be recycled or disposed of by incineration. Biochemie (Germany/Austria), a subsidiary of Novartis, has developed an enzyme-catalyzed process for manufacture of the antibiotic cephalosporin. The efficiency of the enzymes was optimized by genetically modifying the micro-organisms that produce the enzymes. When compared to the conventional chemical process, the enzymatic process produces 100 times less waste solvent to be incinerated and, as a result, the cost of production and the potential environmental impact of the process are both reduced. Metabolic engineering is a technique which involves genetically engineering a microorganism to contain all the enzyme steps for a series of reactions leading to a particular product and then uses the cell metabolism to drive the reaction. In effect, the cell then becomes a highly efficient mini-reactor for synthesizing that product. Hoffmann La-Roche (Germany) now uses a metabolically engineered microorganism to produce vitamin B2. This has enabled the company to reduce a six-step chemical process to one step. As a result, use of non-renewable raw

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Environmental Biotechnology

materials has decreased by 75%, emissions of volatile organic compounds to air and water have decreased by 50% and operating costs have decreased by 50%. Similarly, DSM (Netherlands) has used a metabolically engineered microorganism to reduce the waste produced in the manufacture of cephalexin 3 to 7-fold. This has allowed the company to reduce production costs so that it can compete effectively in international markets. Intermediate Chemicals: Other case studies indicate that, once the underlying biotechnology has been developed and understood, lateral application can occur in other areas. Thus, biotechnologies developed at high cost in the pharmaceutical and fine chemicals segment of the chemical industry can be adapted and applied at lower cost to produce lower value products, such as intermediate chemicals for synthesis of other chemicals or plastics. S-chloropropionic acid is an intermediate chemical used in the synthesis of certain herbicides. The “S” indicates that the molecule is chiral, that is, one of two asymmetric isomers (the other isomer is the “R” form). The “S” isomer is the one that is biologically active. Conventional chemical procedures for separating chiral molecules are often energy-intensive, or require the use of additional chemicals which subsequently require disposal. A biological method for separating chiral molecules involves using a microorganism that selectively degrades one of the two isomers, leaving the other in essentially pure form, once it has been isolated. Avecia (United Kingdom) has developed a bioprocess for producing pure Schloropropionic acid that uses a Pseudomonas bacterium to selectively degrade the “R” form. Mutation, selection and adoption of sophisticated means of fermentation resulted in a four-fold increase in productivity, while use of genetic modification to optimize performance even further resulted in an additional five-fold increase in productivity. The bioprocess results not only in lower production costs but also in less waste byproduct that requires treatment and disposal. Mitsubishi Rayon Company (Japan) produces acrylamide, a chemical used to produce acrylic polymers. The conventional chemical process for producing acrylamide from acrylonitrile involves high temperature and the use of, either a copper catalyst or sulphuric acid. Mitsubishi Rayon has developed a bioprocess which, instead, uses a naturally-occurring enzyme, nitrile hydratase, to catalyze the conversion of acrylonitrile into acrylamide. The performance and yield of this enzyme has been optimized by genetically engineering the micro-organism which naturally produces the enzyme. The enzyme-catalyzed process uses 80% less energy, saves costs and yields higher purity acrylamide than the conventional chemical process. Polymers: The conventional chemical process for producing certain polyesters involves the use of either a titanium or tin-based catalyst with solvents and inorganic acid at high temperature (200°C). Baxenden Chemicals (United Kingdom) has developed a bioprocess that uses the enzyme lipase

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from the yeast Candida antarctica to catalyze the polymerization reaction at a much lower temperature (60°C). The lipase gene was transferred into a genetically engineered industrial strain of E. coli bacterium to reduce the cost of producing the enzyme. The enzyme-catalyzed polymerization process, when compared with the conventional process, eliminates the use of organic solvents and inorganic acids and yields energy savings of about 2000 megawatts annually at full industrial scale operation. The polymer from the bioprocess also has a more uniform polymer chain length. This results in a melting point over a narrower range of temperature than the conventional polyester, making it more valuable for use as a hot-melt adhesive. Thus, there were both environmental and economic benefits from implementing the enzyme-based bioprocess. Cargill Dow LLC (United States) has developed polylactic acid (PLA), a biopolymer that not only involves the use of bioprocesses (developed, using biotechnology) that are energy and materials-efficient but also utilizes a renewable agricultural feedstock, corn4. PLA is not only recyclable, but also biodegradable, and can be composted. It can functionally replace plastics such as nylon, PET, polyester and polystyrene and life cycle analysis shows that, it can do so with a net fossil fuel saving of 20-50% and at a cost which reflects the lower cost of energy and raw material in its manufacture. In the mediumterm, advances in biotechnology will allow PLA to be produced also from the cellulose found in agricultural and forest by-products. The plastic will then become a net sink for carbon sequestered from the air by crops and trees. Cargill-Dow has constructed a plant in Nebraska, USA, that will produce 140,000 tons of PLA annually. Food Processing: Often, food processing uses large quantities of water and produces large quantities of organic waste. Biotechnology can help reduce water usage as well as the production of organic waste. For example, Pasfrost (Netherlands) has developed a biological treatment system for water in its vegetable-processing facility that has reduced water use by 50% and led to significant cost savings. Similarly, Cereol (Germany) has implemented an enzyme-based system for the degumming of vegetable oil during purification after extraction. This bioprocess was compared with the conventional degumming process that used sulphuric acid, phosphoric acid, caustic soda and large quantities of water. The enzyme system eliminated the need for treatment with strong acid and base, reduced water use by 92% and waste sludge by 88% and resulted in an overall cost reduction of 43%. Fibre Processing: Large quantities of energy, water and chemicals are used to bleach and treat natural fibres for making textiles and paper. Enzymes can help reduce some of these input costs and associated environmental impacts. For example, Windel (Netherlands) uses an enzymatic process to reduce the energy and time required to wash hydrogen peroxide bleach from textiles before dyeing. Use of the enzyme made it possible to reduce the temperature

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Environmental Biotechnology

and volume of the second wash from 80 – 95°C to 30 – 40°C, resulting in a 9–14% saving of energy, a 17–18% saving of water and an overall cost saving of 9%. This is very significant in the highly competitive textile industry because margins are generally quite small. Domtar (Canada) has begun to use the enzyme xylanase, supplied by Iogen Corporation (Canada) as an auxiliary brightening agent (this process is called “bio-bleaching”) for wood pulp in papermaking. The enzyme opens up the lignin structure of the wood pulp so that it takes 10–15% less chlorine dioxide to achieve the desired level of brightness. Iogen has reduced the production cost and improved the performance of xylanase by genetically engineering the fungus from which it is extracted. The use of xylanase has helped Domtar reduce the amount of organically-bound chlorine in waste water by 60% and the cost of bleaching chemicals by 10–15%. Oji Paper (Japan) has also used xylanase to achieve similar reductions in the requirement for bleaching chemicals and in levels of organically-bound chlorine in its waste water. In addition, it produces its own xylanase on-site by fermentation, so its input costs are reduced even further. Mining and Metal Refining: Billeton (South Africa) has developed a bioprocess (“bio-leaching”) to liberate copper from sulphide ore. The bioprocess uses naturally-occurring bacteria to oxidize the sulphur and iron present in the ore at ambient temperature. The conventional process for isolating the copper from the ore involves transporting the mined ore to a smelter where the impurities are driven-off at high temperature. The bioleaching process is carried out at the mine site. This saves the cost and energy required to transport the ore and also eliminates the emission of large quantities of sulphur oxides, arsenic and other toxic metals into the atmosphere by the high temperature roasting process. After the copper is extracted from the acidic leach water, the wastewater is neutralized and toxic substances such as arsenic are immobilized in a stable form stored at the mine site. The bioleaching process can be used to process low-grade ores and arsenic containing ores that could not be processed effectively by high temperature smelting. The capital cost requirements of the bio leaching process are 25% less than for building a smelter. Bio-leaching currently accounts for 20–25% of world copper production. Budel Zinc (Netherlands) is a major producer of zinc. The acidic waste water from its zinc refinery contains zinc and other metals (tin, copper, nickel, manganese, chromium, lead and iron). The conventional process for treating this wastewater involves neutralizing it with lime or limestone, which results in large quantities of gypsum contaminated with heavy metals. Budel has developed a bioprocess that uses sulphate-reducing bacteria to capture and recycle zinc and other metals in its wastewater as metal sulphide precipitate. The metal sulphide precipitate is recycled back into the refinery feedstock. This process has resulted in a 10- to 40-fold decrease in the concentration of

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heavy metals in the refinery wastewater and eliminated the production of metal-contaminated gypsum which is a hazardous solid waste by-product. Energy: Examples of biotechnology applications in the energy sector occur in both the conventional fossil-fuel and the renewable energy segments of the industry. Conventional fossil-fuels are usually extracted from deposits buried below the surface of the earth. Drilling of oil wells requires the use of substances called drilling fluids or drilling mud. These substances help lubricate the drill and its pipe as well as hold open the wellbore. Drilling fluids are designed to deposit a low-permeability layer on the surface of the borehole to limit leakage of the drilling fluid into the oil-bearing formation and to prevent invasion of solids into the oil production zones. Once the well is drilled to the desired depth, the low-permeability layer must be removed in order to maximize oil production rates. Traditional drilling fluids are muds—dispersions of clay minerals in water and oil where the clay provides the required viscosity and the oil provides the lubrication. These muds pose two problems: (i) the oil used in their formulation can have negative environmental impacts and requires treatment. (ii) the strong acid required to remove the low-permeability layer is toxic to the environment, corrodes equipment and does not uniformly remove the low-permeability layer. M-I and British Petroleum Exploration (United Kingdom) are now using a drilling fluid containing mixtures of bio-organic polymers such as xanthan gum, which provides viscosity, and starch or cellulose, which acts as a binder. The formulation also contains an inert solid called a bridging agent that has a particle size allowing it to bridge pores in the structure of the rock being drilled. This formulation is non-toxic and avoids the problems of conventional drilling muds: (i) there is no oil or other component which requires treatment before release into the environment; and, (ii) the enzymes used in removing the low-permeability layer not only perform better but also do not corrode equipment or pose environmental hazard. Biotechnology has been used to optimize the characteristics of these enzymes (cellulase, hemicellulase, amylase and pectinase) to work under the conditions found in a borehole. Although the use of bio-organic drilling fluid systems is in its early days, it appears in a number of cases that their performance is satisfactory and permit cost savings of USD 75,000 – 83,000 per well drilled. Ethanol is one renewable fuel whose production is increasing rapidly in response to the need for transportation fuels that produce lower net emissions of greenhouse gases (GHG). Ethanol is produced by fermentation of sugars

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Environmental Biotechnology

(such as glucose) using brewers’ yeast. The sugar can come from cornstarch. It takes considerable energy to produce corn, however, so the net reduction in GHG emissions is around 40–50%, when ethanol from corn is used to replace gasoline (petrol). If wood cellulose and waste materials are used as the source of sugar to produce ethanol, the net reduction in GHG emissions is larger, around 60–70%. Therefore, cellulose-containing materials are, from a GHG perspective, the material of choice for producing ethanol. However, the lignin in woody plant material can prevent full conversion of cellulose into fermentable sugar. Iogen Corporation (Canada) has developed a process utilizing cellulase enzymes that maximize the conversion of cellulose into fermentable sugar. The yield and activity of the cellulose enzymes has been optimized using biotechnology. Iogen is in the scale-up phase of the technology and indications are that the cost of ethanol produced in this manner will be competitive with the cost of gasoline produced from oil costing USD 25 per barrel.

Lessons from the Case Studies It is possible to draw a number of general conclusions from the case studies:

(i) The application of biotechnology in a wide range of industry sectors (chemicals, plastics, food processing, natural fiber processing, mining and energy) has invariably led to both economic and environmental benefits via processes that are less costly and more environmentally friendly than the conventional processes they replace. In effect, the application of biotechnology has contributed to an uncoupling of economic growth from environmental impacts.



(ii) The application of biotechnology to increase the eco-efficiency of industrial products and processes can provide a basis for moving a broad range of industries toward more sustainable production. To achieve this, further development of biotechnology and supporting technologies will be needed, as well as policies that provide incentives for achieving more sustainable production.

(iii) The main driving forces for adoption of more efficient bioprocesses and bio-products are cost savings and improved product quality/ performance. Environmental considerations were (in the case studies, at least) an important but secondary driving force. (iv) Successful biotechnology/bioprocess development requires effective management of technology development by companies and use of tools that assess both the economic and environmental performance of technology during its development. There is a need for improved assessment tools that are easier to use at earlier stages of the technology development process.

(v) Even large companies may not have, in-house, all the expertise required to develop more efficient bio-products and bioprocesses. Collaboration with university and government researchers and other companies is

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an important contributing factor for successful introduction of these products and processes. (vi) Long lead times are often required for introduction of ‘paradigm shift’ technology into a company; but, development times can be reduced considerably in subsequent development cycles. (vii) The application of biotechnology for developing industrial products and processes is still in its infancy. As awareness builds and the technology continues to be developed and diffused through different industry sectors over the next few decades, the economic and environmental benefits are predicted to grow. Setting a Path to a Sustainable Future – The Bio-based Economy: The case studies outlined above show that biotechnology is an effective tool which provides a means of reconciling the need for economic growth with the need for environmental protection. The eco-efficiency of industrial bioproducts and bioprocesses can provide a basis for moving a broad range of industries toward more sustainable production. However, these applications are occurring as a “thousand points of light”, that is, without a guiding principle or a strategic orientation. Such a strategic orientation is needed to avoid investing resources on incremental improvements in the cleanliness of industrial production systems which may never make it to “clean enough”, i.e. sustainable. Shifting toward an economy more extensively based on renewable raw materials—a bio-based economy {The bio-based economy uses renewable bio-resources (agricultural, forestry and marine) and eco-efficient processes (including bioprocesses) to produce sustainable bio-products, jobs and income.}—does provide such an integrating principle. As can be seen in Table 1, continued use of conventional processes that are not eco-efficient in combination with non-renewable feed stocks results in continued pollution and exhaustion of resources. Choice of Process and Feedstock—Implications for Sustainability Contentional Processes

Cleaner Processes

Non-renewable Feedstock

Status quo-pollution; rapid exhaustion of resources

Extended life of resources— “Postponing the inevitable”

Renewable Feedstock (e.g. biomass)

Depletion of renewable resources

Best chance for sustainability

If conventional processes that are not eco-efficient are used in combination with renewable resources, they may lead to depletion of the renewable resource as the global economy grows and demand increases. If cleaner production processes are used on non-renewable resources, they will extend the lifetime of those resources, but only postpone their inevitable exhaustion. Sustainability is most likely to be found in utilizing renewable resources through cleaner

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processes that are eco-efficient. Developing a sustainable economy more extensively based on renewable carbon and eco-efficient bioprocesses (a ‘biobased economy’) is one of the key strategic challenges for the 21st century. At present, the global economy depends to a large extent on energy, chemicals and materials derived from fossil carbon sources, mainly petroleum. Petroleum provides us with fuels for transportation and heating. It also yields synthetic chemicals for producing plastics, paints, dyes, adhesives and a wide range of other useful industrial and consumer products. These developments have contributed to strong economic growth and employment and have literally transformed our global society. But this has come at a cost. The Petrochemical Age has also resulted in massive pollution of air, water and soil as well as emissions of greenhouse gases responsible for climate change. Petroleum is also a finite, diminishing resource now subject to strong price increases and fluctuations. The present level of global energy consumption, production and industrial growth is ultimately not sustainable because it is only made possible by continued withdrawals from the stored “bank” of fossil carbon which is finite and not renewable. The world was not always dependent on petroleum. A traditional biobased economy provided and continues to provide us with food, feed, fiber and wood. Before the 1920s, many of our industrial products were also bio-products, such as fuels, chemicals and materials derived from biomass, primarily wood, and various agricultural crops. Cheap and abundant oil changed that. However, as seen in the case studies outlined above, advances in technology, and biotechnology in particular, are making it economically viable and environmentally attractive to “go back to the future” and begin supplementing, and eventually perhaps, replacing petroleum with biomass, a renewable feedstock derived mostly from plants. Improved understanding of biodiversity, ecology, biology and biotechnology is making it possible both sustainably to increase biomass productivity in forestry and agriculture as well as to utilize that biomass and waste organic materials in a highly efficient and sustainable manner. Without such advances in science and technology, the move to a bio-based economy would result in rapid depletion of renewable resources and environmental degradation. Thus, advances in science and technology are making it possible to have an economy where industrial development and job creation are not in opposition to environmental protection and quality of life. Getting there will be a major challenge, requiring effective tools to assess technology, processes and products for sustainability and also policies that encourage sustainable production and consumption. The life sciences, and in particular, biotechnology, will play a prominent role in meeting that challenge. For example, the Vision (Vision for Plant/ Crop Based Renewable Resources 2020; www.oit.doe.gov/agriculture/pdfs/ vision2020.pdf) and Technology Roadmap (The Technology Roadmap for

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Plant/Crop Based Renewable Resources 2020. Web site: www.oit.doe.gov/ agriculture/pdfs/ag25945.pdf) for Plant/Crop-Based Renewable Resources 2020 provide a view of how this can be conceived, planned and executed through targeting the development of technologies in the near, medium and long term for:

• Selecting and developing value-added crop and tree varieties for conventional and industrial applications.



• High-yield, sustainable crop and tree production.



• Eco-efficient harvesting and processing.



• Sustainable utilization of the resulting products.



• Closing the loop back to the environment to maintain soil organic content and fertility.

The “bio-based economy” offers hope both for developed and developing countries. For developed countries, it presents the opportunity to use their technological capabilities for national energy security to head off major economic and social disruptions which will be caused by fluctuations in the availability and price of energy and petrochemicals as the supply of these finite, non-renewable resources continues to diminish. It will also help them diversify and grow employment in their rural economies. For a number of developing countries, it provides the potential to leapfrog (at least in part) the age of fossil-fuels and petrochemicals to the age of biofuels and bio chemicals. These are less toxic and more easily biodegradable than their petrochemical counterparts and can be derived from locally grown feedstock, leading to local self-sufficiency, an improved economy and a better quality of life. However, if we are to see a move to such a future in the 21st century, then, despite the potential economic, environmental and social benefits, it is not realistic to assume that a new “green revolution” will sweep spontaneously over existing industries. Potentially, the move to a bio-based economy could be at least as big as that caused by the development of the petrochemical age during the 20th century. But, societal values are different in 2001 from those of 1901. The transition, therefore, will need to be carefully managed, not least because it will link such issues as biotechnology and GMOs, preservation of biodiversity, climate change, globalization, economic growth, sustainable development and quality of life. The interplay of these issues could pose complex problems and policy issues for governments, industry and civil society as they try to optimize economic, environmental and societal benefits, while enabling and fostering the development of a bio-based economy in their countries. Visionary thinking is required among stakeholders if we are to identify proactively the key issues and policy decisions that will have to be dealt with along the way. Further work on these issues is underway in a number of countries.

CHAPTER

15

Ethical Issues in Environmental Biotechnology

Ethics are the rules or standards that govern the way people behave and their decisions on the ‘right thing’ to do. It asks basic questions about what is right and wrong, how we should act towards others and what we should do in specific situations.

• It is important to note that ethics discussing  biotechnology  and its applications are not fundamentally different from other situations. Ethics are practiced by everyone, every day.



• One common feature of ethics is that different people with different values often disagree on the ‘right thing’ for individuals and society. One reason for this disagreement is that one thing that benefits some may not be of benefit to others.



• An example is embryonic stem cell research, which some people see as having great potential to develop cures for diseases; but others object to because it involves the destruction of human embryos that have the potential to become a human being.



• There is no clear right or wrong position in ethics, as a person’s individual experience and view of the world often guides the way they make ethical choices.



• For instance, someone who has a strong environmental outlook might see the use of genetically modified (GM) crops as unnatural. But someone who has a strong scientific-based view of the world might see the use of GM crops as a natural extension of traditional crop breeding technologies.

Many new technologies raise ethical concerns that might not be part of the worldview held by those who develop the technologies in the first place. When it comes to developing products for commercial use, the goal is usually to increase sales and increase profits for shareholders. The decision for developing products can be seen as good for industry development,

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but perhaps not as good for some individuals who do not have products developed to suit their needs when there is not enough company profit to be made. Also, in some areas of biotechnology development, the money needed to fund research projects is out of the range of individuals or small groups and can only be undertaken by multinational or overseas companies. For some, this is perceived as acceptable, as it helps local researchers form links with wealthy larger companies. But others do not think it is not acceptable, as local research and development leave the community and are then controlled by international corporations.

• Many people believe that biotechnology products and applications should respond to, and fulfil community needs. For example, some products may be of obvious social benefit (such as a drug that treats cancer), while others may be created by a business by attractive advertising and skilful marketing (for example, unusual coloured flowers for the floral industry or fluorescent fish for the pet industry).



• In a world with decreasing resources, where many people go hungry, is spending research dollars on developing a fluorescent fish an acceptable thing or not? Your answer will differ depending on your worldview.

When looking at ethical positions, it is important to realise that the ‘right thing’ for one person may not be right for others and it can be very difficult to balance these conflicting views.

• There are particular ethical positions that are commonly shared, such as the view that, it is essential for all biotechnology products to be safe for humans and the environment (which is why Australia has developed a sound regulatory system to look at safety). But other ethical positions are diverse, such as an individual’s rights to do what they want with their body.



• There are many different ethical ways to view the world and none of these are inherently right or wrong. There are many approaches, or frameworks, to ethics. Some of these approaches are listed below:

Action-based (whether or not, actions in a particular circumstance are ethical):

• Principalism uses benefit-maximising and harm-reducing principles.



• Consequentialism is based on the greatest good for the greatest number.



• Non-consequentialism (deontology) refers to rights and responsibilities. Agent-based (emphasis on the person rather than the action they perform):



• Virtue-based can acknowledge character traits over consequences.

Situation-based (a broader perspective that takes into account other factors such as time, place and culture):

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• Casuistry considers each situation to be completely unique.



• Feminist concentrates on communication, consultation and sensitivity.



• Geocultural focuses on relativity (cultural, special and time-specific contexts).

Environmental ethics has been called “a triangular affair.” The triangle is composed of the moral consideration of—

(1) only humans,



(2) animals or



(3) ecosystems.

The first is often called “anthropocentrism” and is atomistic in that individuals are the focal point. The final is often called “ecocentrism” and is holistic in, that entire ecosystems, even abiotic components, are considered in moral deliberations on what is good and right. Between the extremes is the ethical framework of the animal welfare movement which is nonanthropocentric in, that the well-being of non-humans is considered but which is still atomistic in individuals, and not systems, are the unit of consideration. Ethics of research involving the environment: Gene technology makes it possible for humans to alter living organisms such as plants, animals, and bacteria to cater for human needs and wants. Such needs could include increased crop yields, bigger, leaner, disease-free animals and new drugs or vaccines. Some biotechnologies are also used for purposes such as veterinary medicine.  Genetically modified organisms  (GMOs) can even benefit the environment by cleaning up waste material or converting oil spills into nontoxic compounds (bioremediation). Although many scientists and agriculture companies argue that genetically modified crops require less pesticide and herbicide than other plants, there is still a great deal of debate about the environmental safety and value of GMOs. For example, organizations are concerned about the following issues with regard to genetic engineering:

• Consumer rights and the labelling of GM products



• The ethics of patenting genes and living organisms



• Who benefits from collecting genes from plants, animals, bacteria or even people?



• Eugenics–the idea that humans can be made perfect



• Human rights



• Animal rights and welfare



• The control of genetic engineering



• Environmental impacts of new organisms

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Other examples of concerns about GMOs in the environment include: • Do we know if they are safe? • Can they be harmful to humans, animals, or plants? • Are there other, more environmentally responsible, ways of producing the effects of genetic modification? • Do GMOs have advantages over natural organisms and farming methods? • Can GMOs independently survive in nature and disturb ecosystems? • Can GMOs transfer genetic material to other organisms? • Are we causing harm to animals or plants by genetically modifying them? The subject of environmental ethics also raises questions about  genetic modification for human or environmental benefit. • Do humans have the right to alter the genetic structure of animals and plants? • How do humans see the environment and their connections to it? • What sort of connections should humans have with other species and the environment? • Do humans have a responsibility to protect other species and the environment? • Does genetic modification cause suffering for animals or the ecosystem? • How might releasing GMOs affect the environment in the future?

15.1  RELEASE OF GENETICALLY MODIFIED ORGANISMS (GMOS) Genetically modified organisms (GMOs), organisms in which genes from another organism are inserted into the targeted organism’s DNA, have the potential to both positively and negatively affect the environment and human health.  Plants can be genetically modified easily because they can be grown from a single cell or small pieces of tissue.  Thus, one only needs to modify a single cell to produce an entire genetically modified organism.  Several methods have been used to insert foreign genes into the target organism.  • In one method, the target organism takes up a vector with cloned DNA from a donor organism and incorporates this foreign DNA into its genome.   • Another method involves removing the wall of the target cell, allowing the introduced DNA to easily penetrate.  • A third method requires using a special gun to inject foreign DNA into the target cell in hopes that the cell will incorporate the new DNA into its genome. Crops have been modified for centuries by humans using selective breeding techniques, but GMO biotechnology is a more specific and rapid selection

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process.  For instance, genes from a different species can be incorporated into the modified crop.  Therefore, GMO technology creates concern over potential environmental and human health impacts. Ethical Issues: The use of genetically modified organisms is a practice still in its infancy.  The long-term effects of this technology are yet to be seen, and thus, we must proceed with caution, as we develop our practices and guidelines.

15.1.1  Effects on the Environment Herbicide Use and Resistance: Effects on the environment are a particular concern with regard to GMO crops and food production.  One area of development involves adding the ability to produce pesticides and resistance to specific herbicides.  These traits are helpful in food production, allowing farmers to use fewer chemicals, and to grow crops in less-than-ideal conditions.   However, herbicide use could be increased, which will have a larger negative effect on the surrounding environment.   Also unintended hybrid strains of weeds and other plants can develop resistance to these herbicides through cross-pollination, thus negating the potential benefit of the herbicide.   Effects on Untargeted Species: Bt corn, which produces its own pesticide, is also in use today.   Concerns have been raised regarding adverse effects on Monarch butterfly populations, which are not the original target of the pesticide.  Although the pesticide can protect crops against unwanted insects, they can also have unintentional effects on neutral or even beneficial species.

15.1.2  Effects on Human Health Allergies: GMO crops could potentially have negative effects on human health, as well.  When splicing genes between species, there are examples in which consumers have developed unexpected allergic reactions.   Long-Term Effects: Because GMO technology has been available for such a short amount of time, there is relatively little research which has been conducted on the long-term effects on health.  The greatest danger lies not in the effects that we have studied, but in those which we cannot anticipate at this point. New Proteins: Proteins, which have never been ingested before by humans, are now part of the foods that people consume every day.   Their potential effects on the human body are as of yet unknown. Food Additives: GMOs also present us with possibilities of introducing additional nutrients into foods, as well as antibiotics and vaccines.   This availability of technology can provide nutrition and disease-resistance to those countries that don’t have the means to provide these, otherwise.   The distribution of these foods is more feasible than mass inoculations for current diseases.   However, even these possibilities carry with them potential negative effects such as the creation of antibiotic and vaccine-resistant strains of

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diseases. It is imperative that we ensure that environmental issues and human health are kept at the forefront of development in this field.  It is important that we not lose sight of the repercussions that could accompany the benefits if we do not carefully investigate and control development. The genetic engineering of animals has increased significantly in recent years, and the use of this technology brings with it ethical issues, some of which relate to animal welfare — defined by the World Organization for Animal Health as “the state of the animal…how an animal is coping with the conditions in which it lives.” These issues need to be considered by all stakeholders, to ensure that all parties are aware of the ethical issues at stake and can make a valid contribution to the current debate regarding the creation and use of genetically engineered animals. In addition, it is important to try to reflect societal values within scientific practice and emerging technology, especially, publicly-funded efforts that aim to provide societal benefits, but that may be deemed ethically contentious. As a result of the extra challenges that genetically engineered animals bring, governing bodies have started to develop relevant policies, often calling for increased vigilance and monitoring of potential animal welfare impacts. Veterinarians can play an important role in carrying out such monitoring, especially, in the research-setting, when new genetically engineered animal strains are being developed. Several terms are used to describe genetically engineered animals: genetically modified, genetically altered, genetically manipulated, transgenic, and biotechnology-derived, amongst others. In the early stages of genetic engineering, the primary technology used was Trans genesis, literally meaning, the transfer of genetic material from one organism to another. However, with advances in the field, new technology emerged that did not necessarily require trans genesis: recent applications allow for the creation of genetically engineered animals via the deletion of genes, or the manipulation of genes, already present. To reflect this progress and to include those animals that are not strictly transgenic, the umbrella term “genetically engineered” has been adopted. Hence the scientist offers the following definition of a genetically engineered animal: “an animal that has had a change in its nuclear or mitochondrial DNA (addition, deletion, or substitution of some part of the animal’s genetic material or insertion of foreign DNA) achieved through a deliberate human technological intervention.” Those animals that have undergone induced mutations (for example, by chemicals or radiation — as distinct from spontaneous mutations that naturally occur in populations) and cloned animals are also considered to be genetically engineered due to the direct intervention and planning involved in creation of these animals.

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Current context of genetically engineered animals: Genetic engineering technology has numerous applications involving, companion, wild, and farm animals, and animal models used in scientific research. The majority of genetically engineered animals are still in the research phase, rather than actually in use for their intended applications, or commercially available. Companion animals: By inserting genes from sea anemone and jellyfish, zebrafish have been genetically engineered to express fluorescent proteins — hence the commonly termed “GloFish.” GloFish began to be marketed in the United States in 2003 as ornamental pet fish; however, their sale sparked controversial ethical debates in California — the only US state to prohibit the sale of GloFish as pets. In addition to the insertion of foreign genes, gene knock-out techniques are also being used to create designer companion animals. For example, in the creation of hypoallergenic cats, some companies use genetic engineering techniques to remove the gene that codes for the major cat allergen. Companion species have also been derived by cloning. The first cloned cat, “CC,” was created in 2002. At the time, the ability to clone mammals was a coveted prize, and after just a few years, scientists created the first cloned dog, “Snuppy”. With the exception of a couple of isolated cases, the genetically engineered pet industry is yet to move forward. However, it remains feasible that genetically engineered pets could become part of day-to-day life for practicing veterinarians, and there is evidence that clients have started to enquire about genetic engineering services, in particular, the cloning of deceased pets. Wild animals: The primary application of genetic engineering to wild species involves cloning. This technology could be applied to either extinct or endangered species; for example, there have been plans to clone the extinct thylacine and the woolly mammoth point out that, “As many conservationists are still suspicious of reproductive technologies, it is unlikely that cloning techniques would be easily accepted. Individuals involved in field conservation often harbour suspicions that hi-tech approaches, backed by high profile publicity, would divert funding away from their own efforts.” However, cloning may prove to be an important tool to be used alongside other forms of assisted reproduction to help retain genetic diversity in small populations of endangered species. Farm animals: There is “an assorted range of agricultural livestock applications [for genetic engineering] aimed at improving animal productivity; food quality and disease resistance; and environmental sustainability.” Productivity of farm animal species can be increased using genetic engineering. Examples include, transgenic pigs and sheep that have been genetically altered to express higher levels of growth hormone. Genetically engineered farm animals can be created to enhance food quality.

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Farm species may be genetically engineered to induce disease-resistance specific examples include, conferring immunity to offspring via antibody expression in the milk of the mother; disruption of the virus entry mechanism (which is applicable to diseases such as pseudorabies); resistance to prion diseases; parasite control (especially, in sheep); and mastitis resistance (particularly, in cattle). Genetic engineering has also been applied with the aim of reducing agricultural pollution. Effort has also been made to generate genetically engineered farm species such as cows, goats, and sheep that express medically important proteins in their milk. Research animals: Biomedical applications of genetically engineered animals are numerous, and include understanding of gene function, modeling of human disease to either understand disease mechanisms or to aid drug development, and xenotransplantation. Through the addition, removal, or alteration of genes, scientists can pinpoint what a gene does by observing the biological systems that are affected. While some genetic alterations have no obvious effect, others may produce different phenotypes that can be used by researchers to understand the function of the affected genes. Genetic engineering has enabled the creation of human disease models that were previously unavailable. Animal models of human disease are valuable resources for understanding how and why a particular disease develops, and what can be done to halt or reverse the process. As a result, efforts have focused on developing new genetically engineered animal models of conditions such as Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, and cancer. However, as scientists points out: “these [genetically engineered animal] models do not always accurately reflect the human condition, and care must be taken to understand the limitation of such models.” The use of genetically engineered animals has also become routine within the pharmaceutical industry, for drug discovery, drug development, and risk assessment. Nowadays “Transgenic and knock out mouse models are extremely useful in drug discovery, especially, when defining potential therapeutic targets for modifying immune and inflammatory responses. Specific areas for which [genetically engineered animal models] may be useful are, in screening for drug induced immunotoxicity, genotoxicity, and carcinogenicity, and in understanding toxicity-related drug metabolizing enzyme systems.”Perhaps, the most controversial use of genetically engineered animals in science is to develop the basic research on xenotransplantation — that is, the transplant of cells, tissues, or whole organs from animal donors into human recipients. In relation to organ transplants, scientists have developed a genetically engineered pig, with the aim of reducing rejection of pig organs by human recipients. This particular application of genetic engineering is currently at the basic research stage, but it shows great promise in alleviating the long waiting lists for organ transplants, as the number of people needing

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transplants currently far outweighs the number of donated organs. However, as a direct result of public consultation, a moratorium is currently in place preventing pig organ transplantation from entering a clinical trial phase until the public is assured that the potential disease transfer from pigs to humans can be satisfactorily managed.

15.1.3  Environmental Biotechnology - Biosafety Management Biosafety is associated with the use of genetically modified organisms (GMOs) and, more generally, with the introduction of non-indigenous species into natural or managed ecosystems. A relatively new concept in environmental research, it tempers the adoption of a new technology by carefully considering its potential effects on human health and the environment.

• Environmental biotechnology has allowed the movement of genetic material across unrelated species, something impossible with the traditional breeding methods. This intentional transfer of genetic material has, in turn, brought biotechnology out from the laboratory to the field.



• Genetically modified organisms (GMO’s) are organisms whose genetic material has been artificially modified to change their characteristics in some way or another. In essence, “genetic modification” or “genetic engineering” techniques enable scientists to find individual genes that control particular characteristics, separate them from the original source, and transfer them directly into the cells of an animal, plant, bacterium, or virus.



• This technology has many potential applications. These new opportunities bring along greater public scrutiny and government regulation. Risk assessment is a common regulatory tool used in the decision-making process for a proposed commercial release of a GMO into the environment.



• Environmental applications of microorganisms are wide and varied, ranging from bio-remediation, bio-pesticides, nitrogen fixation, plant growth promoter, to bio-control of plant diseases, and other such agricultural practices.



• The sensible application of recombinant DNA techniques has shown the potential for genetically improved microorganisms to be used as soil or seed inoculants. However, when introduced into the environment, they could have unintended environmental consequences and may play more pronounced ecological roles than the wild types.



• Genetically improved microorganisms are able to reproduce and establish themselves as persistent populations and may have subtle and long-term effects on biological communities and natural ecosystems. Results of DNA modification may not be limited only to the particular

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characteristics of the replaced gene. It is therefore important to ensure that, when these organisms are released into nature, they do not harm the environment or human health. Such concerns have led to broader interests in the theme of risk assessment in the release of GMOs. A cautious approach is necessary to assess environmental risks which may occur due to introduction of recombinant organisms in the natural environment. Biosafety Guidelines and Regulations: Many countries have formulated the Biosafety Guidelines for rDNA manipulation with the aims

(i) to minimize the probability of occasional release of GMMs, and



(ii) to ban the deliberate release of such organisms into the environment. In India, DBT has evolved “the recombinant DNA safety guidelines” to exercise powers conferred through the Environmental Protection Act, 1986 for the manufacture, use, import, export and storage of hazardous microorganisms / genetically engineered organisms, cell, etc.

These guidelines are being implemented through the following three mechanisms:

• the institutional biosafety committees (IBSCs) monitors the research activity at institutional level,



• the review committee on genetic manipulation (RCGM) functioning in the DBT which allows the risky research activities in the laboratories, and



• the genetic engineering approval committee (GEAC) of the Ministry of Environment and Forest has the power to permit large-scale use of GMOs at commercial level, and open field trials of transgenic materials including agricultural crops, industrial products, health care products, etc.

Genetically modified organisms (GMOs), including GM crops, have been developed for only a short time. Just like many other major changes in human history, biotechnology has involved some concerns and mistrust. The issues concerning environmnetal biotechnology include environmental safety and biodiversity, human health and food safety, trade and economic impacts, social and ethical considerations, and much more. Many of the approaches and policies dealing with these issues are of recent origin, still evolving and largely unresolved. As a developing country, India considers that it is very important to have international cooperation and exchange in the regulation of GMO biosafety. Currently, some of the major challenges facing us are: an appropriate regulatory approach, a science-based safety assessment, capacity building, transparency, communication and information exchange. We believe a good environment is necessary to facilitate the realization of the potential benefits of modern biotechnology.

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15.1.4 Environmental Biotechnology & Intellectual Property Rights (IPRs) Environmental Biotechnology is a field of technology of growing importance in which inventions may have a significant effect on our future, particularly in agriculture, energy and protection of the environment. The science of environmental biotechnology concerns living organisms, such as plants, animals and microorganisms, as well as biological material, such as, enzymes, proteins and plasmids (which are used in “genetic engineering”). In recent times, scientists have developed processes to modify the genetic composition of living organisms (genetic engineering). For example, the modified microorganisms created by Chakrabarty (an inventor in the United States of America) were able to break down components of oil pollution in oceans and rivers, and helped to protect environment. The patent on these microorganisms was the subject of a landmark decision by the United States Supreme Court, in which modified microorganisms were recognized as patentable subject matter. The Court noted that the laws of nature, physical phenomena and abstract ideas were not patentable. The claimed invention, however, was not directed to an existing natural phenomenon but to new bacteria with markedly different characteristics from any found in nature. The invention, therefore, resulted from the inventor’s ingenuity and effort and could be the subject of a patent. The list of industries using biotechnology has expanded to include health care, agriculture, food processing, bioremediation, forestry, enzymes, chemicals, cosmetics, energy, papermaking, electronics, textiles and mining. This expansion of applications has resulted from innovations that have led to significant economic activity and development. Need to protect biotechnological inventions: As in other fields of technology, there is a need for legal protection in respect of biotechnological inventions. Such inventions are creations of the human mind just as much as other inventions, and are generally, the result of substantial research, inventive effort and investment in sophisticated laboratories. Typically, enterprises engaged in research, only make investments, if legal protection is available for the results of their research. As with other inventions and industries, the need for investment in research and development efforts creates an obvious need for the protection of biotechnological inventions. • This need is not only in the interest of inventors and their employers, but also in the public interest of promoting technological progress. Modern, flexible intellectual property systems and policies have contributed to fostering investment needed to establish biotechnology industries creating tangible products. Flexible intellectual property policies can play a role in favoring stable legal environments conducive to public/private partnerships, investment and other economic activity needed to spread biotechnological innovations to more countries.

• The patenting of biotechnology innovations has been accompanied by controversy, as has the use of some of these new innovations. Policy

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makers of all countries, however, have been careful to avoid extending patent rights to things, as they exist in nature or to natural phenomena. A new plant species discovered in the wild, for instance, cannot be patented and neither can laws of nature. In each country, the laws on patentability of biotechnological inventions need to be consulted to learn the availability of patent protection and its scope.

• When considering these issues, one also needs to recognize that legal regimes other than patent systems are typically relied upon to address other public interests, such as the environmental or medical safety of products, efficacy of products, and unfair competition that may occur in the assertion of patent rights. The confluence of this new technology with legal and regulatory systems makes biotechnology an evolving and dynamic component of intellectual property law.

APPENDIX

Useful Terms and their Meanings of Environmental Biotechnology

Abatement Debris: Waste resulting from remediation (clean-up) activities. Abiogenesis: Abiogenesis is the study of how life on earth could have arisen from inanimate matte. It should not be confused with evolution, which is the study of how groups of living things change over time. Abiotic stress: The stress caused (e.g., to crop plants) by non-living, environmental factors such as cold, drought, flooding, salinity, ozone, toxicto-that-organism metals (e.g., aluminum, for plants), and ultraviolet-B light. ABS toxins: ABS toxins are six-component protein complexes secreted by a number of pathogenic bacteria. All share a similar structure and mechanism for entering targeted host cells. Abzyme: See Catalytic antibody. Acid rain: Rain that is more acidic than normal because raindrops have dissolved acid gases and/or dust particles from the atmosphere; the principal gases responsible for increased acidity are oxides of sulfur and nitrogen. Generally, rain with a pH below about 4.5 is considered environmentally harmful. Acidogenesis : Acidogenesis represents the second stage in the four stages of anaerobic digestion: (1) Hydrolysis: A chemical reaction where particulates are solubilized and large polymers converted into simpler monomers; (2) Acidogenesis : A biological reaction where simple monomers are converted into volatile fatty acids; (3) Acetogenesis : A biological reaction where volatile fatty acids are converted into acetic acid, carbon dioxide, and hydrogen; and (4) Methanogenesis : A biological reaction where acetates are converted into methane and carbon dioxide, while hydrogen is consumed. Acidophilic autotrophs: Organisms that are able to live solely on sulphides and in acid conditions. Activated sludge: Sludge particles produced in raw or settled wastewater (primary effluent) by the growth of organisms (including zoogleal bacteria) in

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aeration tanks in the presence of dissolved oxygen. The term “activated” comes from the fact that the particles are teeming with bacteria, fungi and protozoa. Activated sludge is different from primary sludge in that the sludge particles contain many living organisms which can feed on the incoming wastewater Active site: The site on the enzyme at which the substrate binds. Acute toxicity unit: For a given species and a single toxic substance, the 96hr median tolerance limit (TLm). For a mixture of toxicants, any combination of concentrations that would be expected to kill half the individuals of the same species in 96 hr. Acute toxicity: Toxicity resulting from exposure to a toxic substance or stress for a relatively brief period, typically no more than 48–96 hr, but never more than 10% of the natural lifetime of an organism. Acute/chronic ratio: The ratio of the concentration or level of a toxic substance or stress that produces toxic effects after a short period of exposure to the concentration or level of the same substance or stress that produces toxic effects after a long period of exposure. Adaptation: Changes in an organism or population through which they become more suited for living in the current environment. Adaptive radiation: The evolution of new species or sub-species to fill unoccupied ecological niches. Adherent cells: The cells which grow adhering to cell culture vessel and are adherent dependent are called adherent cells. Adverse effect: Any effect that results in functional impairment and/or pathological lesions that may affect the performance of a whole organism or that reduces an organism’s ability to respond to an additional challenge. Aerobe: A microorganism dependent on oxygen for it’s growth. Aerobic composting: A method of composting organic wastes using bacteria that need oxygen. This requires that the waste be exposed to air, either via turning or by forcing air through pipes that pass through the material. Aerosol: Suspension of small solid particles and/or liquid droplets in a gas, usually air. Affinity chromatography: A type of chromatography in which the matrix contains chemical groups that can selectively bind (ligands) to the molecules being purified. Affinity tag: The tagged amino acid sequence which forms a part of the recombinant protein and acts as an identification tag. African trypanosomiasis: African trypanosomiasis is a parasitic disease of people and animals, caused by protozoa of the species Trypanosoma brucei (which includes Trypanosoma gambiense) and transmitted by the tsetse fly. Agarose Gel Electrophoresis: Electrophoresis carried out on agarose gel to separate DNA fragments.

Useful Terms and their Meanings of Environmental Biotechnology

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Agricultural Waste: Solid waste generated by the raising of animals or the production and harvest of crops or trees. Agrobacterium tumefaciens: A rod-shaped bacterium that causes crown gall disease by inserting its DNA into plant cells. Alleles: Alternate forms of a gene or DNA sequence, which occur on either of two homologous chromosomes in a diploid organism. Alternative mRNA splicing: The inclusion or exclusion of different exons to form different mRNA transcripts. Amino acids: The building blocks or monomeric units of protein composed of a free amino (NH2) end, a free carboxyl (COOH) end, and a side group (R). Ampicillin (beta-lactamase): An antibiotic derived from penicillin that prevents bacterial growth by interfering with cell wall synthesis. Amplified Fragment Length Polymorphism (AFLP): A sensitive method for the detection of polymorphism in the genome. It is based on the principle of RFLP and RAPD. Amplify: To increase the number of copies of a DNA sequence, in vivo, by inserting into a cloning vector that replicates within a host cell, or in vitro by polymerase chain reaction (PCR). Anaerobe: An organism that lives and reproduces in the absence of dissolved oxygen, instead, deriving oxygen from the breakdown of complex substances. Anaerobic digestion: A method of composting that does not require oxygen. This composting method produces methane. It is also known as anaerobic composting. It is a stabilization process, reducing odor, pathogens, and mass reduction. Androgenesis: Development of plants from male gametophytes. Aneuploidy: An abnormal condition of chromosomes, differing from the usual diploid constitution. This may be due to a loss or gain of chromosomes. Anneal: The pairing of complementary DNA or RNA sequences, via hydrogen bonding, to form a double-stranded polynucleotide. Most often used to describe the binding of a short primer or probe. Anthrax: Anthrax is an acute disease caused by Bacillus anthracis. It affects both humans and animals and most forms of the disease are highly lethal. Antibiotic resistance : Antibiotic resistance is the ability of a microorganism to withstand the effects of antibiotics. It is a specific type of drug resistance. Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by plasmid exchange. If a bacterium carries several resistance genes, it is called multi-resistant or, informally, a superbug. The term antimicrobial resistance is sometimes used to explicitly encompass organisms other than bacteria.

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Antibiotic resistance: The ability of a microorganism to produce a protein that disables an antibiotic or prevents transport of the antibiotic into the cell. Antibiotic: A class of natural and synthetic compounds that inhibit the growth of or kill other microorganisms. Antibody: An immunoglobulin protein produced by B-lymphocytes of the immune system that binds to a specific antigen molecule. Anticodon: A set of three nucleotides in tRNA molecule that are complementary to a set of three nucleotides (codon) in mRNA. Antigen: Antigen is the protein or polysaccharide which after penetration into a cell can stimulate it to produce antibodies—the proteins (immunoglobulins) which selectively connect with the antigen and inactivate them (antibodies are produced by lymphocyte cells). The microorganisms, and specifically their surface structures, e.g. the O-antigens and endotoxins building the cell wall of gram-negative bacteria, are antigens. Specific antigens are allergens. The name “antigen” is shortened from the Latin anticorporis generator that is literally “the producer of antibodies”. Antigenic determinant: A surface feature of a microorganism or macromolecule, such as a glycoprotein, that elicits an immune response. Antigenic switching: The altering of a microorganism’s surface antigens through genetic rearrangement, to elude detection by the host’s immune system. Antimicrobial agent: Any chemical or biological agent that harms the growth of microorganisms. Antisense RNA: A complementary RNA sequence that binds to a naturally occurring (sense) mRNA molecule, thus blocking its translation. Antisense therapy: The in vivo treatment of a genetic disease by blocking translation (protein synthesis) with a DNA or RNA sequence that is complimentary to specific mRNA. Apoptosis: Programmed cell death ARS: Autonomously Replicating Sequence Asexual reproduction: Non-sexual means of reproduction which can include grafting and budding. Ash: The noncombustible solid by-products of incineration or other burning process. Assisted Reproductive Technology (ART): The manipulations of reproduction in animals and humans. ATP: Adenosine Triphosphate Autoclaving: Sterilization via a pressurized, high-temperature steam process. Autoradiography: The process of detection of radioactively labeled molecules by exposure of an X-ray sensitive film.

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Autosome: A chromosome that is not involved in sex determination. Auxins: A group of plant growth regulators which are involved in cell elongation, root initiation etc., e.g. Indole acetic acid. BAC: Bacterial Artificial Chromosome Bacillus thuringiensis (Bt): A Gram-positive, soil-dwelling bacterium of the genus Bacillus. Additionally, B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surface of plants. B. thuringielnsis was discovered in 1901 in Japan by Ishiwata and 1911 in Germany by Ernst Berliner, who discovered a disease called Schlaffsucht in flour moth caterpillars. B. thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, the cause of anthrax: the three organisms differ mainly in their plasmids. Like other members of the genus, all three are aerobes capable of producing endospores. Zakharyan RA et al. first reported the presence of plasmids in B. thuringiensis and suggested involvement of the plasmids in endospore/crystal formation. They also described the presence of large plasmid in the Cry+ variant of B. thuringiensis Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal dendotoxins (Cry toxins) which are encoded by cry genes. It was determined that the cry genes are harbored in the plasmids in most strains of B. thuringiensis. Cry toxins have specific activities against species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), hymenoptera (wasps, bees, ants and sawflies) and nematodes. Thus, B. thuringiensis serves as an important reservoir of Cry toxins and cry genes for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals the alkaline pH of their digestive tract causes the toxin to become activated. It becomes inserted into the insect’s gut cell membranes forming a pore resulting in swelling, cell lysis and eventually killing the insect. Bacillus: A rod-shaped bacterium. Backcross: Crossing an organism with one of its parent organisms. Backyard Composting: The diversion of food scraps and yard trimmings from the MSW stream through the onsite controlled decomposition of organic matter by micro-organisms (mainly bacteria and fungi) into a humus-like product. Backyard composting is excluded from MSW recycling activities and is considered source reduction because the composted materials never enter the MSW stream. Bacterial Artificial Chromosome (BAC): A vector system based on the F-factor plasmid of E. coli , BAC is used for cloning large (100-300 kb) DNA segments. Bacterial oxidation (BIOX): BIOX is a biohydrometallurgical process developed for precyanidation treatment of refractory gold ores or concentrates. The bacterial culture is a mixed culture of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans. The bacterial oxidation process

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comprises contacting refractory sulfide ROM ore or concentrate with a strain of the bacterial culture for a suitable treatment period under an optimum operating environment. The bacteria oxidise the sulfide minerals, thus liberating the occluded gold for subsequent recovery via cyanidation. Under controlled continuous plant conditions, the number of bacterial cells and their activity is optimized to attain the highest rate of sulfide oxidation. The bacteria require a very acidic environment, a temperature of between 30 and 45°C, and a steady supply of oxygen and carbon dioxide for optimum growth and activity. The unusual operating conditions for the bacteria are not favourable for the growth of most other microbes, thus eliminating the need for sterility during the bacterial oxidation process. Because organic substances are toxic to the bacteria, they are non-pathogenic and incapable of causing disease. The bacteria employed in the process do not, therefore, pose a health risk to humans or animals. The bacterial oxidation of iron sulfide minerals produces iron(III) sulfate and sulfuric acid, and in the case of arsenopyrite, arsenic acid is also produced. The arsenic is removed from the liquor by coprecipitation with the iron and sulfate in a two-stage neutralization process. This produces a solid neutralization precipitate containing largely calcium sulfate, basic iron(III) arsenate and iron(III) hydroxide. The iron(III) arsenate is sufficiently insoluble and stable to allow the neutralisation product to be safely disposed of on a slimes dam. The neutralization liquor, purified to contain an acceptable level of arsenic, can be re-used in the milling, flotation or bacterial oxidation circuits. Bacteriocide: A class of antibiotics that kills bacterial cells. Bacteriocins : Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Bacteriocins were first discovered by A. Gratia in 1925. He was involved in the process of searching for ways to kill bacteria, which also resulted in the development of antibiotics and the discovery of bacteriophage, all within a span of a few years. He called his first discovery a colicine because it killed E. coli. Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. There are several large categories of bacteriocin which are only phenomenologically related. These include the bacteriocins from gram-positive bacteria, the colicins, the microcins, and the bacteriocins from Archaea. The bacteriocins from E. coli are called colicins. They are the longest studied bacteriocins. They are a diverse group of bacteriocins and do not include all the bacteriocins produced by E. coli. For example, the bacteriocins produced by Staphylococcus warneri, are called as warnerin or warnericin. In fact; one of the oldest known so-called colicins was called colicin V and is now known as microcin V. It is much smaller and produced and secreted in a different manner than the

Useful Terms and their Meanings of Environmental Biotechnology

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classic colicins. The bacteriocins of lactic acid fermenting bacteria are called lantibiotics. This naming system is problematic for a number of reasons. First, naming bacteriocins by what they putatively kill would be more accurate if their killing spectrum were contiguous with genus or species designations. The bacteriocins frequently possess spectra that exceed the bounds of their named taxa and almost never kill the majority of the taxa for which they are named. Further, the original naming is generally derived not from the sensitive strain the bacteriocin kills, but instead the organism that produces the bacteriocin. Bacteriophage (phage or phage particle): A virus that infects bacteria. Altered forms are used as vectors for cloning DNA. Bacteriostat: A class of antibiotics that prevents growth of bacterial cells. Bacterium: A single-celled, microscopic prokaryotic organism: a single cell organism without a distinct nucleus. Bacteroidetes : Bacteroidetes is composed of three large classes of bacteria that are widely distributed in the environment, including in soil, in sediments, sea water and in the guts of animals. By far, the Bacteroidales class are the most well-studied, including the genus Bacteroides (an abundant organism in the feces of warmblooded animals including humans), and Porphyromonas, a group of organisms inhabiting the human oral cavity. Members of the genus Bacteroides are opportunistic pathogens. Rarely are members of the other two classes pathogenic to humans. Researcher Jeffrey Gordon and his colleagues found that obese humans and mice had intestinal flora (gut flora) with a lower percentage of Bacteroidetes and relatively more bacteria from the Firmicutes family. However, they are unsure if Bacteroidetes prevent obesity or if these intestinal flora are merely preferentially selected by intestinal conditions in those who are not obese. Bag-house: A combustion plant emission control device that consists of an array of fabric filters through which flue gases pass in an incinerator flue. Particles are trapped and thus prevented from passing into the atmosphere. Baker’s yeast: The living cells of aerobically grown yeast, Saccharomyces cerevisiae, used in bread making. Base Pair (bp): A pair of complementary nitrogenous bases (nucleotides) in a DNA molecule—adenine-thymine and guanine-cytosine. Also, the unit of measurement for DNA sequences. Base ratio: The ratio of A to T, or C to G in a double-stranded DNA. Basel Convention: An international agreement on the control of transboundary movements of hazardous wastes and their disposal, drawn up in March 1989 in Basel, Switzerland, with over 100 countries as signatories. Batch culture: Batch culture is a closed culture system containing limited amount of nutrients. Bergmann’s plating technique: The most widely used method for culture of isolated single plant cells.

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beta-DNA: The normal form of DNA found in biological systems, which exists as a right-handed helix. beta-Lactamase: Ampicillin resistance gene. Bio concentration factor (BCF): The biological accumulation factor associated with direct uptake of a substance from the water in the absence of any possible intake through the food chain. Bioaccumulation: It’s studies address the buildup of bioaccumulative compounds through biomagnification and/or bioconcentration. Bioaccumulation means an increase in the concentration of a chemical in a biological organism over time, compared to the chemical’s concentration in the environment. Compounds accumulate in living things any time they are taken up and stored faster than they are broken down (metabolized) or excreted. Understanding the dynamic process of bioaccumulation is very important in protecting human beings and other organisms from the adverse effects of chemical exposure, and it has become a critical consideration in the regulation of chemicals. Bioaugmentation: It is the introduction of a group of natural microbial strains or a genetically engineered variant to treat contaminated soil or water. Usually, the steps involve studying the indigenous varieties present in the location to determine if biostimulation is possible. If the indigenous variety do not have the metabolic capability to perform the remediation process, exogenous varieties with such sophisticated pathways are introduced. Bioaugmentation is commonly used in municipal wastewater treatment to restart activated sludge bioreactors. At sites where soil and groundwater are contaminated with chlorinated ethenes, such as tetrachloroethylene and trichloroethylene, bioaugmentation is used to ensure that the in situ microorganisms can completely degrade these contaminants to ethylene and chloride, which are non-toxic. Bioaugmentation is typically only applicable to bioremediation of chlorinated ethenes, although there are emerging cultures with the potential to biodegrade other compounds including chloroethanes, chloromethanes, and MTBE. The first reported application of bioaugmentation for chlorinated ethenes was at Kelly Air Force Base, TX. Bioaugmentation is typically performed in conjunction with the addition of electron donor (biostimulation) to achieve geochemical conditions in groundwater that favor the growth of the dechlorinating microorganisms in the bioaugmentation culture. Bioavailability: The availability of chemicals to degradative microorganisms. Biocenosis: Biocenosis (microbiocenosis) is the association of organisms (of microorganisms) which reside in a common biotop (the living environment), between which the various interactions are formed (food web, for example). The biocenosis along with the biotope creates together the ecological system called ecosystem.

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Biochemical Oxygen Demand (BOD): The oxygen required to meet the metabolic needs of aerobic organisms in water containing organic compounds. Biocomposting: It involves combining organic materials under conditions that enables them to decompose more quickly than they would in nature. Think about logs and leaves on the ground in a forest. The leaves will break down and disappear within a year. Logs, of course, will take much longer to crumble away. Composting involves combining organic materials under conditions that enables them to decompose more quickly than they would in nature. Biodegradable material: Any organic material that can be broken down by microorganisms into simpler, more stable compounds. Most organic wastes (e.g., food, paper) are biodegradable. Biodegradable matter: Organic matter that can be broken down by bacteria or other microorganisms to more stable forms which will not create a nuisance or give off foul odors. Biodegradation: It is nature’s way of recycling wastes, breaking down organic matter into nutrients that can be used by other organisms. “Degradation” means decay, and the prefix “bio-” means that the decay is carried out by a huge assortment of bacteria, fungi, maggots, worms, and other organisms that eat dead material and recycle it into new forms. In nature, there is no waste because everything gets recycled. The waste products from one organism become the food for others, providing nutrients and energy while breaking down the waste organic matter. Some organic materials will break down much faster than others, but all will eventually decay. By harnessing these natural forces of biodegradation, people can reduce wastes and clean up some types of environmental contaminants. Through composting, we accelerate natural biodegradation and convert organic wastes to a valuable resource. Biodiesel: Biodiesel is a fuel comprised of mono-alkyl esters of long chain fatty acids derived from biologically produced oils or fats including vegetable oils, animal fats and microalgal oils. It is a cleaner alternative fuel with combustion properties very similar to petroleum diesel, most often used as an additive to improve the lubricity of pure ultra-low sulfur petrodiesel fuel. Biodiversity: The multitude of different living beings in a particular ecosystem or on the whole earth. Bioenergy: In recent decades, efforts were made for evolving were nonpolluting bioenergy sources or energy generation from organic waste or biomass. These are all eco-friendly solution. Biomass energy supply demand balances have become a component of energy sector analysis and planning and assumed greater importance in countries. These are variety of biological energy sources. Biomass, Biogas, Hydrogen are the example of Bioenergy. Bio-enrichment: Adding nutrients or oxygen to increase microbial breakdown of pollutants.

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Biofertilizer: To reduce the impact of excess chemical fertilizers in the field of agriculture the biofertilizer is a potential too; biologically fixed nitrogen is such a source which can supply an adequate amount of Nitrogen to plants and other nutrients to some extent. Many free-living and symbiotic bacteria, which fix atmospheric Nitrogen were used as biofertilizer material as a substitute for Nitrogen fertilizer. In general, two types of biofertilizers are used: 1. Bacterial Biofertilizer, 2. Algal Biofertilizer. Biofilm: A biofilm is a structured community of microorganisms encapsulated within a self-developed polymeric matrix and adherent to a living or inert surface. Biofilms are also often characterized by surface attachment, structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances. Biofilteration: The process of removing complex wastes from domestic and industrial sources by using microorganisms. Biofuel: The term biofuel is attributed to any alternative fuel that derives from organic material, such as energy crops (corn, wheat, sugar cane, sugar beet, cassava, among others), crop residues (e.g. rice straw, rice husk, corn stover, corn cobs) or waste biomass (for instance, food waste, livestock waste, paper waste, construction-derived wood residues and others). Biohazards: The accidents or risks associated with biological materials. Biohydrometallurgy, biomining, bioleaching: A method of mining and extracting metals from ores by using microorganisms. Bioinformatics: Bioinformatics is an interdisciplinary field, which addresses biological problems using computational techniques. The field is also often referred to as computational biology. It plays a key role in various areas such as functional genomics, structural genomics, and proteomics, and forms a key component in the biotechnology and pharmaceutical sector. Bioleaching: The use of bacteria to recover valuable metals from ores. Biolistics: The process of introducing DNA into plants and animal cells, and organelles by bombardment of DNA-coated pellets under pressure at high speed. This is also called as ‘microprojectile bombardment’. Biological accumulation factor: The ratio of the concentration of a substance in one or more tissues of an aquatic organism to the concentration of the same substance in the water in which the organism has been living. Biological warfare (BW): It is the use of pathogens (bacteria, viruses, or other disease causing agents) as biological weapons (or bioweapons). Biologics: Agents, such as vaccines, that gives immunity from diseases or harmful biotic stresses. Biomarker: It is a biological response to a chemical that gives a measure of exposure and, sometimes, of toxic effect. Biological markers found in crude oils and source rock extracts can provide molecular evidence of the correlation among oils and their sources.

Useful Terms and their Meanings of Environmental Biotechnology

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Biomass: The total dry weight of all organisms in a particular sample, population, or area. (or) The organic mass that can be used as a source of energy. Biometry: Application of statistical methods to study biological problems. Biomining: Biomining is an approach to the extraction of desired minerals from ores being explored by the mining industry in the past few years. Microorganisms are used to leach out the minerals, rather than the traditional methods of extreme heat or toxic chemicals, which have a deleterious effect on the environment. Biopesticide: Pest control by biological antagonism appears to be very useful tool in recent years. Bacterial pesticides are being developed. Heliothis complex, which lives in close association with plant roots, consists of two major crop pests-budworm and ball warm. Biological insecticides against both these insects are being prepared by transfer of a gene from Bacillus thuringiensis. Biopesticides: The toxic compounds produced by living organisms that can specifically kill a particular pest species. Bioprocess technology: A more recent usage to replace fermentation technology that involves large scale cultivation of microorganisms for industrial purposes. Bioreactor: A growth chamber or a vessel for cells or microorganisms. The cells or cell extracts carry out biological reactions in a bioreactor. Bioremediation: It is a clean-up technology that uses naturally occurring microorganisms to degrade hazardous substances into less toxic or nontoxic compounds. These microorganisms may: Ingest and degrade organic substances as their food and energy source, degrade organic substances such as chlorinated solvents or petroleum products, that are hazardous to living organisms, including humans, and degrade the organic contaminants into inert products. Because the microorganisms already occur naturally in the environment, they pose no contamination risk. Biosensor: Biosensor represents biophysical devices which will detect the presence and measure the quantities of specific substances in a variety of environments. These specific substances may include sugars, proteins, or humas and a variety of toxins in the industrial effluents. In designing a biosensor, an enzyme or an antibody or even microbial cells are associated with microchip devices which are used for quantitative estimate of a substance. Biosorption: The process of microbial cell surface adsorption of metals. Biosphere: The biosphere is the global sum of all ecosystems. It can also be called the zone of life on Earth. From the broadest biophysiological point of view, the biosphere is the global ecological system integrating all living beings and their relationships, including their interaction with the elements of the lithosphere, hydrosphere, and atmosphere.

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Biostimulation: Addition of specific nutrients to enhance the growth of naturally occurring microorganisms that convert toxic compounds to nontoxic compounds. Biotechnology: The applications of biological principles, organisms and products to practical purposes. Biotic stress: Living organisms which can harm plants, such as viruses, fungi, and bacteria, and harmful insects. Biotin: A non-radioactive label used for labeling probes, detected through a cytochemical reaction Biotransformation: This is a process of biological changes of complex compound to simpler, toxic to non-toxic, or vice-versa. Several microorganisms are capable of transforming a variety of compounds found in nature but generally, with respect to synthetic compounds, they are unable to show any appropriate action. Biotransfer appears to be one of the major detoxication methods known so far. BLAST: Basic Local Alignment Search Tool BOD: Biochemical Oxygen Demand – the amount of oxygen consumed by water microorganisms in breaking down the organic matter. Bottom ash: Relatively coarse, noncombustible, and generally toxic residue of incineration that accumulates on the grate of a furnace. Brewer’s Yeast: A strain of yeast usually belonging to Saccharomyces cerevisiae that is used for the production of beer. Brewing: Brewing is the production of alcoholic beverages and alcohol fuel through fermentation. Broth: Any fluid medium supporting the growth of microorganisms. Bt. Plants: The plants carrying the toxin producing gene from Bacillus thuringiensis, and capable of protecting themselves from insect attack. Bubonic plague: It is the best known manifestation of the bacterial disease plague, caused by the bacterium Yersinia pestis (formerly known as Pasteurella pestis). Bulky waste: Large wastes such as appliances, furniture, and trees and branches, that cannot be handled by normal MSW processing methods. Butanol: Butanol is a four-carbon alcohol. It can be produced via clostridial fermentation. Callus: A mass of undifferentiated plant tissues formed from plant cells or tissue cuttings when grown in culture. Capsid: See Coat protein. Carcinogen: A substance that induces cancer. Carcinogens: Carcinogens is the chemical, physical and biological agents causing cancer (tumour). Most carcinogens are mutagens, because a first step

Useful Terms and their Meanings of Environmental Biotechnology

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of the carcinogenesis (process creating a cancer) is the mutation in the gene controlling cell divisions. Carcinoma: A malignant tumor derived from epithelial tissue, which forms the skin and outer cell layers of internal organs. Casettee mutagenesis: Replacement of a wild type DNA by a synthetic double-stranded oligonucleotide (a small DNA fragment). Catalyst: A substance that promotes a chemical reaction by lowering the activation energy of a chemical reaction, but which itself remains unaltered at the end of the reaction. Catalytic antibody (abzyme): An antibody selected for its ability to catalyze a chemical reaction by binding to and stabilizing the transition state intermediate. Catalytic RNA (ribozyme): A natural or synthetic RNA molecule that cuts an RNA substrate. Cation: A positively charged ion. cDNA Library: A library composed of complementary copies of cellular mRNAs. cDNA: Complimentary DNA i.e. DNA produced by reverse transcription from mRNA by the enzyme reverse transcriptase. Cell culture: The culture of dispersed (or disaggregated) cells obtained from the original tissue, or from a cell line. Cell lines: Animal or plant cells that can be cultivated under laboratory conditions. Cell: The basic unit by which a landfill is developed. It is the general area where incoming waste is tipped, spread, compacted, and covered. Cell-mediated immune response: The activation of the T-lymphocytes of the immune system in response to a foreign antigen. Cellular oncogene (proto-oncogene): A normal gene that when mutated or improperly expressed contributes to the development of cancer. Cellulose: It is a polymer of glucose. Unlike starch, the glucose monomers of cellulose are linked together through â-1-4 glycosidic bonds by condensation resulting in tightly packed and highly crystalline structures that are resistant to hydrolysis. Cellulosic ethanol : Cellulosic ethanol is a biofuel produced from wood, grasses, or the non-edible parts of plants. It is a type of biofuel produced from lignocellulose, a structural material that comprises much of the mass of plants. Lignocellulose is composed mainly of cellulose, hemicellulose and lignin. Corn stover, switchgrass, miscanthus, woodchips and the byproducts of lawn and tree maintenance are some of the more popular cellulosic materials for ethanol production. Production of ethanol from lignocellulose has the advantage of abundant and diverse raw material compared to sources like

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Environmental Biotechnology

corn and cane sugars, but requires a greater amount of processing to make the sugar monomers available to the microorganisms that are typically used to produce ethanol by fermentation. Centers of origin: Usually the location in the world where the oldest cultivation of a particular crop has been identified. Central dogma: Francis Crick’s seminal concept that in nature genetic information generally flows from DNA to RNA to protein. Centrifugal extractor: A method of solvent extraction that uses the principle of centrifugal forces. Centrifugation: Separating molecules by size or density using centrifugal forces generated by a spinning rotor. G forces of several hundred thousand times gravity are generated in ultracentrifugation. Centromere: The central portion of the chromosome to which the spindle fibers attach during mitotic and meiotic division. Chemical Precipitation: Precipitation of metals is achieved by the addition of coagulants such as alum, lime, iron salts and other organic polymers. The large amount of sludge containing toxic compounds produced during the process is the main disadvantage. Chemoautotrophs: Chemoautotrophs generally only use inorganic energy sources. Most are bacteria or archaea that live in hostile environments such as deep sea vents and are the primary producers in such ecosystems. Evolutionary scientists believe that the first organisms to inhabit earth were chemoautotrophs that produced oxygen as a by-product and later evolved into both aerobic, animal-like organisms and photosynthetic, plant-like organisms. Chemoautotrophs generally fall into several groups : methanogens, halophiles, sulfur reducers, nitrifiers, anammoxbacteria and thermoacidophiles. Chemocar: A special vehicle for the collection of toxic and hazardous wastes from residences, shops, and institutions. Chemotherapy: A treatment for cancers that involves administering chemicals toxic to malignant cells. Chemotrophs: Chemotrophs are organisms that obtain energy by the oxidation of electron donating molecules in their environments. These molecules can be organic (organotrophs) or inorganic (lithotrophs). The chemotroph designation is in contrast to phototrophs which utilize solar energy. Chemotrophs can be either autotrophic or heterotrophic. Chimera: A recombinant DNA molecule that contains sequences from different organisms. Chimeric antibodies: Antibodies in which the individual polypeptide chains are composed of segments from two different species (usually man and mouse). Chloramphenicol: An antibiotic that interferes with protein synthesis.

Useful Terms and their Meanings of Environmental Biotechnology

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Chromatid: Each of the two daughter strands of a duplicated chromosome joined at the centromere during mitosis and meiosis. Chromatography: An analytical technique dealing with the separation of closely related compounds from a mixture. Chromosome walking: It is a technique used to identify the overlapping sequences of DNA in a chromosome in order to identify a particular locus of interest. Chromosome: A single DNA molecule, a tightly coiled strand of DNA, condensed into a compact structure, in vivo by complexing with accessory histones or histone-like proteins. Chromosomes exist in pairs in higher eukaryotes. Cistron: A DNA sequence that codes for a specific polypeptide; a gene. Cleaner production : Processes designed to reduce the wastes generated by production. Clone: All the individuals derived by asexual reproduction from a single original individual. In molecular biology, a strain of organism that carries a particular DNA sequence. Cloning vector: A plasmid or a phage that carries an inserted foreign DNA to be introduced into a host cell. Cloning: The mitotic division of a progenitor cell to give rise to a population of identical daughter cells or clones. Coat protein (Capsid): The coating of a protein that encloses the nucleic acid core of a virus. Co-disposal: The disposal of different types of waste in one area of a landfill or dump. For instance, sewage sludges may be disposed of with regular solid wastes. Codon: A triplet nucleotide sequence of mRNA coding for an amino acid in a polypeptide. Coenzyme (Cofactor): An organic molecule, such as a vitamin, that binds to an enzyme and is required for its catalytic activity. Cogeneration: Production of both electricity and steam from one facility, from the same fuel source. Coliform index: It is a rating of the purity of water based on a count of fecal bacteria. Coliform bacteria are microorganisms that primarily originate in the intestines of warm-blooded animals. By testing for coliforms, especially the well known E.Coli, which is a thermotolerant coliform, one can determine if the water has probably been exposed to fecal contamination; that is, whether it has come in contact with human or animal feces. It is important to know this because many disease-causing organisms are transferred from human and animal feces to water, from where they can be ingested by people and infect them. Water that has been contaminated by feces usually contains pathogenic

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Environmental Biotechnology

bacteria, which can cause disease. Some types of coliforms cause disease, but the coliform index is primarily used to judge if other types of pathogenic bacteria are likely to be present in the water. The coliform index is used because it is difficult to test for pathogenic bacteria directly. There are many different types of disease-causing bacteria, and they are usually present in low numbers which do not always show up in tests. Thermotolerant coliforms are present in higher numbers than individual types of pathogenic bacteria and they can be tested for relatively easily. Collection: The process of picking up wastes from residences, businesses, or a collection point, loading them into a vehicle, and transporting them to a processing, transfer, or disposal site. Colony hybridization: A technique that employs nucleic acid probe to identify a bacterial colony with a vector carrying specific gene (s). Colony: Colony is the group of cells visible with the naked eye on the solid medium (e.g. the agar medium) formed by cells originating from the initial unit—which can be one or more cells. If this is from a single cell then the colony is the pure strain. Combustibles: Burnable materials in the waste stream, including paper, plastics, wood, and food and garden wastes. Combustion Ash: Residual substance produced during the burning, combustion or oxidation of waste materials. Combustion: In context of Municipal Solid Waste Management, the burning of materials in an incinerator. Commensalism: The close association of two or more dissimilar organisms where the association is advantageous to one and doesn’t affect the other(s). Commercial Waste: Waste generated by businesses, such as office building, retail and wholesale establishments, and restaurants. Examples include cardboard, food scraps, office paper, disposable tableware, paper napkins and yard trimmings. Commingled Recyclables: A mixture of several recyclable materials. Commingled: Mixed recyclables that are collected together after having been separated from mixed Municipal Solid Waste. Communal collection: A system of collection in which individuals bring their waste directly to a central point, from which it is collected. Compactor vehicle: A collection vehicle using high-power mechanical or hydraulic equipment to reduce the volume of solid waste. Competence: Ability of a bacterial cell to take in DNA Competency: An ephemeral state, induced by treatment with cold cations, during which bacterial cells are capable of up taking foreign DNA. Complementary DNA or RNA. The matching strand of a DNA or RNA molecule to which its bases pair.

Useful Terms and their Meanings of Environmental Biotechnology

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Complementary Nucleotides: Members of the pairs adenine-thymine, adenine-uracil, and guanine-cytosine that have the ability to hydrogen bond to one another. Composite liner: A liner system for a landfill consisting of an engineered soil layer and a synthetic sheet of material. Compost: The material resulting from composting. Compost, also called humus, is a soil conditioner and in some instances is used as a fertilizer. Composting Facility: Offsite facility where the organic component of municipal solid scraps is biologically decomposed under controlled conditions; an aerobic process in which organic materials are ground or shredded and then decomposed to humus in windrow piles or in mechanical digesters, drums or similar enclosures. Composting: Biological decomposition of solid organic materials by bacteria, fungi, and other organisms into a soil-like product. Concatemer: A DNA segment composed of repeated sequences linked end to end. Conjugation: The joining of two bacteria cells when genetic material is transferred from one bacterium to another. Constitutive promoter. An unregulated promoter that allows for continual transcription of its associated gene. (See Promoter.) Construction & Demolition (C&D) Debris: Waste that is generated during construction, remodeling, repair, or demolition of buildings, bridges, pavements and other structures. C&D debris includes concrete, asphalt, lumber, steel girders, steel rods, wiring, dry wall, carpets, window glass, metal and plastic piping, tree stumps, soil and other miscellaneous items related to the activities listed, including natural disaster debris. These efforts are excluded from calculating the MSW recycling rate. Construction and demolition debris: Waste generated by construction and demolition of buildings, such as bricks, concrete, drywall, lumber, miscellaneous metal parts and sheets, packaging materials, etc. Contigs: These are continuous (contiguous) sequences which have overlapping regions on either ends. Contiguous (Contig) Map: The alignment of sequence data from large, adjacent regions of the genome to produce a continuous nucleotide sequence across a chromosomal region. Continuous cell lines: The cell lines that get transformed, and under in vitro conditions grow continuously, are called Continuous cell lines. These cells show no contact inhibition and no anchorage dependence. Controlled dump: A planned landfill that incorporates to some extent some of the features of a sanitary landfill: siting with respect to hydrogeological suitability, grading, compaction I some cases, leachate control, partial gas management, regular (not usually daily) cover, access control, basic record-keeping, and controlled waste picking.

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Environmental Biotechnology

Copy DNA: See cDNA. Cosmid: A hybrid vector of plasmid and phage DNA; contains specific sequence called as cos sites of phage DNA. Cross-Hybridization: The hydrogen bonding of a single-stranded DNA sequence that is partially, but not entirely, complementary to a single-stranded substrate. Often, this involves hybridizing a DNA probe for a specific DNA sequence to the homologous sequences of different species. Crossing-Over: The exchange of DNA sequences between chromatids of homologous chromosomes during meiosis. Cross-Pollination: Fertilization of a plant from a plant with a different genetic makeup. Crumb Rubber: Ground rubber pieces used in rubber or plastic products, or processed further into reclaimed rubber or asphalt products. Cryopreservation: Storage and preservation at very low temperatures (-1960C). Cryoprotectant: A chemical agent or a compound that can prevent damage to cells while they are frozen or defrosted. Culture medium: The nutrients prepared in the form of a fluid (broth) or solid for the growth of cells/tissues in the laboratory. Culture: A population of plant or animal cells/microorganisms that are grown under controlled conditions. Curbside collection: Collection of compostables, recyclables, or trash at the edge of a sidewalk in front of a residence or shop. Curing: Allowing partially composted materials to sit in a pile for a specified period of time as part of the maturing process in composting. Cybridization: The process of formation of cybrids. Cybrids: The cytoplasmic hybrids obtained by the fusion of enucleated and nucleated protoplasts are called Cybrids. Cyclic AMP (Cyclic Adenosine Monophosphate): A second messenger that regulates many intracellular reactions by transducing signals from extracellular growth factors to cellular metabolic pathways. Cystic fibrosis: A disease affecting lungs and other tissues due to defects in ion transport. It is caused by the deficiency of CFTR gene. Cytogenetics: Study that relates the appearance and behavior of chromosomes to genetic phenomenon. Cytokines: Various chemicals produced in the body which mediate immunological responses. Cytotoxicity: The toxic effects on cells that result in metabolic alterations including the death of cells.

Useful Terms and their Meanings of Environmental Biotechnology

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Dalton: A unit of measurement equal to the mass of a hydrogen atom, 1.67 x 10E-24 gram/L (Avogadro’s number). Death phase: The final growth phase, during which nutrients have been depleted and cell number decreases. Decomposers: Decomposers (or saprotrophs) are organisms that consume dead or decaying organisms, and, in doing so, carry out the natural process of decomposition. Like herbivores and predators, decomposers are heterotrophic, meaning that they use organic substrates to get their energy, carbon and nutrients for growth and development. Decomposers use deceased organisms and non-living organic compounds as their food source. The primary decomposers are bacteria and fungi. Bacteria are the primary decomposers of dead animals (carrion) and are the primary decomposers of dead plant matter (litter) in some ecosystems. In soils, active fungal hyphae and bacteria are much more important in the recycling of nutrients. Bacteria can also be very important in agricultural fields, because tillage usually increases the abundance of bacteria relative to fungi. Fungi are the primary decomposers of litter in many ecosystems. Unlike bacteria, which are unicellular, most saprotrophic fungi grow as a branching network of hyphae. While bacteria are restricted to growing and feeding on the exposed surfaces of organic matter, fungi can use their hyphae to penetrate larger pieces of organic matter. Additionally, only fungi have evolved the enzymes necessary to decompose lignin, a chemically complex substance found in wood. These two factors make fungi the primary decomposers in forests, where litter has high concentrations of lignin and often occurs in large pieces. Some animals, like millipedes, woodlice, and various worms are commonly called decomposers, because such animals consume dead organic matter and contribute to the process of decomposition. Scientists, however, refer to such organisms as detritivores. This distinction is made because bacteria and fungi are capable of digesting many complex chemical molecules that animals are incapable of digesting. Additionally, bacteria and fungi digest and decompose organic matter more fully than detritivores, reducing it to inorganic material. For these reasons, bacteria and fungi play a more fundamental role in the processes of decomposition and nutrient recycling than animals. Decomposition: Decomposition refers to the process by which tissues of dead organisms break down into simpler forms of matter. Such a breakdown of dead organisms is essential for new growth and development of living organisms because it recycles the finite chemical constituents and frees up the limited physical space in the biome. Bodies of living organisms begin to decompose shortly after death. It is a cascade of processes that go through distinct phases. It may be categorized in two stages by the types of end products. The first stage is limited to the production of vapors. The second stage is characterized by the formation of liquid materials; flesh or plant matter begin to decompose. The science which studies such decomposition generally is called taphonomy from the Greek word taphos—which means grave.

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Environmental Biotechnology

Denature: To induce structural alterations that disrupt the biological activity of a molecule. Often, refers to breaking hydrogen bonds between base pairs in double-stranded nucleic acid molecules to produce in single-stranded polynucleotides or altering the secondary and tertiary structure of a protein, destroying its activity. Density gradient centrifugation: High-speed centrifugation in which molecules “float” at a point where their density equals that in a gradient of cesium chloride or sucrose. (See Centrifugation.) Diabetes: A disease associated with the absence or reduced levels of insulin, a hormone essential for the transport of glucose to cells. Diazotrophs: The microorganisms involved in diazotrophy. Dideoxynucleotide (didN): A deoxynucleotide that lacks a 3’ hydroxyl group, and is thus unable to form a 3’-5’ phosphodiester bond necessary for chain elongation. Dideoxynucleotides are used in DNA sequencing and the treatment of viral diseases. Digest: To cut DNA molecules with one or more restriction endonucleases. Diploid cell: A cell which contains two copies of each chromosome. Directional cloning: DNA insert and vector molecules are digested with two different restriction enzymes to create non-complementary sticky ends at either end of each restriction fragment. This allows the insert to be ligated to the vector in a specific orientation and prevents the vector from recircularizing. (See Cloning.) Disinfection byproducts: Side reactions can occur in water when chemical oxidants such as chlorine and ozone are used to control potentially pathogenic microorganisms. These reactions can form low levels of disinfection byproducts, several of which have been regulated for potential adverse human health effects. Disinfection of water: Water systems add disinfectants to destroy microorganisms that can cause diseases in humans. Primary methods of disinfection include chlorination, chloramines, chlorine dioxide, ozone, and ultraviolet light. Disposal Facilities: Repositories for solid waste including landfills and combustors intended for permanent containment or destruction of waste material; excludes transfer stations and composting facilities. Disposal: The final handling of solid waste, following collection, processing, or incineration. Disposal most often means placement of wastes in a dump or a landfill. Distillation: Distillation is a method of separating chemical compounds based on their differences in volatility; and volatility is a measure of the speed at which a chemical compound evaporates. Diversion rate: The proportion of waste material diverted for recycling, composting, or reuse and away from landfilling or incineration.

Useful Terms and their Meanings of Environmental Biotechnology

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DMSO: Dimethyl sulfoxide DNA (Deoxyribonucleic acid): An organic acid and polymer composed of four nitrogenous bases—adenine, thymine, cytosine, and guanine linked via intervening units of phosphate and the pentose sugar deoxyribose. DNA is the genetic material of most organisms and usually exists as a double-stranded molecule in which two anti-parallel strands are held together by hydrogen bonds between adenine-thymine and cytosine-guanine. DNA Diagnosis: The use of DNA polymorphisms to detect the presence of a disease gene. DNA Fingerprint: The unique pattern of DNA fragments identified by Southern hybridization (using a probe that binds to a polymorphic region of DNA) or by polymerase chain reaction (using primers flanking the polymorphic region). DNA Fingerprinting: A technique for the identification of individuals based on the small differences in DNA sequences. DNA Hybridization: The pairing of two DNA molecules used to detect the specific sequence in the sample DNA. DNA Marker: A DNA sequence that exists in two or more readily identifiable forms (polymorphic forms) which can be used to mark a mal position on a genome map. DNA Polymorphism: One of two or more alternate forms (alleles) of a chromosomal locus that differ in nucleotide sequence or have variable numbers of repeated nucleotide units. DNA Probe: A segment of DNA that is tagged with a label (i.e. isotope) so as to detect a complementary base sequence in the DNA sample after a hybridization reaction. DNA Profiling: The term used to describe different methods for the analysis of DNA to establish the identity of an individual. DNA Repair: The biochemical processes that correct mutations occurring due to replication errors or as a consequence of mutagenic agents. DNA Sequencing: Procedures for determining the nucleotide sequence of a DNA fragment. DNAse: Deoxyribonuclease Dolly: The first mammal (sheep) cloned by Wilmut and Campbell in 1997. Dominant (-acting) Oncogene: A gene that stimulates cell proliferation and contributes to oncogenesis when present in a single copy. Dominant gene: A gene whose phenotype is when it is present in a single copy. Dominant: An allele is said to be dominant if it expresses its phenotype even in the presence of a recessive allele.

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Dormancy: A period in which a plant does not grow, awaiting necessary environmental conditions such as temperature, moisture, nutrient availability. Double helix: Describes the coiling of the anti-parallel strands of the DNA molecule, resembling a spiral staircase in which the paired bases form the steps and the sugar-phosphate backbones form the rails. Double-stranded complementary DNA (dscDNA): A duplex DNA molecule copied from a cDNA template. Drop-Off Center: Method of collection whereby recyclable or compostable materials are taken by individuals to a collection site and placed in designated containers. These can be staffed or unstaffed. Dump: See controlled dump and open dump. Duplex DNA: Double-stranded DNA. Ecology: The study of the interactions of organisms with their environment and with each other. Ecosystem: The organisms in a plant population and the biotic and abiotic factors which impact on them. Edaphic: (i) Of, or pertaining to the soil. (ii) Resulting from, or influenced by factors inherent in the soil or other substrate, rather than by climatic factors. Edible vaccines: The vaccines produced in plants which can enter the body on eating them. Electrodialysis: In this process, the ionic components (heavy metals) are separated through the use of semi-permeable ion-selective membranes. Application of an electrical potential between the two electrodes causes a migration of cations and anions towards respective electrodes. Because of the alternate spacing of cation- and anion-permeable membranes, cells of concentrated and dilute salts are formed. The disadvantage is the formation of metal hydroxides, which clog the membrane. Electrophoresis: Electrophoresis is the technique of separation of macromolecules (nucleic acids, proteins) within the electric field created between the anode and the cathode. The molecules with the negative charge migrate to the anode, and molecules with the positive charge—to the cathode. The separation is achieved due to differences in the speed of the migration of ions, which in turn depends on their mass, shape and on the charge. Electroporation: The technique of introducing DNA into cells by inducing transient pores by electric pulse. Electrowinning: The final method of extracting the metal, by using an electrochemical cell. ELISA: Enzyme Linked Immunosorbent Assay. A technique for the detection of small quantities of proteins by utilizing antibodies linked to enzymes, which in turn catalyze the formation of coloured products.

Useful Terms and their Meanings of Environmental Biotechnology

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EMBL: European Molecular Biology Laboratory Embryo rescue: The culture of immature embryos to rescue them from unripe or hybrid seeds which fail to germinate. Embryo transfer: The process of implantation of embryos from a donor animal, or developed by in vitro fertilization into the uterus of a recipient animal. Embryonic Stem cells (ES CELLS): The cells of an early embryo that can give rise to all differentiated cells, including germ cells. Emissions: Gases released into the atmosphere. Encapsidation: Process by which a virus’ nucleic acid is enclosed in a capsid. End User: Facilities that purchase or secure recovered materials for the purpose of recycling. Examples include recycling plants and composting facilities; excludes waste disposal facilities. Endolith : Endolith is an organism (archaeum, bacterium, fungus, lichen, alga or amoeba) that lives inside rock, coral, animal shells, or in the pores between mineral grains of a rock. Endonuclease. See Nuclease. Endophyte: An organism that lives inside another. Endosymbiont : It is any organism that lives within the body or cells of another organism, i.e. forming an endosymbiosis. Examples are nitrogenfixing bacteria (called rhizobia) which live in root nodules on legume roots, single-celled algae inside reef-building corals, and bacterial endosymbionts that provide essential nutrients to about 10%-15% of insects. Many instances of endosymbiosis are obligate, that is either the endosymbiont or the host cannot survive without the other such as the gut-less marine worms of the genus Riftia, which get nutrition from their endosymbiotic bacteria. The most common examples of obligate endosymbiosis are mitochondria and chloroplasts. However, not all endosymbioses are obligate. Also, some endosymbioses can be harmful to either of the organisms involved. Energy recovery: The process of extracting useful energy from waste, typically from the heat produced by incineration or via methane gas from landfills. Entrez: This is an integrated data base retrieval system for obtaining comprehensive information on a given biological question Environmental impact assessment (EIA): An evaluation designed to identify and predict the impact of an action or a project on the environment and human health and well-being. Can include risk assessment as a component, along with economic and land use assessment. Environmental Protection Agency (EPA): The U.S. regulatory agency for biotechnology of microbes. The major laws under which the agency has

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regulatory powers are the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); and the Toxic Substances Control Act (TSCA). Environmental risk assessment (EnRA): An evaluation of the interactions of agents, humans, and ecological resources. Comprised of human health risk assessment and ecological risk assessment, typically evaluating the probabilities and magnitudes of harm that could come from environmental contaminants. Enzymes: Proteins that control the various steps in all chemical reactions. Epidemic: An epidemic occurs when new cases of a certain disease occur in a given human population, during a given period, substantially exceed what is “expected,” based on recent experience (the number of new cases in the population during a specified period of time is called the “incidence rate”). (An epizootic is the analogous circumstance within an animal population.) In recent usages, the disease is not required to be communicable; examples include cancer or heart disease. Defining an epidemic can be subjective, depending in part on what is “expected”. An epidemic may be restricted to one locale (an outbreak), more general (an “epidemic”) or even global (pandemic). Because it is based on what is “expected” or thought normal, a few cases of a very rare disease may be classified as an “epidemic,” while many cases of a common disease (such as the common cold) would not. Common diseases that occur at a constant but relatively low rate in the population are said to be “endemic.” An example of an endemic disease is malaria in some parts of Africa (for example, Liberia) in which a large portion of the population is expected to get malaria at some point in their lifetime. The term “epidemic” is often used in a sense to refer to widespread and growing societal problems, for example, in discussions of obesity or drug addiction. It can also be used metaphorically to relate a type of problem like those mentioned above. Epitopes: The specific antigen determinants located on the antigens. EPO: Erythropoietin Escherichia coli: A commensal bacterium inhabiting the human colon that is widely used in biology, both as a simple model of cell biochemical function and as a host for molecular cloning experiments. ESI: Electron Spray Ionization EST: Expressed Sequence Tag Esterification: Esterification is a general name for a chemical reaction between alcohols and acids (carboxylic acids, mineral acids, and acid chlorides) to form compounds called esters. Ethidium bromide: A fluorescent dye used to stain DNA and RNA. The dye fluoresces when exposed to UV light. Eugenics: The science of improving human stock by selective breeding. It involves giving better chances for more suitable people in the society to reproduce than the less suitable people.

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Eukaryote: An organism whose cells possess a nucleus and other membrane-bound vesicles, including all members of the protist, fungi, plant and animal kingdoms; and excluding viruses, bacteria, and blue-green algae. Eutrophicaton: Excess growth of algae (in sewage/wastewaters) which leads to oxygen depletion. Evolution: The long-term process through which a population of organisms accumulates genetic changes that enable its members to successfully adapt to environmental conditions and to better exploit food resources. Exon: A DNA sequence that is ultimately translated into protein. Exonuclease: See Nuclease. Exotoxin: An exotoxin is a toxin excreted by a microorganism, including bacteria, fungi, algae, and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Exotoxins may be secreted, or, similar to endotoxins, may be released during lysis of the cell. Explant: The whole plants can be regenerated virtually from any plant referred to as explant. Exponential phase: This refers to a phase in culture in which the cells divide at a maximum rate. Exports: Garbage and recyclables that are transported outside the state or locality where they originated. Express: To translate a gene’s message into a molecular product. Expressed Sequence Tag (EST): A cDNA that is sequenced in order to gain rapid access to the genes in a genome. Expression library. (See Library.) Extremophile: An extremophile is an organism that thrives in and even may require physically or geochemically extreme conditions that are detrimental to the majority of life on Earth. Fecal coliforms: These are facultative-anaerobic, rod-shaped, gramnegative, non-sporulating bacteria. Fed-Batch culture: In a Fed-Batch culture, the culture is continuously or sequentially fed with fresh medium without removing the growing culture. Fermentation: Fermentation refers to the conversion of sugar to alcohol using yeast under anaerobic conditions. A more general definition of fermentation is the chemical conversion of carbohydrates into alcohols or acids. When fermentation stops prior to complete conversion of sugar to alcohol, a stuck fermentation is said to have occurred. The science of fermentation is known as zymology. Fermentation usually implies that the action of the microorganisms is desirable, and the process is used to produce alcoholic beverages such as wine, beer, and cider. Fermentation is also employed in

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preservation to create lactic acid in sour foods such as pickled cucumbers, kimchi and yogurt. Fermenter: A containment system for the cultivation of prokaryotic cells. Ferrous Metals: Magnetic metals derived from iron (steel). Products made from ferrous metals include large and small appliances, furniture and containers and packaging (steel drums and barrels). Examples of recycling include processing steel cans, strapping and ferrous metals from appliances into new products. Filtration: Filtration is the process of removing suspended solids from water by passing the water through a permeable fabric or porous bed of material. The most common filtration process employs a granular media (e.g., sand, anthracite coal). Filtration is usually a combination of physical and chemical processes. Finite Cell Lines: Finite cell lines are those which have a limited life span and they grow through a limited number of cell generations. FISH (Fluorescent in situ Hybridization): The method of employing fluorescent labels for locating markers on chromosomes by detecting the hybridization positions. Flanking region: The DNA sequences extending on either side of a specific locus or gene. Flaring: The burning of methane emitted from collection pipes at a landfill. Flavr savr: Transgenic tomato developed by using antisense technology. Flow cytometry: A method used to sort out cells, organelles or biological materials by passing through apertures of defined sizes. Fluidized-bed incinerator: A type of incinerator in which the stoker grate is replaced by a bed of limestone or sand that can withstand high temperatures. The heating of the bed and the high air velocities used cause the bed to bubble, which gives rise to the term fluidized. Fly ash: The highly toxic particulate matter captured from the flue gas of an incinerator by the air pollution control system. Food and Drug Administration (FDA): Agency responsible for regulation of biotechnology food products. The major laws under which the agency has regulatory powers include the Food, Drug, and Cosmetic Act; and the Public Health Service Act. Food Processing Waste: Food residues produced during agricultural and industrial operations. Food Scraps: Uneaten food and food preparation waste from residences and commercial establishments (grocery stores, restaurants and produce stands), institutional sources (school cafeterias) and industrial sources (employee lunchrooms). Excludes food-processing waste from agricultural

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and industrial operations. Includes offsite composting but excludes source reduction activities such as backyard (onsite) composting and use of food items for human consumption. Free radicals: Free radicals are labile, extremely reactive forms of molecules with unpaired electrons, e.g. peroxyl radical O2-. Free radicals form eg. during UV radiation. Their high oxidative reactivity can cause serious damage to cellular structures. Antioxidants (e.g. â-carotene, vitamins C, E) neutralize free radicals. Fulvic acids: Yellow organic material that remains in solution after removal of humic acid by acidification. Fungicide: An agent, such as a chemical, that kills fungi. Fusion gene: A hybrid gene created by joining portions of two different genes (to produce a new protein) or by joining a gene to a different promoter (to alter or regulate gene transcription). Fusion protein: A protein that is formed by fusion of two polypeptides, normally coded by separate genes Fusobacterium : Fusobacterium is a genus of filamentous, anaerobic, Gramnegative bacteria, similar to Bacteroides. Fusobacterium contribute to several human diseases, including periodontal diseases, Lemierre’s syndrome, and topical skin ulcers. Fusogen: An agent that induces fusion of protoplasts in somatic hybridization. Gamete: A haploid sex cell, egg or sperm, that contains a single copy of each chromosome. Gametoclonal variations: The variations observed in the regenerated plants from gametic cells (e.g. anther culture). Garbage: In everyday usage, refuse, in general. Some MSWM manuals use garbage to mean “food wastes,” although this usage is not common. Gasification: Gasification involves a group of processes that turn biomass into combustible gas by breaking apart the biomass using heat and pressure to produce a combustible gas, volatiles, char, and ash. The gases can then be used as a fuel or feedstock chemical. GEM: Genetically Engineered Microorganism. Gene Amplification: The presence of multiple genes. Amplification is one mechanism through which proto-oncogenes are activated in malignant cells. Gene Bank: A library of genes or clones of an entire genome of a species. Gene cloning: The process of synthesizing multiple copies of a particular DNA sequence using a bacteria cell or another organism as a host. Gene expression: The process of producing a protein from its DNA- and mRNA-coding sequences.

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Gene flow: The exchange of genes between different, but (usually) related populations. Gene frequency: The percentage of a given allele in a population of organisms. Gene insertion: The addition of one or more copies of a normal gene into a defective chromosome. Gene linkage: The hereditary association of genes located on the same chromosome. Gene modification: The chemical repair of a gene’s defective DNA sequence. Gene pool: The totality of all alleles of all genes of all individuals in a particular population. Gene splicing: Combining genes from different organisms into one organism. Gene therapy: Treatment of diseases by use of genes or DNA sequences. Gene translocation: The movement of a gene fragment from one chromosomal location to another, which often alters or abolishes expression. Gene: A locus on a chromosome that encodes a specific protein or several related proteins. It is considered the functional unit of heredity. Genetic assimilation: Eventual extinction of a natural species as massive pollen flow occurs from another related species and the older crop becomes more like the new crop. Genetic code: The three-letter code that translates nucleic acid sequence into protein sequence. The relationships between the nucleotide base-pair triplets of a messenger RNA molecule and the 20 amino acids that are the building blocks of proteins. Genetic disease: A disease that has its origin in changes to the genetic material, DNA. Usually refers to diseases that are inherited in a Mendelian fashion, although non-inherited forms of cancer also result from DNA mutation. Genetic drift: Random variation in gene frequency from one generation to another. Genetic engineering: The manipulation of an organism’s genetic endowment by introducing or eliminating specific genes through modern molecular biology techniques. A broad definition of genetic engineering also includes selective breeding and other means of artificial selection. Genetic library: A collection of clones representing the entire genome of an organism. Genetic linkage map: A linear map of the relative positions of genes along a chromosome. Distances are established by linkage analysis, which

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determines the frequency at which two gene loci become separated during chromosomal recombination. Genetic maps: Maps giving relative distance and position of one gene with respect to the other, wherein the distances are based on recombination values. Genetic marker: A gene or group of genes used to “mark” or track the action of microbes. Genetic Modification (GM): Introduction of isolated genes or pieces of DNA into another organism. Synonymous terms are gene technology, and genetic engineering Genetically Engineered Microorganisms (GEMS): The microorganisms with genetic modifications are collectively referred to as GEMs. Genetically Modified (GM) FOODs: The entry of transgenic plants and animals into the food chain represents GM foods. Genetically Modified Organisms (GMOS): A term used to represent organisms that are genetically engineered. It usually describes the transgenic plants and transgenic animals. Genotype: The structure of DNA that determines the expression of a trait. Genome: The total content of DNA represented by the genes contained in a cell. Genomic DNA: The DNA of an organism containing the essential genes of the organism. Genomic library: A library composed of fragments of genomic DNA. Genomics: Genomics is the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis, epistasis, pleiotropy and other interactions between loci and alleles within the genome. In contrast, the investigation of the roles and functions of single genes is a primary focus of molecular biology and is a common topic of modern medical and biological research. Research of single genes does not fall into the definition of genomics unless the aim of this genetic pathway, and functional information analysis is to elucidate its effect on place in response to the entire genome’s networks. For the United States Environmental Protection Agency, “the term “genomics” encompasses a broader scope of scientific inquiry associated technologies than when genomics was initially considered. A genome is the sum total of all an individual organism’s genes. Thus, genomics is the study of all the genes of a cell, or tissue, at the DNA (genotype), mRNA (transcriptome), or protein (proteome) levels.” Genomics: The study of the structure and functions of genomes. Genus: A category including closely related species. Interbreeding between organisms within the same category can occur.

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GEO: Genetically engineered organism. Geobacter : Geobacter is a genus of Proteobacteria. Geobacter are an anaerobic respiration bacterial species which have capabilities that may make them useful in bioremediation. The Geobacter was found to be the first organism with the ability to oxidize organic compounds and metals, including iron, radioactive metals and petroleum compounds into environmentally benign carbon dioxide while using iron oxide or other available metals as electron acceptor. Geobacter metallireducens was first isolated by Derek Lovley in 1987 in sand sediment from the Potomac River in Washington D.C. The first strain was deemed strain GS-15. Geobacter have been found in anaerobic conditions in soils and aquatic sediment. Research on the potential of the Geobacter is underway and ongoing. The Geobacter’s ability to consume oil-based pollutants and radioactive material with carbon dioxide as waste by-product has already been used in environmental clean-up for underground petroleum spills and for the precipitation of uranium out of groundwater. The Geobacter metabolizes the material by creating “pili,” columns with width of a 3-5 nanometers that act as conduits to pass electrons between the food material and the Geobacter. This manner of consumption has also led scientists to theorize that the Geobacter could act as a natural battery. Geomicrobiology: Geomicrobiology is a subset of the scientific discipline microbiology. The field of geomicrobiology concerns the role of microbes, and microbial processes in geological and geochemical processes. The field is especially important when dealing with microorganisms in aquifers and public drinking water supplies. Another area of investigation in geomicrobiology is the study of extremophile organisms—the microorganisms that thrive in environments normally considered hostile. Such environments may include extremely hot (hot springs or mid-ocean ridge black smoker) environments, extremely saline environments, or even space environments such as Martian soil or comets. Germ Cell (GERM LINE) Gene Therapy: The repair or replacement of a defective gene within the gamete-forming tissues, which produces a heritable change in an organism’s genetic constitution. Germ cell: Reproductive cell. Germ Line: Reproductive cells that produce gametes which, in turn, give rise to sperms and eggs. Germplasm: Germplasm refers to the sum total of all genes present in a crop and its related species. Glucan: Glucan is the anhydrous form of D-glucose as found within a polysaccharide such as starch or cellulose that has 1 molecule of Water (18 g/ mol) less mass due to a condensation reaction forming the polymer, C6H10O5 GMO: Genetically modified organism. A synonymous term is transgenic organism.

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Golden rice: The genetically engineered rice with provitamin A (betacarotene) enrichment. Gram staining: Gram staining is the method of the differentiation of microorganisms (mostly bacteria) by staining them with two dyes. Usually, crystal violet and safranin are used. At first, the cells are stained with the crystal violet and then are decolorized with alcohol. Those bacteria which do not decolorize and stay violet are named gram-positive. Those which decolorize and stain with the second dye (safranin) become pink and are named gram negative. GRAS: Generally Regarded as Safe, and is in use in some countries to represent the safety (no history of causing illness to humans) of foods, drugs, and other materials. GRAS is also used to represent the host organisms employed in genetic engineering experiments. Green biotechnology: Green biotechnology is biotechnology applied to agricultural processes. An example is the designing of transgenic plants to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. An example of this would be Bt corn. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate. Green revolution: Advances in genetics, petrochemicals, and machinery that culminated in a dramatic increase in crop productivity during the third quarter of the 20th century. Greenhouse effect: Trapping of heat from the sun by the atmosphere, in the same manner as the sun’s heat is trapped by the glass walls and roof of a greenhouse. The atmosphere, like the glass, is largely transparent to the sun’s radiation, but it absorbs the longer-wavelength radiation from the earth’s surface into which the sun’s radiation is converted. The principal gases responsible for the absorption are carbon dioxide, water vapor, and ozone. Groundwater: Water beneath the earth’s surface that fills underground pockets (known as aquifers), supplying wells and springs. Growth factor: A serum protein that stimulates cell division when it binds to its cell-surface receptor. Growth phase (curve): The characteristic periods in the growth of a bacterial culture, as indicated by the shape of a graph of viable cell number versus time. GST: Genomic Sequence Tag GT-Food: Food produced from GMOs.

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Habitat: A habitat (which is Latin word for “it inhabits”) is an ecological or environmental area that is inhabited by a particular animal or plant species. It is the natural environment in which an organism lives, or the physical environment that surrounds (influences and is utilized by) a species population. The term “species population” is preferred to “organism” because, while it is possible to describe the habitat of a single black bear, we may not find any particular or individual bear but the grouping of bears that comprise a breeding population and occupy a certain biogeographical area. Further, this habitat could be somewhat different from the habitat of another group or population of black bears living elsewhere. Thus, it is neither the species nor the individual for which the term habitat is typically used. A microhabitat is a physical location that is home to very small creatures, such as woodlice. Microenvironment is the immediate surroundings and other physical factors of an individual plant or animal within its habitat. Half-life: The length of time required for one-half the original radioactive material to decay to new atoms. Haploid cell: A cell containing only one set, or half the usual (diploid) number, of chromosomes. Hazardous solid waste (HSW): A solid waste that meets any one of the following four criteria: (1) the waste is specifically listed as a hazardous waste by regulation, (2) the waste is a mixture containing a hazardous waste, (3) the waste is derived from the treatment, storage, or disposal of a hazardous waste (e.g., the ash residue resulting from incineration of a hazardous waste is also a hazardous waste), or (4) the waste exhibits the characteristics of ignitability, corrosivity, reactivity, or toxicity. Hazardous waste: Waste that is reactive, toxic, corrosive, or otherwise dangerous to living things and/or the environment. Many industrial byproducts are hazardous. Heavy metals: Metals of high atomic weight and density, such as mercury, lead, and cadmium, that are toxic to living organisms. Hela cells: A pure cell line of human cancer cells used for the cultivation of viruses. Hemicellulose: Hemicellulose is a highly branched and substituted polymer comprised mainly of xylose and arabinose, with minor amounts of galactose and glucose. In the plant cell walls, hemicellulose holds crystalline microfibers of cellulose in place. Hemophilia: An X-linked recessive genetic disease, caused by a mutation in the gene for clotting factor VIII (hemophilia A) or clotting factor IX (hemophilia B), which leads to abnormal blood clotting. HEPA: High Efficiency Particulate Air Herbicide: Any substance that is toxic to plants; usually used to kill specific unwanted plants.

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Heterochromatin: Dark-stained regions of chromosomes thought to be for the most part genetically inactive. Heteroduplex: A double-stranded DNA molecule or DNA-RNA hybrid, where each strand is of a different origin. Heterogeneous nuclear RNA (hnRNA). The name originally given to large RNA molecules found in the nucleus, which are now known to be unedited mRNA transcripts, or pre-mRNAs. Heterokaryon: A cell in which two or more nuclei of different genetic make-up are present. Heterologous: These are gene sequences that are not identical, but show variable degrees of similarity. High throughput: Fast rate of sequencing. Histotypic cultures: The growth and propagation of cells in three dimensional matrix to high cell density. Homogenation: Mechanical grinding of cells or tissues. Homologous chromosomes: Chromosomes that have the same linear arrangement of genes—a pair of matching chromosomes in a diploid organism. Homologous recombination: The exchange of DNA fragments between two DNA molecules or chromatids of paired chromosomes (during crossing over) at the site of identical nucleotide sequences. Homozygote: An organism whose genotype is characterized by two identical alleles of a gene. Household hazardous waste: Products used in residences, such as paints and some cleaning compounds, that are toxic to living organisms and/or the environment. HPLC: High Performance/Pressure Liquid Chromatography Human Genome Project (HGP): An international mega project for the identification of human genome sequences, the genes and their functions. This project is coordinated by the National Institutes of Health (NIH) and the Department of Energy (DOE). Human Growth Hormone (HGH, Somatotrophin): A protein produced in the pituitary gland that stimulates the liver to produce somatomedins, which stimulate growth of bone and muscle. Humic substances: Series of relatively high-molecular-weight, brown-toblack substances formed by secondary synthetic reactions. The term is generic in a sense that it describes the coloured material or its fractions obtained on the basis of solubility characteristics, such as humic acid or fulvic acid. Humification: Process whereby the carbon of organic residues is transformed and converted to humic substances by biochemical and chemical processes.

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Humulin: Human insulin used for the treatment of diabetic patients. It was developed by Eli Liply company and was approved for human use in 1982. Humus: Total organic compounds in soil exclusive of undecayed plant and animal tissues, their “partial decomposition” products, and the soil biomass. The term is often used synonymously with soil organic matter. Hybrid DNA: DNA composed of sequences from two different organisms, also called as Recombinant DNA. Hybrid: The offspring of two parents differing in at least one genetic characteristic (trait). Also, a heteroduplex DNA or DNA-RNA molecule. Hybridization: The hydrogen bonding of complementary DNA and/or RNA sequences to form a duplex molecule. Hybridoma: A clone of hybrid cells produced by fusion of a myeloma cell with an antibody-producing cell. Each hybridoma produces only one type of monoclonal antibody. Hydrogen bond: A relatively weak bond formed between a hydrogen atom (which is covalently bound to a nitrogen or oxygen atom) and a nitrogen or oxygen with an unshared electron pair. Hydrogenolysis: Hydrogenolysis is the process of cleaving a molecule or compound with the addition of hydrogen atoms. Hydrolysis: A reaction in which a molecule of water is added at the site of cleavage of a molecule into two products. IEF: Isoelectric Focusing Immobilized enzymes: An enzyme physically localized in a defined region enabling it to be reused in a continuous process. Immortalizing oncogene: A gene that upon transfection enables a primary cell to grow indefinitely in culture. Immunoglobulins: The special group of proteins, commonly referred to as antibodies, produced by B-lymphocytes, and involved in humoral immunity. Immunology: Immunology is the science that deals with the resistance of organisms. Imports: Garbage and recyclables that have been transported to a state or locality for processing or final disposition, but did not originate in that state. In situ Hybridisation: The process of annealing a probe in order to screen a DNA library. In situ: Refers to performing assays or manipulations with intact tissues. In vitro GENE BANKS: In vitro gene banks have been made to preserve the genetic resources by non-conventional methods, i.e. cell and tissue culture methods.

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In vitro: Literally means “in glass” refers to biological activities/reactions carried out in the test tube rather than the living cell or organism. In vivo GENE BANK: In vivo gene bank have been made to preserve the genetic resources by conventional methods e.g. seeds, vegetative propagules etc. In vivo GENE THERAPY: The direct delivery of gene(s) to a tissue or an organ to alleviate genetic disorders. In vivo: Refers to biological processes that take place within a living organism or cell. Incineration: The process of burning solid waste under controlled conditions to reduce its weight and volume, and often to produce energy. Incinerator: A furnace for burning solid waste under controlled circumstances. Incomplete dominance: A condition where a heterozygous offspring has a phenotype that is distinctly different from, and intermediate to, the parental phenotypes. Indicator bacteria: These are certain species of bacteria used by health authorities to detect contaminated water. Each gram of human feces contains approximately 12 billion bacteria; among them may include pathogenic bacteria, such as Salmonella, associated with gastroenteritis. In addition, feces may contain pathogenic viruses, protozoa and parasites. If ingested, these organisms would cause disease. When testing drinking water for contamination, the variety and often low concentrations of pathogens makes them difficult to test for individually. Health authorities, therefore, use the presence of other more abundant and more easily detected fecal bacteria as indicators of the presence of fecal contamination. Indicator bacteria are not themselves dangerous to the health but are used to indicate the presence of a health risk.The most popularized known indicator bacteria are fecal coliforms, which are found in the intestinal tracts of warm-blooded animals. Another less commonly used group of indicator organisms are hydrogen sulfide producing bacteria, which are also found in humans as well as the intestinal tracts of birds and reptiles—known carriers of Salmonella. Industrial MSW: Non-hazardous wastes discarded at industrial sites from packaging and office/administrative sources. Examples of MSW recycling efforts include cardboard, plastic film, wood pallets, lunchroom wastes and office paper excludes industrial process wastes from manufacturing operations. Industrial Process Waste: Residues and materials produced during manufacturing operations. Industrial Sludge: The semi liquid residue remaining after the treatment of industrial water and wastewater.

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Informal sector: The part of an economy that is characterized by private, usually small-scale, labor-intensive, largely unregulated, and unregistered manufacturing or provision of services. Initiation codon: The mRNA sequence AUG, coding for methionine, which initiates translation of mRNA. Inorganic waste: Waste composed of material other than plant or animal matter, such as sand, dust, glass, and many synthetics. Inositol lipid: A membrane-anchored phospholipid that transduces hormonal signals by stimulating the release of any of several chemical messengers. Insertion mutations: Changes in the base sequence of a DNA molecule resulting from the random integration of DNA from another source. Institutional MSW: Waste generated at institutions, such as schools, libraries, hospitals and prisons. Examples of MSW recycling include cafeteria and restroom trashcan wastes, office paper, classroom wastes and yard trimmings. Insulin: A peptide hormone secreted by the islets of Langerhans of the pancreas that regulates the level of sugar in the blood. Integrated solid waste management: Coordinated use of a set of waste management methods, each of which can play a role in an overall MSVVM plan. Interferons: A group of glycoproteins that resist viral infection and regulate immune responses. Intergenic regions: DNA sequences located between genes that comprise a large percentage of the human genome with no known function. Interleukins: A group of lymphokines important for the function of immune system. International NGO: An organization that has an international headquarters and branches in major world regions, often with the purpose of undertaking development assistance. Introgression: Backcrossing of hybrids of two plant populations to introduce new genes into a wild population. Intron: A non-coding DNA sequence within a gene that is initially transcribed into messenger RNA but is later snipped out. See Coding, DNA, Messenger RNA, Transcription. Invasiveness: Ability of a plant to spread beyond its introduction site and become established in new locations where it may have a deleterious effect on organisms already existing there. In-vessel composting: Composting in an enclosed vessel or drum with a controlled internal environment, mechanical mixing, and aeration.

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Ion: A charged particle. Ion-exchange: In this process, metal ions from dilute solutions are exchanged with ions held by electrostatic forces on the exchange resin. The disadvantages include: high cost and partial removal of certain ions. Isotope: One of two or more forms of an element that have the same number of protons (atomic number) but differing numbers of neutrons (mass numbers). Radioactive isotopes are commonly used to make DNA probes and metabolic tracers. Itinerant waste buyer: A person who moves around the streets buying (or bartering for) reusable and recyclable materials. Joining (J) Segment: A small DNA segment that links genes to yield a functional gene encoding an immunogobulin. Junk DNA: The intergenic content of DNA is also referred to as junk DNA. Kanamycin: An antibiotic of the aminoglycoside family that poisons translation by binding to the ribosomes. Karyotype: All of the chromosomes in a cell or an individual organism, visible through a microscope during cell division. Knock out mouse: A genetically altered mouse lacking the genes for an entire organ or organ system. Korarchaeota: These are a group of Archaea that have been found only in high temperature hydrothermal environments. Analysis of their 16S rRNA gene sequences suggests that they are a deeply-branching lineage that does not belong to the main archaeal groups, Crenarchaeota and Euryarchaeota. Analysis of the genome of one korarchaeote that was enriched from a mixed culture revealed a number of both Crenarchaeota and Euryarchaeota like features and supports the hypothesis of a deep-branching ancestry. Labelling: Attaching radioactive or non-radioactive molecules to specific substances in order to detect them. Lag phase: The initial growth phase, during which cell number remains relatively constant prior to rapid growth. Land farming: A technique for the bioremediation of hydrocarboncontaminated soils. Landfill gases: Gases arising from the decomposition of organic wastes; principally methane, carbon dioxide, and hydrogen sulfide. Such gases may cause explosions at landfills. Landfilling: The final disposal of solid waste by placing it in a controlled fashion in a place intended to be permanent. The Source Book uses this term for both controlled dumps and sanitary landfllls. Lawn: A uniform and uninterrupted laver of bacterial growth, in which individual colonies cannot be observed.

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Leachate pond: A pond or tank constructed at a landfill to receive the leachate from the area. Usually, the pond is designed to provide some treatment of the leachate, by allowing settlement of solids or by aeration to promote biological processes. Leachate: Liquid (which may be partly produced by decomposition of organic matter) that has seeped through a landfill or a compost pile and has accumulated bacteria and other possibly harmful dissolved or suspended materials. If uncontrolled, leachate can contaminate both groundwater and surface water. Leaching solution: A solution that is used for solubilisation and removal of metals from an ore by microbial attack. Lead-Acid Batteries: Batteries used in automobiles, trucks and motorcycles. They contain plastic, lead (a toxic metal) and sulfuric acid; excludes leadacid batteries from large equipment, heavy-duty trucks and tractors, aircraft, military vehicles and boats. Legume: A member of the pea family that possesses root nodulescontaining nitrogen-fixing bacteria. Library: A collection of cells, usually bacteria or yeast, that have been transformed with recombinant vectors carrying DNA inserts from a single species. (See cDNA library, Expression library, Genomic library.) Lift: The completed layer of compacted waste in a cell at a landfill. Ligand exchange solvent extraction: A method of extracting a metal from a solution by using ligands. Ligase (DNA ligase): An enzyme that catalyzes a condensation reaction that links two DNA molecules via the formation of a phosphodiester bond between the 3’ hydroxyl and 5’ phosphate of adjacent nucleotides. Ligate: The process of joining two or more DNA fragments. Lineage: A chart that traces the flow of genetic information from generation to generation. Liner: A protective layer, made of soil and/or synthetic materials, installed along the bottom and sides of a landfill to prevent or reduce the flow of leachate into the environment. Linkage: The frequency of coinheritance of a pair of genes and/or genetic markers, which provides a measure of their physical proximity to one another on a chromosome. Linked Genes/Markers: Genes and/or markers that are so closely associated on the chromosome that they are coinherited in 80% or more of cases. Linker: A short, double-stranded oligonucleotide containing a restriction endonuclease recognition site, which is ligated to the ends of a DNA fragment. Lipoplexes: The lipid-DNA complexes also referred to as liposomes.

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Liposomes: Membrane-bound vesicles constructed in the laboratory to transport biological molecules. Lithotroph: A lithotroph is an organism that uses an inorganic substrate (usually of mineral origin) to obtain reducing equivalents for use in biosynthesis (e.g., carbon dioxide fixation) or energy conservation via aerobic or anaerobic respiration. Locus (plural = loci): A specific location or site on a chromosome. Logarithmic phase (log or exponential growth phase): The steepest slope of the growth curve—the phase of vigorous growth during which cell number doubles every 20-30 minutes. Lysis: The destruction of the cell membrane. Lysogen: A bacterial cell whose chromosome contains integrated viral DNA. Lysogenic: A type or phase of the virus life cycle during which the virus integrates into the host chromosome of the infected cell, often remaining essentially dormant for some period of time. Lytic cycle: The replication cycle of bacteria that ultimately results in the lysis of host cells. Lytic: A phase of the virus life cycle during which the virus replicates within the host cell, releasing a new generation of viruses when the infected cell lyses. MALDI: Matrix Assisted Laser Desorption /Ionization Malignant: Having the properties of cancerous growth. Manual landfill: A landfill in which most operations are carried out without the use of mechanized equipment. Mapping: Determining the physical location of a gene or genetic marker on a chromosome. Marker gene: A gene which detects insertion of DNA by its inactivation. Market waste: Primarily organic waste, such as leaves, skins, and unsold food, discarded at or near food markets. Mass-burn incinerator: A type of incinerator in which solid waste is burned without prior sorting or processing. Material Recovery Facility (MRF): A facility where recyclables are sorted into specific categories and processed, or transported to processors, for remanufacturing. Materials recovery: Obtaining materials that can be reused or recycled. Medical Waste: Any solid waste generated in the diagnosis, treatment, or immunization of humans or animals, in research pertaining to humans or animals, or in the production or testing of biologicals.

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Megabase cloning: The cloning of very large DNA fragments. Meiosis: The reduction division process by which haploid gametes and spores are formed, consisting of a single duplication of the genetic material followed by two mitotic divisions. Membrane filtration: Membrane separation processes use semipermeable membranes to separate impurities from water. The membranes are selectively permeable to water and certain solutes. A driving force is used to force the water to pass through the membrane, leaving the impurities behind as a concentrate. The amount and type of material removed depends upon the type of membrane, the type and amount of the driving force and the characteristics of the water. Meristem: A localized region of actively dividing cells in plants i.e., tips of stems and roots. Messenger RNA (mRNA): The class of RNA molecules that copies the genetic information from DNA, in the nucleus, and carries it to ribosomes, in the cytoplasm, where it is translated into protein. Metabolism: The biochemical processes that sustain a living cell or organism. Metagenomics: It is the study of genetic material recovered directly from environmental samples. Traditional microbiology and microbial genome sequencing rely upon cultivated clonal cultures. This relatively new field of genetic research enables studies of organisms that are not easily cultured in a laboratory as well as, studies of organisms in their natural environment. Earlier, environmental gene sequencing cloned specific genes (often the 16S rRNA gene) to produce a profile of diversity in a natural sample. Such work revealed that the vast majority of microbial diversity had been missed by cultivation-based methods. Recent studies use “shotgun” Sanger sequencing or chip-based pyro sequencing to get (mostly) unbiased samples of all genes from all members of sampled communities. Metallothionein: A protective protein that binds heavy metals, such as cadmium and lead. Methane: An odorless, colorless, flammable, explosive gas, CH, produced by anaerobically decomposing MSW at landfills. Methanogenesis: It is the formation of methane by microbes known as methanogens. Organisms capable of producing methane have been identified only from the kingdom Archaea a group phylogenetically distinct from both eukaryotes and bacteria, although many live in close association with anaerobic bacteria. The production of methane is an important and widespread form of microbial metabolism. In most environments, it is the final step in the decomposition of biomass. Recently, some experiments have suggested that leaf tissues of living plants emit methane. Other research has indicated that the plants are not actually generating methane; they are just absorbing

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methane from the soil and the emitting it through their leaf tissues. There may still be some unknown mechanism by which plants produce methane, but that is by no means certain. Methanogenesis in microbes is a form of anaerobic respiration. Methanogens do not use oxygen to breathe; in fact, oxygen inhibits the growth of methanogens. The terminal electron acceptor in methanogenesis is not oxygen, but carbon. The carbon can occur in a small number of organic compounds, all with low molecular weights. Methanogens: These are archaea that produce methane as a metabolic byproduct in anoxic conditions. They are common in wetlands, where they are responsible for marsh gas, and in the guts of animals such as ruminants and humans, where they are responsible for the methane content of flatulence. Microarray: Large number of DNA spots present on a glass slide representative of the total mRNA of a cell, used for detecting expression patterns. Microbial food web: Refers to the combined trophic interactions among microbes in aquatic environments. These microbes include viruses, bacteria, algae, heterotrophic protists (such as ciliates and flagellates). In aquatic environments, microbes constitute the base of the food web. Single celled photosynthetic organisms such as diatoms and cyanobacteria are generally the most important primary producers in the open ocean. Many of these cells, especially cyanobacteria, are too small to be captured and consumed by small crustaceans and planktonic larvae. Instead, these cells are consumed by phagotrophic protists which are readily consumed by larger organisms. Viruses can infect and break open bacterial cells and (to a lesser extent), planktonic algae (a.k.a phytoplankton). Therefore, viruses in the microbial food web act to reduce the population of bacteria and, by lysing bacterial cells, release particulate and dissolved organic carbon (DOC). DOC may also be released into the environment by algal cells. One of the reasons phytoplankton release DOC is limited availability of essential nutrient (N22P) essential nutrients (e.g. nitrogen and phosphorus) are limiting. Therefore, carbon produced during photosynthesis is not used for the synthesis of proteins (and subsequent cell growth), but is limited due of a lack of the nutrients necessary for macromolecules. Excess photosynthate, or DOC is then released, or exuded. The microbial loop describes a pathway in the microbial food web where DOC is returned to higher trophic levels via the incorporation into bacterial biomass. Microbial mats (biofilms): Layered groups or communities of microbial populations. Microenterprise: A synonym for small-scale enterprise: a business, often family-based or a cooperative that usually employs fewer than ten people and may operate “informally.” Microinjection: A means to introduce a solution of DNA, protein, or other soluble material into a cell using a fine micro capillary pipet.

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Micronutrient: An element required by plants and bacteria, in relatively small amounts, for survival and growth. Micronutrients include: Iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B), and Molybdenum (Mo). Micropropagation: This method of tissue culture utilizes the culture of apical shoots, auxiliary buds, and meristems. Mining Waste: Residues resulting from the extraction of raw materials from the earth. Mitosis: The replication of a cell to form two daughter cells with identical sets of chromosomes. Mixed waste: Unsorted materials that have been discarded into the waste stream. MoAB/MAb: Monoclonal Antibodies. A specific and single type of antibody that is produced by hybridoma cells. MAb is directed against a specific antigenic determinant (epitope). Modern Biotechnology: Biotechnology, including the use of genetic modification of the producer cells and other newer procedures. Modular incinerator: A relatively small type of prefabricated solid waste combustion unit. Molecular biology: The study of the biochemical and molecular interactions within living cells. Molecular breeding: Breeding assisted by molecular (nucleic acid) markers is known as molecular breeding. Molecular cloning: The biological amplification of a specific DNA sequence through mitotic division of a host cell into which it has been transformed or transfected. Molecular genetics: The study of the flow and regulation of genetic information between DNA, RNA, and protein molecules. Molecular pharming: Use of transgenic animals to obtain products of medicinal commercial purposes through recombinant DNA technology Monellin: A protein found in the fruits of an African plant Discorephyllum cumminsii which is about 100,000 times sweeter than sucrose. Monoclonal antibodies: Immunoglobulin molecules of single-epitope specificity that are secreted by a clone of B cells. Monoculture: The agricultural practice of cultivating crops consisting of genetically similar organisms. Monofill: A landfill intended for one type of waste only. Monogenic: Controlled by, or associated with, a single gene. Morphogenesis: The growth and development of an undifferentiated structure to a differentiated structure or form. MTCC: Microbial Type Culture Collection

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Mulching: The process by which the volume of organic waste is reduced through shredding or grinding. Multicellular Tumor spheroids (MCTs): In vitro cellular three-dimensional proliferating models for the study of tumor cells. Multi-Locus probe: A probe that hybridizes to a number of different sites in the genome of an organism. Municipal Sludge: The semi-liquid residue remaining after the treatment of municipal water and wastewater. Municipal solid waste (MSW): All solid waste generated in an area except industrial and agricultural wastes. Sometimes, includes construction and demolition debris and other special wastes that may enter the municipal waste stream. Generally, excludes hazardous wastes except to the extent that they enter the municipal waste stream. Sometimes, defined to mean all solid wastes that a city authority accepts responsibility for managing in some way. Municipal solid waste management implementation of systems to handle MSW.

(MSWM):

Planning

and

Mushrooms: The fungi belonging to the class Basidiomycetes; some of them are edible e.g. Agaricus bisporus (button mushroom). Mutagenesis: The changes in the nucleotides of DNA of an organism by physical or chemical treatments. Mutagens: Mutagens are chemical or physical agents causing mutations— that are the changes in the DNA structure—passed to the next generations (hereditary). Some pollutants are mutagens, e.g. some aromatic hydrocarbons and their derivatives (benzo-a-pyrene), and also some fungal toxins (aflatoxins). Mutation: An alteration in DNA structure or sequence of a gene. Muteins: The second-generation recombinant therapeutic proteins are collectively referred to as muteins. Mycelium: A mass of interwoven thread-like filaments of a fungus or bacteria. Mycofiltration: It is the process of using mushroom mycelium mats as biological filters. The term was coined by mycologist Paul Stamets. Stamets originally carne up with the technique to control E. coli in the water outflow from his property. After planting a mushroom bed in the gulch where the water was leaving, within a year the coliform count had decreased to nearly undetectable levels. He discovered that the mushroom produced crystalline entities advancing in front of the growing mycelium, disintegrating when they encountered E. coli. As they did so, a chemical signal was sent back to the mycelium that, in tum, generated what appeared to be a customized macro-crystal which attracted the motile bacteria by thousands, summarily stunning them. The advancing mycelium then consumed the E. coli, effectively eliminating them from the environment. Another mushroom, Polyporus

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umbellata, has been demonstrated to inhibit Plasmodium falciparum, a parasite that causes the most dangerous type of malaria infection. One industrial application of mycofiltration has been to prevent erosion due to water runoff. Its primary application has been on abandoned logging roads. The approach here has been to place bark and wood chips onto logging roads, and inoculate this wood debris with mycelia of native fungal species. As the wood chips decompose, the mycelial networks develop and they act as filters to prevent silt-flow. In the process, they also renew top soils, spurring the growth of native flora and fauna. Mycoremediation : It is form of bioremediation, comprising the process of using fungi to return an environment (usually soil) contaminated by pollutants to a less contaminated state. The term Mycoremediation was coined by Paul Stamets and refers specifically to the use of fungal mycelia in bioremediation. One of the primary roles of fungi in the ecosystem is decomposition, which is performed by the mycelium. The mycelium secretes extracellular enzymes and acids that break down lignin and cellulose, the two main building blocks of plant fiber. These are organic compounds composed of long chains of carbon and hydrogen, structurally similar to many organic pollutants. The key to mycoremediation is determining the right fungal species to target a specific pollutant. Mycorrhizae: Fungi that form symbiotic relationships with roots of more developed plants. Myeloma: A tumor cell line derived from a lymphocyte which usually produces a single type of immunoglobulin. National Science Foundation (NSF): A non-regulatory agency which has oversight of biotechnology research activities that the agency funds. Natural Disaster Debris: Wastes resulting from earthquakes, floods, hurricanes, tornados, ice storms, natural disasters and other major weather events; excludes wastes resulting from heavy storms. Natural disaster debris is classified as C&D debris. Natural selection: The differential survival and reproduction of organisms with genetic characteristics that enable them to better utilize environmental resources. NCBI : National Centre of Biotechnology Information NGO: Non-governmental organization. May be used to refer to a range of organizations from small community groups, through national organizations, to international ones. Frequently, these are not-for-profit organizations. Nick translation: A procedure for making a DNA probe in which a DNA fragment is treated with DNase to produce single-stranded nicks, followed by incorporation of radioactive nucleotides from the nicked sites by DNA polymerase I. Nicked circle (relaxed circle): During extraction of plasmid DNA from the bacterial cell, one strand of the DNA becomes nicked. This relaxes the

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torsional strain needed to maintain super-coiling, producing the familiar form of plasmid. Night soil: Human excreta. NIMBY: “Not In My Back Yard.” An expression of resident opposition to the siting of a solid waste facility based on the particular location proposed. Nitrocellulose: A membrane used to immobilize DNA, RNA, or protein, which can then be probed with a labeled sequence or antibody. Nitrogen cycle: The nitrogen cycle is the biogeochemical cycle that describes the transformations of nitrogen and nitrogen-containing compounds in nature. It is a cycle which includes gaseous components. Nitrogen fixation: Nitrogen fixation is the process by which nitrogen is taken from its relatively inert molecular form (N2) in the atmosphere; and converted into nitrogen compounds (such as ammonia, nitrate and nitrogen dioxide). This is an essential process for life because fixed nitrogen is needed to make nucleotides which are needed to make DNA and also to make amino acids which in turn are needed to produce proteins. Nitrogen fixation is performed naturally by a number of different prokaryotes, including bacteria, actinobacteria, and certain types of anaerobic bacteria. Microorganisms that fix nitrogen are called diazotrophs. Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Nitrogenous bases: The purines (adenine and guanine) and pyrimidines (thymine, cytosine, and uracil) that comprise DNA and RNA molecules. Nitrosomonas europaea: Is a Gram-negative obligate chemolithoautotroph that can derive all its energy and reductant for growth from the oxidation of ammonia to nitrite and lives in several places such as soil, sewage, freshwater, the walls of buildings and on the surface of monuments, especially in polluted areas where the air contains high levels of nitrogen compounds. Nodule: The enlargement or swelling on roots of nitrogen-fixing plants. The nodules contain symbiotic nitrogen-fixing bacteria. Nonferrous Metals: Nonmagnetic metals such as aluminum, lead and copper. Products made from nonferrous metals include containers and packaging such as beverage cans, food and other nonfood cans; nonferrous metals found in appliances, furniture, electronic equipment; and nonpackaging aluminum products (foil, closures and lids from bimetal cans). These excludes lead-acid batteries and nonferrous metals from industrial applications and C&D debris. Non-hazardous Industrial Process Waste: Waste that is neither MSW nor considered a hazardous waste under Subtitle C of the Resource Conservation and Recovery Act, such as certain types of manufacturing wastes and wastewaters. Non-target organism: An organism which is affected by an interaction for which it was not the intended recipient.

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Northern hybridization (Northern blotting): A procedure in which RNA fragments are transferred from an agarose gel to a nitrocellulose filter, where the RNA is then hybridized to a radioactive probe. NSF: National Science Foundation. Nuclease: A class of enzymes that degrades DNA and/or RNA molecules by cleaving the phosphodiester bonds that link adjacent nucleotides. In deoxyribonuclease (DNase), the substrate is DNA. In endonuclease, it cleaves at internal sites in the substrate molecule. Exonuclease progressively cleaves from the end of the substrate molecule. In ribonuclease (RNase), the substrate is RNA. In the S1 nuclease, the substrate is single-stranded DNA or RNA. Nucleic acids: The two nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are made up of long chains of molecules called nucleotides. Nuclein: The term used by Friedrich Miescher to describe the nuclear material, he discovered in 1869, which today is known as DNA. Nucleocapsid: Nucleocapsid is the complex formed by viral nucleic acid and capsid, the protein coat that encloses the nucleic acid. Some viruses (e.g. influenza viruses) are additionally surrounded by a glycoprotein and lipidcontaining membrane called an envelope. Nucleoid: A term used to represent the DNA containing region of a prokaryotic cell. Nucleoside analog: A synthetic molecule that resembles a naturally occurring nucleoside, but that lacks a bond site needed to link it to an adjacent nucleotide. Nucleoside: A building block of DNA and RNA, consisting of a nitrogenous base linked to a five-carbon sugar. Nucleotide: A building block of DNA and RNA, consisting of a nitrogenous base, a five-carbon sugar, and a phosphate group. Together, the nucleotides form codons, which when strung together form genes, which in turn link to form chromosomes. Nucleus: The membrane-bound region of a eukaryotic cell that contains the chromosomes. Nylon-eating bacteria: These are a strain of Flavobacterium that is capable of digesting certain byproducts of nylon-6 manufacture. This strain of Flavobacterium, Sp. KI72, became popularly known as nylon-eating bacteria, and the enzymes used to digest the man-made molecules became collectively known as nylonase. Obligate anaerobes: These are anaerobic organisms which fail to grow in the presence of oxygen. Obligate (strict) anaerobes die in presence of oxygen due to the absence of the enzymes superoxide dismutase and catalase which would convert the lethal superoxide formed in their cells due to the presence of

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oxygen. Instead of oxygen, obligate anaerobes use alternate electron acceptors for respiration such as sulfate, nitrate, iron, manganese, mercury, and carbon monoxide. The energy yield of these respiratory processes is less than oxygen respiration, and not all of these electron acceptors are created equal. Oligonucleotide-directed mutagenesis: A technique to alter one or more specific nucleotides in a gene (DNA sequence) so that a protein with specific amino acid change is produced. Oncogene: A gene that contributes to cancer formation when mutated or inappropriately expressed. Oncogenesis: The progression of cytological, genetic, and cellular changes that culminate in a malignant tumor. Oncomouse: The animal model of mouse for cancer which was granted U.S. patent in 1988, the first animal to be patented. Open dump: An unplanned “landfill” that incorporates few, if any, of the characteristics of a controlled landfill. There is typically no leachate control, no access control, no cover, no management, and many waste pickers. Open pollination: Pollination by wind, insects, or other natural mechanisms. Open Reading Frame (ORF): A long DNA sequence that is uninterrupted by a stop codon and encodes part or all of a protein. Operator: A prokaryotic regulatory element that interacts with a repressor to control the transcription of adjacent structural genes. Organ culture: The in vitro culture of an organ so as to achieve the development and/or preservation of the original organ. Organelle: A cell structure that carries out a specialized function in the life of a cell. Organic pump: The uptake of vast quantities of water by plant roots whereby the water is transpired by the plant into the atmosphere. Organic waste: Technically, waste containing carbon, including paper, plastics, wood, food wastes, and yard wastes. In practice, in MSWM, the term is often used in a more restricted sense to mean material that is more directly derived from plant or animal sources, and which can generally be decomposed by microorganisms. Organogenesis: The process of morphogenesis that finally results in the formation of organs e.g. shoots, roots. Origin of replication: The nucleotide sequence at which DNA synthesis is initiated. OSHA: Occupational Safety and Health Administration. Overlapping Reading Frames: Start codons in different reading frames generate different polypeptides from the same DNA sequence.

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Ovum: A female gamete. Ozone: Ozone is a colorless gas that is extremely unstable and is a strong oxidizing agent that is capable or reacting with a wide variety of organic and inorganic solutes in water. Effectiveness of ozone disinfection is a function of the pH, temperature of water and method for ozone application. Paleobiology: Is a growing and comparatively new discipline which combines the methods and findings of the natural science biology with the methods and findings of the earth science paleontology. It is occasionally referred to as “geobiology.” Paleobiological research uses biological field research of current biota and of fossils, millions of years, old to answer questions about the molecular evolution and the evolutionary history of life. In this scientific quest, macrofossils, microfossils and trace fossils are typically analyzed. However, the 21st-century biochemical analysis of DNA and RNA samples offer much promise, as does the biometric construction of phylogenetic trees. Paleontology: The study of the fossil record of past geological periods and of the phylogenetic relationships between ancient and contemporary plant and animal species. Palindromic sequence: A DNA locus whose 5’-to-3’ sequence is identical on each DNA strand. The sequence is the same when one strand is read left to right and the other strand is read right to left. Recognition sites of many restriction enzymes are palindromic. PAM: Point Accepted Mutation pAMP: Ampicillin-resistant plasmid developed for the laboratory course. Parasitism: Is a type of symbiotic relationship between two different organisms where one organism, the parasite, takes favor from the host, sometimes for a prolonged time. In general, parasites are much smaller than their hosts, show a high degree of specialization for their mode of life, and reproduce more quickly and in greater numbers than their hosts. Parasitology: Parasitology is the study of parasites, their hosts, and the relationship between them. As a biological discipline, the scope of parasitology is not determined by the organism or environment in question, but by their way of life. This means, it forms a synthesis of other disciplines, and draws on techniques from fields such as cell biology, bioinformatics, biochemistry, molecular biology, immunology, genetics, evolution and ecology. The parasitic mode of life is the most common on the planet, with representatives from all major taxa, from the simplest unicellular organisms to complex vertebrates. Every free-living species has its own unique species of parasite, so the number of parasitic species greatly exceeds the number of free living species. The study of these diverse organisms means that the subject is often broken up into simpler, more focused units, which use common techniques, even if they are not studying the same organisms or diseases. Much research in parasitology

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falls somewhere between two or more of these definitions. In general, the study of prokaryotes fall under the field of bacteriology rather than parasitology. Patent: A government issued document that provides the holder the exclusive rights to manufacture, use, or sell an invention for a defined period, usually 20 years. Pathogen: An organism capable of causing disease. pBR322: A derivation of ColE1, one of the first plasmid vectors widely used. Pedigree: A diagram mapping the genetic history of a particular family. PEG: Polyethylene glycol Persistence: Ability of an organism to remain in a particular setting for a period of time after it is introduced. Pesticide: A substance that kills harmful organisms (for example, an insecticide or fungicide). PGDF: Platelet-derived growth Factor Phage ecology: Phage ecology is the study of the interaction of bacteriophages with their environments. Phage ecology is increasingly an important component of sessions and symposiums associated with phage meetings as well as general microbiological meetings. Phage: A virus infecting bacterium Phagocytosis: It is the process of taking up small particles (e.g. bacteria) by some cells of the immune system of the organism (e.g. macrophages). Phenotype: The observable characteristics of an organism, the expression of gene alleles (genotype) as an observable physical or biochemical trait. Pheromone: A hormone-like substance that is secreted into the environment. Phosphatase: An enzyme that hydrolyzes esters of phosphoric acid, removing a phosphate group. Phosphinothricin (glufosinate): A broad-spectrum herbicide Phosphodiester bond: A bond in which a phosphate group joins adjacent carbons through ester linkages. A condensation reaction between adjacent nucleotides results in a phosphodiester bond between 3’ and 5’ carbons in DNA and RNA. Phospholipid: A class of lipid molecules in which a phosphate group is linked to glycerol and two fatty-acyl groups. A chief component of biological membranes. Phosphorylation: The addition of a phosphate group to a compound. Photoautotrophs or Phototroph: These are organisms (usually plants) that carry out photosynthesis to acquire energy. Energy from sunlight, carbon dioxide and water are converted into organic materials to be used in cellular

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functions such as biosynthesis and respiration. In an ecological context, they provide nutrition for all other forms of life (besides other autotrophs such as chemotrophs). In terrestrial environments, plants are the predominant variety, while aquatic environments include a range of phototrophic organisms such as algae (e.g. kelp), other protists (such as euglena) and bacteria (such as cyanobacteria). One product of this process is starch, which is a storage or reserve form of carbon, which can be used when light conditions are too poor to satisfy the immediate needs of the organism. Photosynthetic bacteria have a substance called bacteriochlorophyll, they live in lakes and pools, using the hydrogen from hydrogen sulfide awakward construction, for the chemical process. (The bacteriochlorophyll pigment absorbs light in the extreme UV and infrared parts of the spectrum, which is outside the range used by normal chlorophyll). Cyanobacteria live in fresh water, seas, soil and lichen, and use a plant-like photosynthesis. A photolithotrophic autotroph is an autotrophic organism that uses light energy, and an inorganic electron source (eg. H2O, H2, H2S), and CO2 as its carbon source. Examples include plants. The depth to which sunlight or artificial light can penetrate into water, so that photosynthesis may occur, is known as the phototrophic zone. Photochemical smog: A form of air pollution caused by chemical reactions of oxides of nitrogen and volatile organic compounds in sunshine, characterized by the formation of elevated concentrations of ozone and of secondary particles, the latter causing a reduction in visibility. Photoheterotrophs: These are heterotrophic organisms which use light for energy, but cannot use carbon dioxide as their sole carbon source. Consequently, they use organic compounds from the environment to satisfy their carbon requirements. They use compounds such as carbohydrates, fatty acids and alcohols as their organic “food”. Examples are purple non-sulfur bacteria, green non-sulfur bacteria and heliobacteria. Phyotovolatilization: The use of plants to volatilize pollutants from polluted soils and water Physical map: A map showing physical locations on a DNA molecule, such as restriction sites, and sequence-tagged sites. Phytoalexins: The secondary metabolites produced in plants in response to infection. Phytodegradation: The process where plants are able to metabolically degrade organic pollutants. Phytoextraction: The use of plants to extract contaminants from the environment. Phytomining: The use of plants to extract metal compounds of high economic value Phytoremediation: The use of plants to remediate polluted soil and/or groundwater.

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Phyto stabilization: The use of plants to reduce bioavailability and migration of contaminants. pI : Isoelectric point PITC: Phenyl iso thiocyanate Plant pathology: It is the scientific study of plant diseases caused by pathogens (infectious diseases) and environmental conditions (physiological factors). Plantlet: Small-rooted shoot or germinated embryo. Plaque: A clear spot on a lawn of bacteria or cultured cells where cells have been lysed by viral infection. Plasmid (p): A circular DNA molecule, capable of autonomous replication, which typically carries one or more genes encoding antibiotic resistance proteins. Plasmids can transfer genes between bacteria and are important tools of transformation for genetic engineers. Plating Efficiency: The percentage of cells plated which produce cell colonies. Pleiotrophy: The effect of a particular gene on several different traits. Point mutation: A change in a single base pair of a DNA sequence in a gene. Pollution: The contamination of soil, water, or the atmosphere by the discharge of waste or other offensive materials. Poly(A) polymerase: Catalyzes the addition of adenine residues to the 3’ end of pre-mRNAs to form the poly(A) tail. Poly(A) tail: A series of A- nucleotides attached to the 3’ end of eukaryotic mRNA. Polyacrylamide Gel Electrophoresis (PAGE) : Electrophoresis through a matrix composed of a synthetic polymer, used to separate proteins, small DNA, or RNA molecules of up to 1000 nucleotides. Used in DNA sequencing. Polyclonal antibodies: A mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. Polycyclic aromatic hydrocarbons (PAH): A group of chemical substances comprising two or more fused benzene rings. Some members of the group are carcinogenic. Polygenic: Controlled by, or associated with, more than one gene. Polyhydroxyalkanoates (PHA): Intracellular carbon and energy storage compounds. They are biodegradable polymers. Polylinker: A short DNA sequence containing several restriction enzyme recognition sites that is contained in cloning vectors. Polymer: A molecule composed of repeated subunits.

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Polymerase (DNA): Synthesizes a double-stranded DNA molecule using a primer and DNA as a template. (See Poly(A) polymerase, Polymerase chain reaction, RNA polymerase, Taq polymerase.) Polymerase chain reaction (PCR): Is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Polymorphism: The allelic variations in the genomes that results in different phenotypes. Polynucleotide: A DNA polymer composed of multiple nucleotides. Polypeptide (protein): A polymer composed of multiple amino acid units linked by peptide bonds. Polyploid: A multiple of the haploid chromosome number that results from chromosome replication without nuclear division. Polysaccharide: A polymer composed of multiple units of monosaccharide. Polyvalent vaccine: A recombinant organism into which has been cloned antigenic determinants from a number of different disease-causing organisms. Population: A local group of organisms belonging to the same species and capable of interbreeding. Post-consumer materials: Materials that a consumer has finished using, which the consumer may sell, give away, or discard as wastes. Primary cell culture: The maintenance of growth of cells dissociated from the parental tissue in culture medium is known as primary cell culture. Primary cell: A cell or cell line taken directly from a living organism, which is not immortalized. Primary material: A commercial material produced from virgin materials used for manufacturing basic products. Examples include wood pulp, iron ore, and silica sand. Primer: A short sequence of oligonucleotides that hybridizes with template strand and provides initiation for the nucleic acid synthesis. Prion: Proteinaceous infectious particle. Privatization: A general term referring to a range of contracts and other agreements that transfer the provision of some services or production from the public sector to private firms or organizations.

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Probe: A sequence of DNA or RNA, labeled or marked with a radioactive isotope, used to detect the presence of complementary nucleotide sequences. Processing: Preparing MSW materials for subsequent use or management, using processes such as baling, magnetic separation, crushing, and shredding. The term is also sometimes used to mean separation of recyclables from mixed MSW. Processors: Intermediate operators that handle recyclable materials from collectors and generators for the purpose of preparing materials for recycling (material recovery facilities, scrap metal yards, paper dealers, and glass beneficiation plants). Processors act as intermediaries between collectors and end users of recovered materials. Producer responsibility: A system in which a producer of products or services takes responsibility for the waste that results from the products or services marketed, by reducing materials used in production, making repairable or recyclable goods, and/ or reducing packaging. Prokaryote: A cell lacking a true nucleus; its DNA is usually in one long strand (E.g. Bacterial Cell). Promoter: A region of DNA extending 150-300 bp upstream from the transcription initiation site that contains binding sites for RNA polymerase and a number of proteins that regulate the rate of transcription of the adjacent gene. Pronucleus: Either of the two haploid gamete nuclei just prior to their fusion in the fertilized ovum. Protease: An enzyme that cleaves peptide bonds that link amino acids in protein molecules. Protein engineering: Production and modification of proteins for medicinal, industrial, and research purposes. Protein kinase: An enzyme that adds phosphate groups to a protein molecule at serine, threonine, or tyrosine residues. Protein targeting: The process of transport of proteins from one compartment to other with in a cell. Also called as protein sorting. Protein: A polymer of amino acids linked via peptide bonds and which may be composed of two or more polypeptide chains. Proteinaceous infectious particle (Prion): A proposed pathogen composed only of protein with no detectable nucleic acid and which is responsible for Creutzfeldt-Jakob disease and kuru in humans and scrapie in sheep. Proteolytic: The ability to break down protein molecules. Proteome: The complete protein complement of cells, tissues, and organisms is referred to as its proteome. Proteomics: Large-scale characterization of the entire protein complement of cells, tissues, and organisms is called proteomics.

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PTFE: Poly Tetra Fluoro Ethylene pUC: A widely used expression plasmid containing a galactosidase gene. Putrescible: Subject to decomposition or decay. Usually, used in reference to food wastes and other organic wastes that decay quickly. Pyrolysis: Chemical decomposition of a substance by heat in the absence of oxygen, resulting in various hydrocarbon gases and carbon-like residue. Quorum sensing: It is a type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacteria use quorum sensing to coordinate their gene expression according to the local density of their population. Similarly, some social insects use quorum sensing to make collective decisions about where to nest. In addition to its function in biological systems, quorum sensing has several useful applications for computing and robotics. R- HuEPO: Recombinant human erythropoietin Radioactive waste: Waste material containing radioactive elements in amounts greater than those normally present in the environment. Such waste is generated in large amounts by nuclear reactors used for production of electric power or of plutonium for weapons manufacture. Much low-level waste also results from uranium and phosphate mining and milling, industrial processes, laboratory research, and discarded materials that were used in medical diagnosis and therapy. Raffinate: The aqueous solution that is taken off after solvent extraction. RAPD: Randomly Amplified Polymorphic DNA—A PCR based method of DNA profiling. It basically involves the amplification of DNA sequences using random primers, and use of genetic fingerprints to identify individual organisms (mostly plants). RBS: Ribosome Binding site. Reading frame: A series of triplet codons beginning from a specific nucleotide. Depending on where one begins, each DNA strand contains three different reading frames. Recessive (-acting) Oncogene (anti-oncogene): A single copy of this gene is sufficient to suppress cell proliferation; the loss of both copies of the gene contributes to cancer formation. Recessive gene: Characterized as having a phenotype expressed only when both copies of the gene are mutated or missing. Recognition sequence (site): A nucleotide sequence composed typically of 4, 6, or 8 nucleotides—that is recognized by a restriction endonuclease. Type II enzymes cut (and their corresponding modification enzymes methylate) within or very near the recognition sequence. Recombinant DNA (rDNA) technology: The techniques involved in the construction, and use of recombinant DNA molecules.

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Recombinant DNA: The process of cutting and recombining DNA fragments from different sources as a means to isolate genes or to alter their structure and function. Recombinant Protein: A protein that is produced by the expression of a cloned gene of a recombinant DNA molecules. Recombinant: A cell that results from recombination of genes. Recombination frequency: The frequency at which crossing over occurs between two chromosomal loci—the probability that two loci will become unlinked during meiosis. Recyclables: Those materials recovered from the solid waste stream and transported to a processor or end user for recycling. Recycling: The process of transforming materials into raw materials for manufacturing new products, which may or may not be similar to the original product. Red biotechnology: Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genomic manipulation. Refuse: A term often used interchangeably with solid waste. Refuse-derived fuel (RDF): Fuel produced from MSW that has undergone processing. Processing can include separation of recyclables and noncombustible materials, shredding, size reduction, and pelletizing. Regulatory gene: A gene whose protein controls the activity of other genes or metabolic pathways. Relaxed plasmid: A plasmid that replicates independently of the main bacterial chromosome and is present in 10-500 copies per cell. Renature: The reannealing (hydrogen bonding) of single-stranded DNA and/or RNA to form a duplex molecule. Replicon: A chromosomal region containing the DNA sequences necessary to initiate DNA replication processes. Repressor: A DNA-binding protein in prokaryotes that blocks gene transcription by binding to the operator. Residential Waste: Waste generated by single or multi-family homes including newspapers, clothing, disposable tableware, food packaging, cans and bottles, food scraps and yard trimmings. Excludes food wastes and yard trimmings diverted to backyard composting (onsite). Residues: The materials remaining after processing, incineration, composting or recycling activities that have been completed. Usually, disposed of in a landfill. Resource recovery: The extraction and utilization of materials and energy from wastes.

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Restriction endonuclease (enzyme): A class of endonucleases that cleaves DNA after recognizing a specific sequence, such as BamH1 (GGATCC), EcoRI (GAATTC), and HindIII (AAGCTT). Type I. Cuts nonspecifically a distance greater than 1000 bp from its recognition sequence and contains both restriction and methylation activities. Type II. Cuts at or near a short, and often symmetrical, recognition sequence. A separate enzyme methylates the same recognition sequence. Type III. Cuts 24-26 bp downstream from a short, asymmetrical recognition sequence. Requires ATP and contains both restriction and methylation activities. Restriction-Fragment-Length Polymorphism (RFLP): Differences in nucleotide sequence between alleles at a chromosomal locus result in restriction fragments of varying lengths detected by Southern analysis. Retro virus: A member of a class of RNA viruses that utilizes the enzyme reverse transcriptase to reverse copy its genome into a DNA intermediate, which integrates into the host-cell chromosome. Many naturally occurring cancers of vertebrate animals are caused by retroviruses. Reuse: The use of a product more than once in its original form, for the same or a new purpose. Examples include refilling glass or plastic bottles, repairing wood pallets, using cardboard or plastic containers for storage and returning milk containers. Reverse genetics: Using linkage analysis and polymorphic markers to isolate a disease gene in the absence of a known metabolic defect, and then using the DNA sequence of the cloned gene to predict the amino acid sequence of its encoded protein. Reverse Osmosis: It is a process in which heavy metals are separated by a semi-permeable membrane at a pressure greater than osmotic pressure caused by the dissolved solids in wastewater. The disadvantage of this method is that it is expensive. Reverse transcriptase (RNA-dependent DNA polymerase): An enzyme isolated from retrovirus-infected cells that synthesizes a complementary (c) DNA strand from an RNA template. Reverse transcription: The process of synthesis of DNA from RNA. RFLP: Restriction Fragment Length Polymorphism. A restriction fragment with variable lengths due to the presence of polymorphic restriction sites at one or both ends. Rhizobaceria : These are root-colonizing bacteria that form a symbiotic relationship with many legumes. Rhizobia: These are soil bacteria that fix nitrogen (diazotrophy) after becoming established inside root nodules of legumes (Fabaceae). Rhizobia require a plant host; they cannot independently fix nitrogen. Morphologically, they are generally gram-negative, motile, non-sporulating rods. The first species of rhizobia, R. leguminosarum, was identified in 1889, and all further

Useful Terms and their Meanings of Environmental Biotechnology

A.57

species were initially placed in the Rhizobium genus. However, more advanced methods of analysis have revised this classification, and now, there are many in other genera. Most research has been done on crop and forage legumes such as clover, beans, and soy. However, recently more work is occurring on North American legumes. Although much of the nitrogen is removed when proteinrich grain or hay is harvested, significant amounts can remain in the soil for future crops. This is especially important when nitrogen fertilizer is not used, as in organic rotation schemes or some less-industrialized countries. Nitrogen is the most commonly deficient nutrient in many soils around the world and it is the most commonly supplied plant nutrient. Supply of nitrogen through fertilizers has severe environmental concerns. Nitrogen fixation by Rhizobium is also beneficial to the environment. Rhizofiltration: The uptake of contaminants by the roots of plants which are immersed in water. Rhizosphere: Zone of soil immediately adjacent to plant roots in which the kinds, numbers, or activities of microorganisms differ from that of the bulk soil. Ribosomal RNA (rRNA): The RNA component of the ribosome. Ribosome: Cellular organelle that is the site of protein synthesis during translation. Ribosome-binding site: The region of an mRNA molecule that binds the ribosome to initiate translation. RNA (ribonucleic acid): An organic acid composed of repeating nucleotide units of adenine, guanine, cytosine, and uracil, whose ribose components are linked by phosphodiester bonds. RNA polymerase : Transcribes RNA from a DNA template. RNA vaccines: RNA molecules which can synthesize antigenic proteins and offer immunity. RNAse: Ribonuclease Root nodule: Specialized structure occurring on roots, especially of leguminous plants, in which bacteria fix nitrogen and make it available for the plant. rRNA: Ribosomal RNA RT-PCR: Reverse transcriptase Polymerase Chain Reaction Rubbish: A general term for solid waste. Sometimes used to exclude food wastes and ashes. Saccharification: It is the process of hydrolyzing a complex carbohydrate into a simple soluble fermentable sugar. Starch or oligosaccharides can be saccharified to produce glucose using glucoamylase enzyme. Saccharomyces cerevisiae: It is a species of budding yeast. It is perhaps the most useful yeast owing to its use since ancient times in baking and brewing.

A.58

Environmental Biotechnology

It is believed that it was originally isolated from the skins of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model prokaryote. It is the microorganism behind the most common type of fermentation. Saccharomyces cerevisiae cells are round to ovoid, 5-10 micrometers in diameter. It reproduces by a division process known as budding. Many proteins important in human biology were first discovered by studying their homologs in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. Salmonella: A genus of rod-shaped, gram-negative bacteria that are a common cause of food poisoning. Sanitary landfill: An engineered method of disposing of solid waste on land, in a manner that meets most of the standard specifications, including sound silting, extensive site preparation, proper leachate and gas management and monitoring, compaction, daily and final cover, complete access control, and record-keeping. Saprophytic microorganisms: Microbes feeding on organic matter from dead organisms (as opposed to parasitic microorganisms, feeding on living organisms and causing diseases). Bacteria and fungi which lead to decay of organic debris participate in the circulation of matter in nature. Satellite DNA: Repetitive DNA that forms a satellite band in a density gradient. Satellite RNA (viroids): A small, self-splicing RNA molecule that accompanies several plant viruses, including tobacco ring spot virus. Scale up : The expansion of laboratory experiments to full-sized industrial processes. Scrubber: Emission control device in an incinerator, used primarily to control acid gases, but also to remove some heavy metals. SDS-PAGE: Sodium Dodecyl Sulphate - Polyacrylamide gel Electrophoresis. Secondary material: A material recovered from post-consumer wastes for use in place of a primary material in manufacturing a product. Secondary metabolite: A metabolite that is not required for the growth and maintenance of cellular functions. Secure landfill: A disposal facility designed to permanently isolate wastes from the environment. This entails burial of the wastes in a landfill that includes clay and/or synthetic liners, leachate collection, gas collection (in cases where gas is generated), and an impermeable cover. Selectable marker: A gene whose expression allows one to identify cells that have been transferred or transfected with a vector containing the marker gene.

Useful Terms and their Meanings of Environmental Biotechnology

A.59

Self-pollination: Pollen of one plant is transferred to the female part of the same plant or another plant with the same genetic makeup. Semi-conservative replication: During DNA duplication, each strand of a parent DNA molecule is a template for the synthesis of its new complementary strand. Thus, one half of a preexisting DNA molecule is conserved during each round of replication. Septage: Sludge removed from a septic tank (a chamber that holds human excreta). Septic tanks: Anaerobic digesters of solids of the sewage settled at the bottom of tanks. Sequence hypothesis: Francis Crick’s seminal concept that genetic information exists as a linear DNA code; DNA and protein sequence are colinear. Sequence-Tagged Site (STS): A unique (single-copy) DNA sequence used as a mapping landmark on a chromosome. Setout container: A box or bucket used for residential waste that is placed outside for collection. Sewage sludge: A semi-liquid residue that settles to the bottom of canals and pipes carrying sewage or industrial wastewaters, or in the bottom of tanks used in treating wastewaters. Sewage treatment: It is the process of removing contaminants from wastewater and household sewage, both runoff (effluents) and domestic. It includes physical, chemical, and biological processes to remove physical, chemical and biological contaminants. Its objective is to produce a waste stream (or treated effluent) and a solid waste or sludge suitable for discharge or reuse back into the environment. Sewage: The liquid waste arising mainly from domestic and industrial sources. Sexual reproduction: The process where two cells (gametes) fuse to form one hybrid, fertilized cell. Shotgun approach: A technique for sequencing of genome in which the molecules to be sequenced are randomly broken down into fragments, which are then individually sequenced. Shuttle vectors: The plasmid vectors that are designed to replicate in two different hosts e.g. E. coli and Streptomyces sp. Siderophore: A low molecular weight Fe- chelating protein synthesized by several soil microorganisms. Signal peptide: A short sequence of amino acids at the N terminal end of some proteins that facilitates the protein to cross membrane. Signal transduction: The biochemical events that conduct the signal of a hormone or growth factor from the cell exterior, through the cell membrane,

A.60

Environmental Biotechnology

and into the cytoplasm. This involves a number of molecules, including receptors, proteins, and messengers. Single Cell Protein (SCP): Cells or protein extracts of microorganisms produced in large quantities for use as human or animal protein supplement. Site remediation: Treatment of a contaminated site by removing contaminated solids or liquids or treating them on-site. Site-directed mutagenesis: The technique used to produce a specified mutation at a predetermined position in a DNA molecule. Sludge: The semi-solid mass produced during the course of sewage/waste water treatment processes. Small Nuclear RNA (snRNA). Short RNA transcripts of 100-300 bp that associate with proteins to form small nuclear ribonucleoprotein particles (snRNPs), which participate in RNA processing. SNPs: Single Nucleotide Polymorphisms Somaclonal variation: The genetic variations found in the cultured plant cells when compared to a pure breeding strain. Somatic cell gene therapy: The repair or replacement of a defective gene within somatic tissue. Somatic cell: Any body cell as opposed to germ cell. Somatic cell is nonreproductive and divides by mitosis. Somatic embryogenesis: Formation of embryos from asexual cells. Somatotrophin: Human growth hormone. Source reduction: The design, manufacture, acquisition, and reuse of materials so as to minimize the quantity and/or toxicity of waste produced. Source separation: Setting aside of compostable and recyclable materials from the waste stream before they are collected with other MSW, to facilitate reuse, recycling, and composting. Southern hybridization (Southern blotting): A procedure in which DNA restriction fragments are transferred from an agarose gel to a nitrocellulose filter, where the denatured DNA is then hybridized to a radioactive probe (blotting). Sparger: A device that introduces air into a bioreactor in the form of a fine stream. Special wastes: Wastes that are ideally considered to be outside the MSW stream, but which sometimes enter it and must often be dealt with by municipal authorities. These include household hazardous waste, medical waste, construction and demolition debris, war and earthquake debris, oils, wet batteries, sewage sludge, human excreta, slaughterhouse waste, and industrial waste. Species: A classification of related organisms that can freely interbreed.

Useful Terms and their Meanings of Environmental Biotechnology

A.61

Spore: A form taken by certain microbes that enables them to exist in a dormant stage. It is an asexual reproductive cell. Staphylococcus aureus: Is the most common cause of staph infections. It is a spherical bacterium, frequently found in the nose and skin of a person. Stationary phase: The plateau of the growth curve after log growth, during which cell number remains constant. New cells are produced at the same rate as older cells die. Stem cells: A progenitor cell that is capable of dividing continuously through out the life of an organism. Sticky end: A protruding, single-stranded nucleotide sequence produced when a restriction endonuclease cleaves off center in its recognition sequence. Stirred tank fermenter: A fermentation vessel in which the cells or microorganisms are mixed by mechanically driven impellers. Stringency: Reaction conditions — notably temperature, salt, and pH— that dictate the annealing of single-stranded DNA/DNA, DNA/RNA, and RNA/RNA hybrids. At high stringency, duplexes form only between strands with perfect one-to-one complementarity; lower stringency allows annealing between strands with some degree of mismatch between bases. Stringent plasmid: A plasmid that only replicates along with the main bacterial chromosome and is present as a single copy, or at most several copies, per cell. (See plasmid.) Structure-functionalism: The scientific tradition that stresses the relationship between a physical structure and its function, for example, the related disciplines of anatomy and physiology. STS: Sequence-tagged site. Sub-cloning: The process of tranferring a cloned DNA fragment from one vector to another. Sub-culturing: Sub-culturing involves removing the growth media, washing the plate, disassociating the adherent cells, usually enzymatically or removing by using pipette, and diluting the cell suspension into fresh media. Subsidy: Direct or indirect payment from government to businesses, citizens, or institutions to encourage a desired activity. Sub-unit vaccine: A vaccine composed of a purified antigenic determinant that is separated from the virulent organism. Superbug: The first genetically engineered organism (bacterial strain of Pseudomonas) that was patented. It carries different hydrocarbon-degrading genes on plasmids. Super-coiled plasmid: The predominant in vivo form of plasmid, in which the plasmid is coiled around histone-like proteins. Supporting proteins are stripped away during extraction from the bacterial cell, causing the plasmid molecule to supercoil around itself in vitro.

A.62

Environmental Biotechnology

Super-gene: A group of neighboring genes on a chromosome that tend to be inherited together and are sometimes functionally related. Supernatant: The soluble liquid & action of a sample after centrifugation or precipitation of insoluble solids. Super-ovulation: The process of inducing more ovarian follicles to ripen and produce more eggs. Suspension cultures: Cells which do not attach to the surface of the culture vessel and grow in a suspended manner in the culture medium are called suspension cultures. Symbiosis: The term symbiosis commonly describes close and often longterm interactions between different biological species. The term was first used in 1879 by the German mycologist Heinrich Anton de Bary, who defined it as “the living together of unlike organisms”. The definition of symbiosis is in flux, and the term has been applied to a wide range of biological interactions. The symbiotic relationship may be categorized as being mutualistic, parasitic, or commensal in nature. Others define it more narrowly, as only those relationships from which both organisms benefit, in which case it would be synonymous with mutualism. Synapsis: The pairing of homologous chromosome pairs during prophase of the first meiotic division, when crossing over occurs. Taq Polymerase: A heat-stable DNA polymerase isolated from the bacterium Therrnus aquaticus, used in PCR. (See Polymerase.) TATA box: An adenine- and thymine-rich promoter sequence located 2530 bp upstream of a gene, which is the binding site of RNA polymerase. T-DNA (transfer DNA, tumor-DNA: The transforming region of DNA in the Ti plasmid of Agrobacterium tumefaciens. Telomere: The end of a chromosome. Template: An RNA or single-stranded DNA molecule upon which a complementary nucleotide strand is synthesized. Teratogens: Chemical or biological factors causing disturbances in embryonic development leading sometimes to serious malformations in embryos. Teratogenic properties can be found in some pesticides, fungal toxins and viruses (e.g. the rubella virus during early pregnancy). Termination codon: Any of three mRNA sequences (UGA, UAG, UAA) that do not code for an amino acid, and thus, signal the end of protein synthesis. Also known as stop codon. Terminator Region: A DNA sequence that signals the end of transcription. Tetracycline: An antibiotic that interferes with protein synthesis in prokaryotes. Thaumatin: A protein extracted from berries which is about 3000 times sweeter than sucrose.

Useful Terms and their Meanings of Environmental Biotechnology

A.63

Thiobacillus ferroxidans: Micro-organisms that can get all their energy from oxidising Fe2+ to Fe3+ and are able to live solely on sulphides and in acid conditions. Thymidine kinase (tk): An enzyme that allows a cell to utilize an alternate metabolic pathway for incorporating thymidine into DNA. Used as a selectable marker to identify transfected eukaryotic cells. Ti (tumor-inducing) PLASMID: A giant plasmid of Agrobacterium tumefaciens that is responsible for tumor formation in infected plants. Ti plasmids are used as vectors to introduce foreign DNA into plant cells. TIGR: The Institute of Genomic Research Tipping fee: A fee for unloading or dumping waste at a landfill, transfer station, incinerator, or recycling facility. Tipping floor: Unloading area for vehicles that are delivering MSW to a transfer station or incinerator. Tissue culture: A process where individual cells, or tissues of plants or animals, are grown artificially. Tissue engineering: The application of the principles of engineering to cell culture for the construction of functional anatomical units. T-Lymphocytes (T cells): The lymphocytes that are dependent on the thymus for their differentiation, and are involved in cell-mediated immune response. Tm: Melting temperature Totipotent: A term used to describe a cell that is not committed to a single developmental pathway, and thus it is capable of forming all types of differential cells. Traditional (old) biotechnology: The age-old practices for the preparation of foods and beverages, based on the natural capabilities of microorganisms. Trans-capsidation: The partial or full coating of the nucleic acid of one virus with a coat protein of a differing virus. Transcription: The process of creating a complementary RNA copy of DNA. Transduction: The transfer of DNA sequences from one bacterium to another via lysogenic infection by a bacteriophage (transducing phage). Transfection: The uptake and expression of a foreign DNA sequence by cultured eukaryotic cells. Transfer point: A designated point, often at the edge of a neighborhood, where sma collection vehicles transfer waste to larger vehicles for transport to disposal sites. Transfer station: A major facility at which MSW from collection vehicles is consolidated into loads that are transported by larger trucks or other means to more distant final disposal facilities, typically landfills.

A.64

Environmental Biotechnology

Transfer: The act of moving waste from a collection vehicle to a larger transport vehicle. Transformant: In prokaryotes, a cell that has been genetically altered through the uptake of foreign DNA. In higher eukaryotes, a cultured cell that has acquired a malignant phenotype. Transformation efficiency: The number of bacterial cells that uptake and express plasmid DNA divided by the mass of plasmid used (in transformants/ microgram). Transformation: In prokaryotes, the natural or induced uptake and expression of a foreign DNA sequence—typically a recombinant plasmid in experimental systems. In higher eukaryotes, the conversion of cultured cells to a malignant phenotype—typically through infection by a tumor virus or transfection with an oncogene. Transforming oncogene: A gene that upon transfection converts a previously immortalized cell to the malignant phenotype. Transgenic animal: Genetically engineered animal or offspring of genetically engineered animals. The transgenic animal usually contains material from at lease one unrelated organism, such as from a virus, plant, or other animal (See Transgenic). Transgenic plant: Genetically engineered plant or offspring of genetically engineered plants. The transgenic plant usually contains material from at least one unrelated organisms, such as from a virus, animal, or other plant. (See Transgenic). Transgenic: An organism in which a foreign DNA gene (a transgene) is incorporated into its genome early in development. The transgene is present in both somatic and germ cells, is expressed in one or more tissues, and is inherited by offspring in a Mendelian fashion. Transition-state intermediate: In a chemical reaction, an unstable and high-energy configuration assumed by reactants on the way to making products. Enzymes are thought to bind and stabilize the transition state, thus lowering the energy of activation needed to drive the reaction to completion. Translation: The process of converting the genetic information of an mRNA on ribosomes into a polypeptide. Transfer RNA molecules carry the appropriate amino acids to the ribosome, where they are joined by peptide bonds. Translocation: The movement or reciprocal exchange of largechromosomal segments, typically between two different chromosomes. Transposition: The movement of a DNA segment within the genome of an organism. Transposon (transposable, or movable genetic element): A relatively small DNA segment that has the ability to move from one chromosomal position to another.

Useful Terms and their Meanings of Environmental Biotechnology

A.65

tRNA (transfer RNA): The class of small RNA molecules that transfer amino acids to the ribosome during protein synthesis. See Transfer RNA. Trypsin: A proteolytic enzyme that hydrolyzes peptide bonds on the carboxyl side of the amino acids arginine and lysine. TSCA: The Toxic Substances Control Act Tumor virus: A virus capable of transforming a cell to a malignant phenotype. Ultrafiltration: They are pressure-driven membrane operations that use porous membranes for the removal of heavy metals. The main disadvantage of this process is the generation of sludge. Ultraviolet (UV): UV light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV. It is so named because the spectrum consists of electromagnetic waves with frequencies higher than those that humans identify as the color violet. UV light is found in sunlight and is emitted by electric arcs and specialized lights such as black lights. As an ionizing radiation, it can cause chemical reactions, and causes many substances to glow or fluoresce. Most people are aware of the effects of UV through the painful condition of sunburn, but the UV spectrum has many other effects, both beneficial and damaging, on human health. The discovery of UV radiation was intimately associated with the observation that silver salts darken when exposed to sunlight. In 1801, the German physicist Johann Wilhelm Ritter made the hallmark observation that invisible rays just beyond the violet end of the visible spectrum were especially effective at darkening silver chloride-soaked paper. He called them “de-oxidizing rays” to emphasize their chemical reactivity and to distinguish them from “heat rays” at the other end of the visible spectrum. The simpler term “chemical rays” was adopted shortly thereafter, and it remained popular throughout the 19th century. The terms chemical and heat rays were eventually dropped in favor of ultraviolet and infrared radiation, respectively. Ultraviolet light: Ultraviolet light is electromagnetic energy that is located in the electromagnetic spectrum at wavelengths between those of X-rays and visible light. UV light, that is effective is destroying microbial entities, is located in the 200- to 310-nm range of the energy spectrum. Most typical applications of UV at water treatment plants apply UV light in the wavelength range of 250 to 270 nm. Vaccine: A preparation of dead or weakened pathogen, or of derived antigenic determinants, that is used to induce formation of antibodies or immunity against the pathogen. Vaccinia: The cowpox virus used to vaccinate against smallpox and, experimentally, as a carrier of genes for antigenic determinants cloned from other disease organisms.

A.66

Environmental Biotechnology

Variable Surface Glycoprotein (VSG): One, of a battery of antigenic determinants expressed by a microorganism, to elude immune detection. Variation: Differences in the frequency of genes and traits among individual organisms within a population. Vector: A vehicle for carrying cloned DNA. Vectors: Organisms that carry disease causing pathogens. At landfills, rodents, flies, and birds are the main vectors that spread pathogens beyond the landfill site. Vegetative propagation: The asexual propagation of plants from the detached parts of the plants. Vermiculture: Worm culture. Viral oncogene: A viral gene that contributes to malignancies in vertebrate hosts. Virgin materials: Any basic material for industrial processes that has not previously been used, for example, wood-pulp trees, iron ore, crude oil, bauxite. Viroid: A plant pathogen that consists of a naked RNA molecule of approximately 250-350 nucleotides, whose extensive base pairing results in a nearly correct double helix. (See Satellite RNA.) Virulence: Is the degree of pathogenicity of an organism, or in other words, the relative ability of a pathogen to cause disease. The word virulent, which is the adjective for virulence, derives from the Latin word virulentus, which means “full of poison.” From an ecological point of view, virulence can be defined as the host’s parasite-induced loss of fitness. Virus: An infectious particle composed of a protein capsule and a nucleic acid core, which is dependent on a host organism for replication. A doublestranded DNA copy of an RNA virus genome that is integrated into the host chromosome during lysogenic infection. VSG: Variable surface glycoprotein Waste characterization study: An analysis of samples from a waste stream to determine its composition. Waste collector: A person employed by a local authority or a private firm to collect waste from residences, businesses, and community bins. Waste dealer: A middleman who buys recyclable materials from waste generators and itinerant buyers and sells them, after sorting and some processing, to wholesale brokers or recycling industries. Waste Generation: The amount (weight or volume) of materials and products that enter the waste stream before recycling, composting, landfilling or combustion takes place.

Useful Terms and their Meanings of Environmental Biotechnology

A.67

Waste management hierarchy: A ranking of waste management operations according to their environmental or energy benefits. The purpose of the waste management hierarchy is to make waste management practices as environmentally sound as possible. Waste picker:A person who picks out recyclables from mixed waste wherever it may be temporarily accessible or disposed of. Waste reduction: All means of reducing the amount of waste that is produced initially and that must be collected by solid waste authorities. This ranges from legislation and product design to local programs designed to keep recyclables and compostables out of the final waste stream. Waste Stream: The total flow of solid waste from homes, businesses, institutions and manufacturing plants that must be recycled, incinerated or disposed of in landfills; or any segment thereof, such as the “residential waste stream” or the “recyclable waste stream.” Waste-to-energy (WTE) plant: A facility that uses solid waste materials (processed or raw) to produce energy. WTE plants include incinerators that produce steam for district heating or industrial use, or that generate electricity; they also include facilities that convert landfill gas to electricity. Waste-To-Energy Facility/Combustor: A facility where recovered MSW is converted into a usable form of energy, usually through combustion. SC identifies waste-to-energy activities (not incineration) as recycling. Wastewater: The used water from households and industrial plants. Storm runoff and infiltration water may also be included in the wastewater. Water table: Level below the earth’s surface at which the ground becomes saturated with water. Weathering: Disintegration and chemical decomposition of rocks by slow reaction with air and water at or near the earth’s surface. Weed: An undesirable plant. Weediness: Unwanted effects of a plant. Wetland: An area that is regularly wet or flooded and has a water table that stands at or above the land surface for at least part of the year. White biotechnology: White biotechnology, also known as grey biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. White biotechnology tends to consume less in resources than traditional processes used to produce industrial goods. Wild type: An organism as found in nature; the organism before it is genetically engineered. Windrow: An elongated pile of aerobically composting materials that are turned periodically to expose the materials to oxygen and to control the temperature to promote biodegradation.

A.68

Environmental Biotechnology

Worin castings: The material produced from the digestive tracts of worms as they live in earth or compost piles. The castings are rich in nitrates, potassium, phosphorous, calcium, and magnesium. Working face: The length and width of the row in which waste is being deposited at a landfill. Also known as the tipping face. Worm culture: A relatively cool, aerobic composting process that uses worms and microorganisms. Also known as vermiculture. Xenobiotic: Compound foreign to biological systems. Often refers to human-made compounds that are resistant or recalcitrant to biodegradation and decomposition. Xerophiles: These are extremophilic organisms that can grow and reproduce in conditions with a low availability of water, also known as water activity. Xerophyte: Is a plant which is able to survive in an environment with little available water or moisture, usually in environments where potential evapotranspiration exceeds precipitation for all or part of the growing season. X-Linked disease: A genetic disease caused by a mutation on the X chromosome. In X-linked recessive conditions, a normal female “carrier” passes on the mutated X chromosome to an affected son. X-RAY crystallography: The diffraction pattern of X-rays passing through a pure crystal of a substance. YAC: Yeast Artificial Chromosome Yard waste: Leaves, grass clippings, prunings, and other natural organic matter discarded from yards and gardens. Yoghurt: Is a dairy product produced by bacterial fermentation of milk. Fermentation of the milk sugar (lactose) produces lactic acid, which acts on milk protein to give yoghurt its texture and its characteristic tang. Z-DNA: A region of DNA that is “flipped” into a left-handed helix, characterized by alternating purines and pyrimidines, and which may be the target of a DNA-binding protein. Zoogleal film: A complex population of organisms that form a “slime growth” on trickling-filter media and break down the organic matter in wastewater. Zygote: The fertilized egg formed by the fusion of two gametes. Parts Per Million (ppm): Indicates the number of units of an element or compound contained in a million units of soil. 2-DE: 2-Dimensional Gel Electrophoresis.

Index

A Agricultural productivity, 1.5 Algae-biotechnology, 9.1 Ammensalism, 2.18 Animal biotechnology, 11.1 Antagonism, 2.16 Apiculture

B

Conservation of biodiversity, 1.34 Cryopreservation, 11.3 Cytotoxicity, 8.24

D Decomposition, 1.21 DNA hybridization, 3.35

E Ecosystem, 1.2

Beneficial microorganisms, 2.36

Ecosystem modeling, 1.13

Biochemical oxygen demand, 3.12

Ectotrophic mycorrhiza, 2.21

Biodegradation, 3.12

Effective microorganisms, 5.1

Biofertilizers, 1.2

Endotrophic mycorrhiza, 2.21

Biofuels, 9.3 Biological reprocessing, 7.10 Biomedical waste, 7.2 Biomimicry, 14.5 Bioremediation, 1.2 Bioresource technology Bio-safety, 14.6 Biosafety management, 15.9 Biotransformation, 1.2

C Cellulose decomposition, 2.11 Cellulosic ethanol, A.10

Environmental biotechnology, 1.1 Environmental microbiology, 2.1 Environmental monitoring, 1.2 Environmental pollution, 1.2 Environmental protection agency, 1.4 Esterification, A.24 Eugenics, 15.3 Eutrophicaton, A.25

F Fecal coliforms, A.25 Fermentation, 1.5 Food processing waste, A.26

Chemoautotrophs, 3.2

G

Coliform index, A.15

Genetically engineered microbes, 1.2

Commensalisms, 2.17 Composting , 1.17

Geomicrobiology, A.30 Green biotechnology, A.31

I.2

Index

H

O

Hazardous solid waste, 14.11

Oil spillage, 1.6

Hazardous waste, 4.25

Organic matter, 1.14

Human activities , 1.2

Organic pollutants, 3.22

Humus decomposition, 2.12 Hydraulic control, 6.2

I Indicator organisms, 4.22 Industrial sludge, A.35 Industrial solid waste , 7.2 Industrial sustainability , 14.1 Intellectual property rights, 15.11

P Parasitism, 2.16 Pest management, 2.31 Phosphorus cycle, 2.52 Photoheterotrophs, A.50 Phytoextraction, 6.2 Phytoremediaiton, 6.5 Phytostabilization 6.6

L

Phytostimulation , 6.4

Lignin decomposition, 2.11

Phytotransformation , 6.3

M Membrane filtration, 7.26 Metagenomics, A.40 Metal accumulator plant species, 6.8 Metal excluders, 6.8 Metal indicators,6.8 Metal transformations, 1.12 Methanogenesis, A.1 Microalgae, 1.20 Microbial ecology, 2.33 Microbial mats (biofilms), A.41 Municipal solid waste, 1.19 Municipal waste reduction, 7.17

Plant biotechnology, 10.1 Pollutants, 1.2 Polymerase chain reaction, 3.35 Predation, 2.16 Proto-cooperation, 2.16 Putrefaction, 5.13

Q Quorum sensing, A.54

R Reclaimed water, 7.29 Reverse osmosis, 3.24

Mutualism, 2.16

Rhizodegradation, 6.4

Mycofiltration, A.43

Rhizofiltration, 6.2

Mycoremediation, A.44

Rhizosphere, 1.23

N

Risk assessment, 1.22

Nature, 1.1

S

Nitrogen cycle, 2.14

Sericulture

Nucleic acid-based techniques, 3.33

Silk synthesis

I.3

Index Soil fertility, 1.11

T

Soil microorganisms, 1.22

Treated wastewater, 7.29

Soil phytoedaphon, 2.6 Soil quality, 5.2

W

Solid waste disposal , 7.1

Wastewater management, 7.7

Sulphur cycle, 2.51 Sustainable agriculture, 1.9

Wastewater treatment, 3.21 Water purification , 4.22 Waterweeds , 7.25