Industrial Biotechnology: Principles and Applications (Biotechnology in Agriculture, Industry and Medicine) 1634638476, 9781634638470

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

INDUSTRIAL BIOTECHNOLOGY PRINCIPLES AND APPLICATIONS

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE

INDUSTRIAL BIOTECHNOLOGY PRINCIPLES AND APPLICATIONS

LOVELEEN KAUR AND ROBINKA KHAJURIA School of Biotechnology and Biosciences, Lovely Professional University

New York

Copyright © 2015 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us:[email protected]

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CONTENTS Preface

ix

Section I: Principles of Industrial Biotechnology

1

Chapter 1

Industrial Biotechnology: Introduction and History

3

Chapter 2

Isolation and Screening of Industrially Important Microorganisms

17

Chapter 3

Media Design

53

Chapter 4

Fermenter Design

79

Chapter 5

Downstream Processing

105

Section II: Applications of Industrial Biotechnology

127

Chapter 6

Production of Microbial Metabolites I

129

Chapter 7

Production of Microbial Metabolites II

137

Chapter 8

Biotransformation

155

Chapter 9

Biomass Products and Single Cell Proteins

171

Chapter 10

Fermented Foods

193

Chapter 11

Biological Waste Treatment

215

Authors' Contact Information

231

Index

233

PREFACE Industrial biotechnology can be defined as the use of modern biological life sciences in process of industries. This field encompasses the use of living organisms which are manipulated particularly at the molecular level through recombinant DNA Technology or genetic engineering to form useful products. Biotechnology has existed since time immemorial, much before any other technology subjects came into practice, even though the term was not used. Setting of curd from milk and production of alcoholic beverages from fruit juices were known to the ancient people even though the science behind it was unknown.Biotechnology today, has myriad applications in our day to day life such as brewing of beer, use of enzymes in detergents, production of fermented food, production of antibiotics, nutritional supplements etc. In addition to this, industrial biotechnology also includes techniques and processes (production of biofuels, treatment of effluents) that contribute to creating an efficient, eco-friendly environment. This book discusses the different aspects of bioprocesses: media design, fermenter design and the economics of it. It also explains in detail the processes and techniques involved in the production of commercially important products. Most work in this area has been carried out using microorganisms isolated from nature or modified through mutations or genetic engineering. This book is an up-to-date collection of latest practices being followed in the field of industrial biotechnology for students both at undergraduate and postgraduate level. This book will also be equally beneficial to the faculty teaching the subject as this book aims to combine the theory and practical applications of the subject. This book is an effort to put forth the advancements of Industrial Biotechnology in addition to laying the foundation of the subject. Industrial biotechnology is an ever widening field in which improvements of products and processes takes place at a very rapid pace. Keeping in view that the subject requires the basic knowledge in order to impart practical understanding of the concepts to the students, the proposed publication is divided into two sections: Principles of Industrial Biotechnology and Applications of Industrial Biotechnology. Thus, the publication has two segments: One dealing with the basic concepts of Bioprocess such as isolation procedures, fermenter designs, screening techniques, scale up techniques, control processes while the second segment will emphasize on the practical applications: the processes involved in production of commercially viable products such as organic acids, antibiotics, enzymes, fermented foods, biofuels etc. This book has been divided into 11 units which cover the following topics essential to Industrial Biotechnology. The content of this book has been designed by taking into consideration the syllabus of Industrial Biotechnology

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Loveleen Kaur and Robinka Khajuria

course taught in most of the leading Universities at both Undergraduate and Postgraduate levels. Chapter 1 introduces the readers to the early, intermediate and late history of Industrial Biotechnology and also gives an overview of the products of Industrial Biotechnology. Chapter 2 describes the techniques pertaining to isolation, purification, selection and screening of industrially important microorganisms. In addition, it also acquaints the readers to the fundamental methods of preservation of cultures and strain improvement of industrially relevant microorganisms. A section on use of culture independent methods for studying microbial populations and another section on culturing techniques for ‗unculturable‘ bacteria is a special highlight. Chapter 3 deals with the various requirements for the growth of microorganisms, how they are met with by various culture media, strategies of media design,media components and their sources, sterilization and media economics. Chapter 4 stresses upon the various types of fermentation processes, design of a typical fermenter and different types of fermenters used for commercially relevant products. Chapter 5, downstream processing, discusses the different unit operations employed for extraction, purification and polishing of the desired product. Chapter 6 gives an insight into the processes followed for the production of industrially important microbial metabolites such as alcohol, enzymes, brewery etc. Chapter 7 includes the production processes for some more commercially relevant metabolities mainly antibiotic production, organic acids, amino acids etc. Chapter 8 deals with some typical biotransformation processes such as vinegar, steroids and gluconic acidproduction. Chapter 9 deals with production technologies and significance of single cell proteins produced from bacteria, yeast, fungi and algae. Chapter 10 pertains to discussion on various fermented foods and their significance in health. Different types of fermented foods such as fermented dairy, vegetable and meat products are highlighted. Chapter 11 is based upon the treatment of waste water. It discusses the primary, secondary and tertiary treatments given to industrial effluents before they are discharged into the water bodies. A large number of people have been a constant source of encouragement for writing this text. We would like to take this opportunity to express our heartfelt gratitude to Dr. S. Kapoor and Dr. Priya Batra, Department of Microbiology, Punjab Agricultural University, Ludhiana for their constant support and encouragement. The untiring efforts, understanding and support extended to us by our families have helped us to work on this book. Special thanks to Mr. Tajinder Singh Walia and Dr. Robinder Khajuria for their unconditional love and motivation. The Authors are also thankful to Nova Science Publishers, Inc. for providing us an opportunity to publish this work. Loveleen Kaur & Robinka Khajuria

SECTION I: PRINCIPLES OF INDUSTRIAL BIOTECHNOLOGY

Chapter 1

INDUSTRIAL BIOTECHNOLOGY: INTRODUCTION AND HISTORY Historically, the introduction of many new key technologies has fundamentally changed societies. The enormous impact of inventions like steam power, the internal combustion engine and electricity is well known. Modern World has seen the growth of two innovations in the form of Information technology (IT) and Biotechnology which have had a gigantic impact on the ways of the modern man. Biotechnology has been successful in offering solutions to major problems challenging the globe such as healthcare, environmental degradation, food security and safety, and energy supply (See Table 1.1).The reason being that biotechnology has the potential to allow truly sustainable development and also contribute to value creation in all sectors of society. According to the UN Convention on Biological Diversity (Article 2), Biotechnology is any technological application that uses biological systems, living organisms or their derivatives, to make or modify products or processes for specific use. Industrial Biotechnology, also known as White Biotechnology (in European countries), is a branch of Biotechnology that deals with the use and application of biotechnology for the sustainable processing and production of chemicals, materials and fuels. Thus, it encompasses the biological principles for the production of goods and services by manipulations of living organisms themselves or the products that they make or the processes they carry out. Biotechnological processing uses enzymes, micro-organisms and plants to make products in a wide range of industrial sectors including chemicals, pharmaceuticals, food and feed, paper and pulp, textiles, energy, materials and polymers. It is often referred to as the third wave in biotechnology. Industrial biotechnology involves working with nature to maximize and optimize existing biochemical pathways that can be used in manufacturing. The industrial biotechnology revolution rides on a series of related developments in three fields of study of detailed information derived from the cell: genomics, proteomics, and bioinformatics. As a result, scientists can apply new techniques to a large number of microorganisms ranging from bacteria, yeasts, and fungi to marine diatoms and protozoa.

4

Loveleen Kaur and Robinka Khajuria Table 1.1. Various applied areas of Biotechnology Field Medical Biotechnology Industrial Biotechnology Food Biotechnology Aquatic Biotechnology Agricultural Biotechnology Geochemical Biotechnology

Applied Area Causative agent of disease in humans, plants and animals; diagnostic procedures, therapies Production of products of human value in large quantities; used in enzyme, amino acid, antibiotics, alcohol, organic acids, growth regulators, pharmaceuticals etc; bioremediation, waste water treatment Food processing; prevention of food spoilage; pasteurization, etc. Water purification; biodegradation of waste and related ecological aspects Fertility of soil; plant and animal diseases; soil health Coal and mineral gas formation; recovery of low grade minerals, bioleaching etc.

The role of many bacteria, yeast, molds and other lower fungi was recognized since the prehistoric times. These microorganisms were found to have a direct relationship with certain types of economic processes. During the later part of the 19th century, the field of microbiology grew with leaps and bounds. Contributions of various scientists, philosophers, thinkers and researchers led to evolution of new technologies and a much deeper understanding of cell metabolism and materials science. There was an increased understanding in the scientific community that the biological and biochemical properties of various organisms were the chief direct cause of chemical transformations of substrates into desired industrial products. This led to identification of many new opportunities and others are continuing to emerge since then. The processes which were carried out at small scale as households were eventually replaced by large scale operations in big manufacturing units due to this knowledge explosion. For example, the tremendous growth in industrial biotechnology is a direct consequence of knowledge of organisms specifically involved in a process and the ones that could be detrimental to that process that might lead to economical losses. The numerous new findings each year have helped to maintain an interest in the sustainability of industrial processes and have also contributed to biotechnology‘s attractiveness. It is believed that Industrial Biotechnology can make a major contribution in sustainability by: 1. making agriculture more competitive and sustainable by creating new non-food markets for crops; 2. improving the quality of life by developing innovative products at affordable costs; and 3. helping industry increase its economic and environmental efficiency (eco-efficiency) and sustainability. There are many examples of products already on the market, such as biopolymer fibers for household applications (e.g. carpeting), biodegradable plastics made from corn, biofuels, lubricants and industrial enzymes used in detergents and in the paper and food processing

Industrial Biotechnology: Introduction and History

5

industries. Biotechnology also forms the basis for the manufacture of some antibiotics, vitamins, amino acids and other fine chemicals.

PRODUCTS OF INDUSTRIAL BIOTECHNOLOGY Biotechnology can replace existing chemical processes and also allow the production of new products. The high specificity and mild reaction conditions of enzymes and cellular processes provide many quality and efficiency advantages. However, these biocatalysts are sensitive to high substrate and product concentrations, and their preference for an aqueous environment, when the majority of chemical products have very limited solubility in water. The following paragraphs give a brief review of some important biotechnological processing applications: Bulk chemicals: Many very high volume chemicals are being produced by industrial fermentation processes such as L-glutamic acid, citric acid and Vitamin C. The list is increasing in size owing to an increase in cost of global petrochemical feedstock. New bulk polymers such as bio-degradable plastics, monomers etc are being developed and there is almost an unlimited scope for further development based on tailored enzymes and microorganisms which is not possible by conventional processes. Bio-fuels and bio-energy: Biotechnological processes to use cellulosic, lignocellulosic and starchy waste materials such as straw and corn cobs as a fermentation source for bioethanol or biodiesel production are an enormous step forward. Biomass for the generation of methane is a wonderful bioresource. Fine and specialty chemicals: Specialized, high-value chemicals often require complex processes for their production. Simpler and more efficient biological processes can offer numerous advantages of quality for such chemicals. These fine chemicals include adhesives, agrochemicals, cleaning materials, cosmetic additives, construction chemicals, elastomers, flavors, food additives, fragrances, lubricants, industrial gases, polymers, surfactants, and textile auxiliaries. New materials: Bio-based performance and nano-composite materials which derive their properties from their specific nano- (or micro-) scale structure can prove to be of tremendous significance. Though microorganisms belonging to bacteria, fungi and yeasts are extensively used in these fermentations, few fermentations are also based on algae, plants and animal cells. Several cellular activities contribute to fermentation products such as: 1. Primary metabolites: Ethanol, vitamins, lactic acid, citric acide, acetic acid etc. (Figure 1.1). 2. Energy storage compounds: Glycerol, polymers and polysaccharides. 3. Proteins: Single Cell Proteins (SCPs), enzymes of both extra and intracellular nature (Table 1.2) and foreign proteins. 4. Intermediate metabolites: Amino acids, vitamins and organic acids. 5. Secondary metabolites: Antibiotics etc (Table 1.3). 6. Whole cell products: SCPs, baker‘s yeast, brewer‘s yeast, bioinsecticides.

6

Loveleen Kaur and Robinka Khajuria Table 1.2. List of industrially important enzymes produced in recombinant microorganisms

Some of the products such as ethanol, lactic acid and cell mass products are generally growth associated, while secondary metabolites, energy storage compounds, and polymers are non-growth associated. Other products, such as proteins depend on the cellular or metabolic function. Unlike primary metabolites which are essential for growth and reproduction, secondary metabolites are not essential for the growth and development of reproducing organism and are produced only in conditions when excess of food is available and the organism is not under any kind of stress. Figure 1.2 gives an account of various stages of growth of microorganisms in a closed system. Primary metabolites are associated with

Industrial Biotechnology: Introduction and History

7

exponential phase (trophophase) while secondary metabolite production takes place in stationary phase (idiophase).

HISTORY The major milestones in the history of Industrial Biotechnology and Microbiology as a science have been discussed in Table 1.4. The practice of industrial biotechnology has its roots deep in antiquity. Long before their discovery, microorganisms were exploited to serve the needs and desires of humans, for example to preserve milk, fruits, and vegetables, and to enhance the quality of life by producing beverages, cheeses, bread, pickled foods, and vinegar. The use of yeasts dates back to ancient days. The oldest fermentation knowhow – the conversion of sugar to alcohol by yeasts, was used to make beer in Sumeria and Babylonia as early as 7000 BC. By 4000 BC, the Egyptians had discovered that carbon dioxide generated by the action of brewer‘s yeast could leaven bread. Ancient people are also known to have made cheese with molds.

Figure 1.1. Primary metabolites produced by various biochemical pathways.

8

Loveleen Kaur and Robinka Khajuria Table 1.3.List of antibiotics produced by different microorganisms and the year of their discovery Name of the antibiotic Penicillin Tyrothricin Griseofulvin Streptomycin Bacitracin Chloramphenicol Polymyxin Chlortetracycline Cephalosporin, C, N, P Neomycin Oxytetracycline Nystatin Erythromycin Novobiocin Kanamycin Fusidic Acid Ampicillin Cephalothin Lincomycin Gentamycin Cephalexin Clindamycin

Name of the discoverer Alexander Fleming S.A. Waksman et al. Johnson et al. Paul Ehrlich Duggar Brolzu

Year of discovery 1929 1939 1939 1943 1945 1947 1947 1948 1948

Waksman et al. Finley et al. Clark -

1949 1950 1950 1952 1955 1957 1960 1961 1962 1962 1963 1967 1968

Figure 1.2. Microbial Growth Curve in a closed system.

Producing organism Penicillium chrysogenum Bacillus sp. Penicillium griseofulvum Bacillus licheniformis Streptomyces griseus S. venezuelae Bacillus polymyxa S. aureofacieus Cephalosporium acremonium S. fradiae S. rimosus S. noursei S. erythreus S. niveus S. kanamyceticus Furidium calcineurin Semi synthetic Semi synthetic S. lincolensis Micromonospora purpurea Semi synthetic Semi synthetic

Industrial Biotechnology: Introduction and History Table 1.4. Major historical developments in the field of Industrial Biotechnology Year B.C. 8000 4000-2000 500 A.D. 100 1590 1663 1675 1783 1797 1823 1830 1833 1835 1837-1838 1840 1857 1859

1861 1864 1865-1866

1870-1890 1874 1876 1877 1879 1895 1897 1902 1906 1911 1914

Major Developments Humans domesticate crops and livestock.Potatoes first cultivated for food. Biotechnology first used to leaven bread and ferment beer, using yeast (Egypt). Production of cheese and fermentation of wine (Samar, China and Egypt). First antibiotic: moldy soybean curds used to treat boils (China) First insecticide: powdered chrysanthemums (China) Janssen invents the first microscope. Hooke discovers existence of the cell. Leeuwenhoek discovers bacteria. Spallanzani observs protease action Jenner inoculates a child with a viral vaccine to protect him from smallpox. Immobilized bacteria used for acetic acid production Proteins discovered First enzyme discovered and isolated Schleiden and Schwann propose that all organisms are composed of cells, and Virchow declares, "Every cell arises from a cell." Schwann and Cagniard Latour give experimental observation of living yeast as agent in alcohol fermentation Industrial enzymatic dextrin production by Payen Pasteur proposes microbes cause fermentation Charles Darwin publishes the theory of evolution by natural selection. The concept of carefully selecting parents and culling the variable progeny greatly influences plant and animal breeders in the late 1800s despite their ignorance of genetics. Detection of anaerobic fermentation by Pasteur Concept of Pasteurization given Science of genetics begins: Austrian monk Gregor Mendel studies garden peas and discovers that genetic traits are passed from parents to offspring in a predictable way-the laws of heredity. Using Darwin's theory, plant breeders crossbreed cotton, developing hundreds of varieties with superior qualities. Christian Hansen‘s lab produces rennet (chymosin) for cheese manufacture. Koch gives agar plate method A technique for staining and identifying bacteria is developed by Koch. Fleming discovers chromatin, the rod-like structures inside the cell nucleus that later came to be called chromosomes Wehmer studies Lactic Acid production Buchner said that fermentation is due to enzyme action; first waste disposal biogas reactor (Bombay) The term immunology first appears. The term genetics is introduced. The first cancer-causing virus is discovered by Rous; Fernbach and stranger carry out microbial acetone and butanol fermentation. Bacteria are used to treat sewage for the first time in Manchester, England.

9

10

Loveleen Kaur and Robinka Khajuria Table 1.4. (Continued)

Year 1915 1916 1919 1920 1928

1933

1938 1940

1941 1942

1944 1947 1953

1955-1956 1958

1960-1961 1964

1965 1966 1967 1969 1970 1971 1973 1975

Major Developments Phages discovered; Production of glycerol for producing explosives and Citric acid production First use of the word biotechnology in print. Evans and Long discover the human growth hormone. Penicillin discovered as an antibiotic by Alexander Fleming. A small-scale test of formulated Bacillus thuringiensis (Bt) for corn borer control begins in Europe. Commercial production of this biopesticide begins in France in 1938. Hybrid corn, developed by Henry Wallace in the 1920s, is commercialized. Growing hybrid corn eliminates the option of saving seeds. Vitamin C production at industrial scale carried out. The term molecular biology is coined. Florey and Chain resume research on penicillin; protein structure revealed; Walksman extends research on antibiotics: actinomycin, streptomycin discovered during 1940s. The term genetic engineering is first used by Danish microbiologist A. Jost in a lecture on reproduction in yeast at the technical institute in Lwow, Poland. The electron microscope is used to identify and characterize a bacteriophage: a virus that infects bacteria. Penicillin mass-produced in microbes. DNA is proven to carry genetic information – Avery et al. Waksman isolates streptomycin, an effective antibiotic for tuberculosis. McClintock discovers transposable elements, or "jumping genes," in corn. The scientific journal Nature publishes James Watson and Francis Crick's manuscript describing the double helical structure of DNA, which marks the beginning of the modern era of genetics; Sanger gives sequence of insulin. Kornberg discovers the enzyme DNA polymerase I, leading to an understanding of how DNA is replicated. DNA is made in a test tube for the first time; First Biotechnology Journal: Journal of Microbiological and Biochemical Engineering (now called Biotechnology and Bioengineering) publishes its first volume. USDA registers first biopesticide: Bacillus thuringiensis; one gene one enzyme concept given. The International Rice Research Institute in the Philippines starts the Green Revolution with new strains of rice that double the yield of previous strains if given sufficient fertilizer. Harris and Watkins successfully fuse mouse and human cells. The genetic code is cracked The first automatic protein sequencer is perfected. An enzyme is synthesized in vitro for the first time. Discovery of restriction enzymes First complete synthesis of a gene by Khorana; Southern Blotting technique Stanley Cohen and Herbert Boyer give technology for gene cloning, industrial production of 6-APA started. The first monoclonal antibodies are produced; Maxam-Gilbert give method of DNA sequencing.

Industrial Biotechnology: Introduction and History Year 1976 1978 1979 1980

1982 1983 1986 1988 1990

1994 1997 2000 2001 2003

11

Major Developments Yeast genes are expressed in E. coli bacteria. Recombinant human insulin first produced. Human growth hormone first synthesized. The U.S. Supreme Court, in the landmark case Diamond vs. Chakraborty, approves the principle of patenting recombinant life forms, which allows the Exxon oil company to patent an oil-eating microorganism. The U.S. patent for gene cloning is awarded to Cohen and Boyer. The first gene-synthesizing machines are developed. Researchers successfully introduce a human gene-one that codes for the protein interferon-into a bacterium. First recombinant DNA vaccine for livestock developed. The polymerase chain reaction (PCR) technique is conceived, first genetic transformation of plant cells by Ti plasmids is performed. First recombinant vaccine for humans: hepatitis B produced, First anti-cancer drug produced through biotech: interferon. U.S. Patent granted for oncomouse, Human genome project idea conceived. Chy-Max™, an artificially produced form of the chymosin enzyme for cheesemaking, is introduced. It is the first product of recombinant DNA technology in the U.S. food supply. The Human Genome Project – an international effort to map all the genes in the human body is launched. The first experimental gene therapy treatment is performed successfully First FDA approval for a whole food produced through biotechnology: FLAVRSAVR™ tomato. First animal cloned from an adult cell: a sheep named Dolly in Scotland. First complete map of a plant genome developed: Arabidopsis thaliana; Golden Rice produced by genetic engineering. Scientific journals publish first draft of human genome sequence. The Human Genome Project is completed

The use of microorganisms in food has a long history. As a method of preservation, milk was fermented to lactic acid to make yogurt and also converted into kefir and koumiss using Kluyveromyces species in Asia. The use of molds to saccharify rice in the koji process dates back to around AD 700. By the fourteenth century AD, the distillation of alcoholic spirits from fermented grain, a practice thought to have originated in China or the Middle East, was common in many parts of the world. Vinegar manufacture began in Orleans, France, at the end of the fourteenth century and the surface technique used is known as the Orleans method.

Fermentation Mysteries Before seventeenth century, the role of microorganisms in human health and other fields was purely philosophical because the invention of microscope to observe microorganisms came much later in the 16th century. In the seventeenth century, Antonie van Leeuwenhoek (1632-1723), a Dutch cloth merchant from Delft (Holland) with no scientific training but a keen amateur interest in the construction of microscopes devised simple lenses. These were

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Loveleen Kaur and Robinka Khajuria

used by him for examination of water, decaying matter, and scrapings from his teeth. He compiled his observations and reported the presence of tiny ―animalcules,‖ moving organisms less than a thousandth the size of a grain of sand. Leeuwenhoek‘s observations were communicated to the Royal British Society in a series of letters. Leeuwenhoek, though was the first person to discover ‗animalcules‘ in droplets of liquids, however, did not associate them with fermentation. Most scientists at the time thought that microbes arose spontaneously from non living matter. This debate went on for about 100 years. However, the theory of spontaneous generation, originally postulated by Aristotle, among others, was discredited with respect to higher forms of life when Italian physician, Francesco Redi disapproved the claim that maggots can arise spontaneously from decaying meat. So the proponents shifted their focus towards abiogenesis of bacteria and claimed that tiny microorganisms could arise spontaneously as clear broth can spontaneously became cloudy. However, another group of scientists believed that microorganisms only came from previously existing microbes and that their ubiquitous presence in air was the reason that they would develop in organic infusions. In the second half of the eighteenth century, Lazzaro Spallanzani undertook microscopic investigations of various samples and helped to disapprove spontaneous generation and studied microbial growth. By the end of the eighteenth and beginning of the nineteenth centuries, respectively, Lavoisier and Gay-Lussac had elaborated quantitative correlations for alcoholic fermentation, without giving explanations for the process underlying it. From the mid-1830s evidence began to accumulate which pointed to the biological nature of fermentation. In the early nineteenth century, three independent investigators viz. Charles Cagniard de la Tour of France, Theodor Schwann, and Friedrich Traugott Kützing of Germany proposed that the products of fermentation, chiefly ethanol and carbon dioxide, were created by a microscopic forms of life. They observed the beer yeast to be little globular bodies able to reproduce themselves by a process called budding. This concept was bitterly opposed by the leading chemists of the period including Justus von Liebig who believed fermentation to be strictly a chemical reaction. Other scientists, including Kützing and Turpin confirmed that living organisms were involved in additional fermentation processes such as acetic acid fermentation. Knapp (1847) reported that brewing was performed in Germany at the level of handicraft, whereas in the UK it was carried out on an industrial scale in large factories with fermenters of up to 240,000 L. In particular, beer, wine, acetic and lactic acid production were reported to contribute significantly to the national economies. Many enzymes such as diastase, emulsion and pepsin were studied in detail (Payen and Persoz 1833). The first industrial processes that used enzymes (diastase) to produce dextrins were established from the 1830s onwards in France.

The Emergence of Microbiology As a Science Interest in the mechanisms of these fermentations resulted in the later investigations by Louis Pasteur, which not only advanced microbiology as a distinct discipline, but also led to the development of vaccines. Louis Pasteur, a French chemist, ended the scientific debate on the nature of fermentation in favor of living microorganisms, starting from hypotheses based

Industrial Biotechnology: Introduction and History

13

on empirical results provided by sophisticated experiments and ingenious theoretical conclusions. Pasteur was called on by the distillers of Lille, France, to find out why the contents of their fermentation vats were turning sour. After numerous microscopic observations, he observed yeast buds in normal fermentation runs, but found rod shaped microorganisms in fermentations that went sour due to the formation of acetic or lactic acid. He investigated lactic acid fermentation in detail. In his research paper, Pasteur (1857a) elaborated the essentials of fermentation processes. He presented the means with which to isolate microorganisms in a pure culture (Buchholz and Collins 2013). In his discussion he introduced: (1) The biological conception of fermentation as the result of the activity of living microorganisms. (2) The practice of inoculation for starting a reliable fermentation. (3) The notion of specificity, according to which all fermentations could be traced to a specific microbe. (4) The essential experimental factor that the fermentation medium must provide the nutrients for the microorganism. (5) Specific chemical features characterized by the main fermentation products and byproducts (Pasteur 1857a, b). Pasteur suggested that the souring of wine could be prevented by a mild heat treatment, which is now known as pasteurization, and is a standard procedure for removing unwanted microorganisms in many industries. Other investigators like Berthelot (1860, 1864) and Béchamp (1864) published a range of relevant papers on fermentation, which mainly dealt with substrates other than sugar. In the 1870s, John Tyndall, Louis Pasteur, and William Roberts (a British physician), directly observed the antagonistic effects of one microorganism on another and suggested probable therapeutic potential of the phenomenon. For the next 50 years, various microbial preparations were tried as medicines, but they were either too toxic or inactive in live animals. However, ultimately in the year 1927, Alexander Fleming discovered penicillin paving way for the discovery of many classes of antibiotics that are obtained from microorganisms. A rapid increase in alcohol production was also observed during this time. The alcohol production in Germany was estimated up to 3.7 million hL in 1893-1894. Here, a process using starch as the raw material was operated at high pressure to ensure gelatinization and then hydrolysis was achieved by adding diastase to stirred tank reactors, followed by fermentation for 72 h, using yeast that had been produced separately. Yeast as a commercial product was mainly generated in high yield in distilleries (as pressed yeast) and was then sold for use in other industrial processes such as bread manufacture (Payen 1874). In Denmark, Hansen was able to obtain pure yeast culture by working with solid culture media which became the basis for pure yeast fermentation and commercial applications. In 1877, Moritz Traube proposed that some material which was protein in nature had an ability to catalyze fermentation and other chemical reactions. This opened the new and exciting era of enzymology. He also proposed that fermentation was carried out via multistage reactions in which the transfer of oxygen occurred from one part of a sugar molecule to another, finally forming some oxidized compound (e.g., carbon dioxide) and a

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reduced compound (e.g., alcohol). The field of biochemistry became established in 1897 when Eduard Buchner found that cell free yeast extracts, lacking whole cells, could convert sucrose into ethanol suggesting that fermentation could also be carried out in the absence of living cells. His evidences suggested that enzyme catalysis, including complex phenomena like that of alcoholic fermentation, was a chemical process not necessarily linked to the presence and action of living cells. Since that time, industrial biotechnology has paved way for the production of enzymes for use in our daily lives and for the manufacturing sector. For instance, meat tenderizer is an enzyme and some contact lens cleaning fluids contain enzymes to remove sticky protein deposits. Fernbach carried out systematic investigations at the Institut Pasteur in Paris on metabolic intermediates during alcoholic fermentation by various microorganisms, e.g. yeast and Tyrothrix tenuis. This included the formation of acids, notably acetic, succinic and pyruvic acids. This helped to elucidate the mechanism of fermentation. He also obtained patents on the fermentation of starch for the production of acetone and higher alcohols (Fernbach 1910, Fernbach and Strange 1911). Around 1907–1910, a shortage of rubber in the world market led to increase in efforts by a team of chemists and bacteriologists, for the production of butanol which could be converted into butadiene which in turn could be polymerized to yield synthetic rubber (Perkin Jr. 1912). During World War I, the need for glycerol, used to manufacture ammunition, resulted in the application of yeast to convert sugars into glycerol. Also during World War I, Chaim Weizmann at the University of Manchester applied the butyric acid bacteria, Clostridium used for centuries for the retting of flax and hemp, for production of acetone and butanol. This fermentation also set a tempo for the development of large scale cultivation of fungi for production of citric acid. Soon after World War I, an aerobic process was devised in which Aspergillus niger was used. Not too many years later, the discoveries of penicillin and streptomycin and their commercial development heralded the start of the antibiotic era. Though discovered in 1928, there was rather a long delay before research and development aiming at production of penicillin was undertaken. Florey, Heatley and Chain, towards the end of the 1930s, began to investigate penicillin in the course of their systematic study of antibacterial substances at Oxford University. Early yields and recovery, however, were very discouraging. Tremendous efforts on the part of Oxford team, National Research Council (USDA) and industrial giants Merck, Squibb, Pfizer and Lederle led to increase in yield of penicillin, particularly when strain improvement was carried out using mutations. Later in 1940s, Waksman isolated actinomycyin in 1940, streptotricin in 1942, and streptomycin in 1944 from cultures of actinomycetes (Ohno et al. 2000). By the 1950s, large-scale production not only of traditional goods, for example, beer, alcohol, cheese, but also new products, including citric acid and pharmaceuticals and other products of particularly high social and economic relevance, had become well established. High cost low volume secondary metabolites, e.g. steroids obtained by biotransformation attracted attention of fermentation industries. Waste water treatment became more wide-spread, due to legislation, and gained great attention. This resulted in new developments, and capital investment, both in the public and industrial sectors (Jördening and Winter 2005). With the advent of Biotechnology in the 1970s, the fermentation industry grew with leaps and bounds. Berg, Cohen and Boyer in 1972 introduced recombinant DNA (rDNA) technology when they constructed the first recombinant plasmids and viruses and introduced them into bacteria, or animal cells respectively, where they were autonomously propagated.

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New methods and tools played a key role in the rapid expansion of recombinant technologies. These include: gel electrophoresis, centrifugation, restriction endonucleases, plasmid cloning. The approval of human insulin as the first recombinant DNA product on the market was followed by a series of other recombinant products, mostly drugs, which could not be produced by other technical means. The products included human growth hormone in 1983, β-interferon, and a hepatitis B vaccine in 1986, tissue plasminogen activator (tPA) in 1987, and erythropoietin in 1989. The exploitation of genetic engineering coincided with the production of monoclonal antibodies. These are being raised in animal cell cultures on commercial scale. Completion of Human Genome Project in 2002 paved way for the possible discovery and production of hundreds of novel pharmaceuticals, many of which are natural human gene products previously not available in significant amounts or as virus-free preparations, significantly improving diagnosis and eventually revolutionizing medicine. In the following years, the public and private sector R&D has become more focused towards improving the products belonging to both low cost high volume as well as high cost low volume products, with latter getting the maximum attention. Metabolic engineering has been used successfully for the optimization of yields, e.g. for the production of amino acids (Buchholz and Collins 2010).

FUTURE OF BIOTECHNOLOGY INDUSTRY Industrial biotechnology is one of the most promising new approaches to pollution prevention, resource conservation, and cost reduction. It offers businesses a way to reduce costs and create new markets while protecting the environment. The application of biotechnology to industrial processes is not only transforming how we manufacture products but is also providing us with new products that could not have been imagined a few years ago. From the beginning, industrial biotechnology has integrated product improvements with pollution prevention. Nothing illustrates this better than the way industrial biotechnology solved the phosphate water pollution problems in the 1970s caused by the use of phosphates in laundry detergent. Biotechnology companies developed enzymes that removed stains from clothing better than phosphates, thus enabling replacement of a polluting material with a nonpolluting biobased additive while improving the performance of the end product. This innovation dramatically reduced phosphate-related algal blooms in surface waters around the globe, and simultaneously enabled consumers to get their clothes cleaner with lower wash water temperatures and concomitant energy savings. Industrial biotechnology is currently aiming to produce different varieties and increased quantities of platform products (which are produced in large volumes). The development and use of Industrial Biotechnology is essential for the sustainable society of the future. In the coming years, an increasing number of chemicals and materials will be produced using biotechnology in one or more of the processing steps. Biotechnological processes will be used to produce chemicals and materials which are hard or impossible to produce conventionally, or to make existing products in a more efficient way. Biotechnology will allow increasingly eco-efficient use of renewable resources as industrial raw materials. Industrial Biotechnology

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will enable a range of industries to manufacture products in an economically and environmentally sustainable way.

REFERENCES Béchamp, M. A. (1864). Sur la fermentation alcoolique. Bulletin de la Société Chimique de Paris,1, 391–392. Berthelot, M. (1860). Sur la fermentation glucosique du sucre de cannes. Les Comptes rendus del’Académie des Sciences,50, 980–984. Berthelot, M. (1864). Remarques sur la note de M. Béchamp relative à la fermentation alcoolique. Bulletin de la Société Chimique de Paris,1, 392-393. Buchholz, K. & Collins, J. (2013). The roots—a short history of industrial microbiology and biotechnology. Appl Microbiol Biotechnol,97, 3747–3762. DOI 10.1007/s00253-0134768-2 Fernbach, A. (1910). Sur la dégradation biologique des hydrates de carbonne. Comptes rendus des séances de l’Académie des sciences (Paris),151, 1004–1006. Fernbach, A. & Strange, E. H. (1911). Fermentation process producing amyl, butyl, or ethyl alcohol, butyric, propionic, or acetic acid, etc. BritishPatent,15, 203. Jördening, H. J. &Winter, J. (2005). Environmental Biotechnology. Wiley- VCH, Weinheim Jr Perkin WH (1912) The production and polymerization of butadiene, isoprene, and their homologues. J Soc Chem Ind,31, 616–623. Knapp, F. (1847). Lehrbuch der chemischen Technologie. Vieweg F, Braunschweig S (eds.) Vol 2, 1-10 Ohno, M.,Otsuka, M., Yagisawa, M., Kondo, S., Heinz Oppinger, H., Hoffmann, H., Sukatsch, D., Hepner, D. & Male, C. (2000). Antibiotics. In: Ullmann’s encyclopedia of industrial chemistry, Wiley-VCH, Weinheim, New York. Pasteur, L. (1857a). Mémoire sur la fermentation appelée lactique. Comt Rend XLV (22): 913–916; also: (1858) Ueber dieMilchsäuregährung. J Prakt Chem,73, 447–451. Pasteur, L. (1857b). Mémoire sur la fermentation alcoolique. Compt Rend 45: 1032–1036; also: (1858) Ueber die alkoholische Gährung. J Prakt Chem,73, 451–455. Payen, A. (1874). Handbuch der Technischen Chemie. Nach A Payen‗s Chimie industrielle, II. Band, F. Stohmann and C. Engler (eds), E. Schweizerbartsche Verlagsbuchhandlung, Stuttgart. Payen, A. & Persoz, J. F. (1833). Mémoire sur la diastase, les principaux produits de ses réactions, et leurs applications aux arts industriels (Memoir on diastase, the principal products of its reactions, and their applications to the industrial arts). Annales de Chimie et de Physique,53, 73–92.

Chapter 2

ISOLATION AND SCREENING OF INDUSTRIALLY IMPORTANT MICROORGANISMS Our environment is formed of a large and complex microbial population which inhabits various parts of our environment. The air, the soil and the water around us, all contain extremely large numbers of mixed populations of microorganisms including bacteria, fungi etc. In natural environments, a single kind of bacterium occurs as a component of a mixed population of bacteria. A sound knowledge of the characteristics of the microorganisms inhabiting different environments are required in order to exploit the industrially relevant microorganisms. In order to study a particular type of bacterial species in such a complex population as well as for the study of production of economically important products, the bacterial species must be isolated and maintained indefinitely in a pure and genetically stable form. But before proceeding to isolation of these microorganisms, it is important that these microorganisms be enriched in the source sample. This is because a particular bacterial or microbial species of interest is often present in small numbers as compared to the total population of microorganisms present in a mixed culture. Some of these microbial species may proliferate very quickly on simple laboratory media. In contrast, there are certain fastidious species of microorganisms which grow less rapidly on ordinary culture media and therefore require specially designed media with certain absolute media requirements for growth.

SELECTION OF INDUSTRIALLY IMPORTANT MICROORGANISMS In order to make a species become a numerically dominant component of a population, a variety of selective methods can be employed. These methods increase the growth of desired species of microorganisms either by discouraging the growth of other unwanted microorganisms present in mixed population (enrichment) or by killing them (selection). Following criteria are considered important while selecting the desired organism (Bull et al. 1979):

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Loveleen Kaur and Robinka Khajuria 1. Nutritional characteristics: The microorganism should be able to grow best on a cheap medium. Thus, the nutritional characteristics of the microorganism need to be studied and its selection should be carried out on a diverse range of cheap and readily available medium components. 2. Optimum temperature of growth: If the microorganism is capable of growing at a higher temperature, say above 40°C, it could considerably reduce the costs involved in the cooling of large scale industrial fermentation process. 3. Suitability: The microorganism should be suitable for the industrial process and additionally should not undergo any kind of reaction with the fermentation vessel or equipment. 4. Stability: The microorganism should be genetically stable under the reaction conditions. At the same time, the ease of genetic manipulation in the organism needs to be considered. 5. Yield: The microorganism should provide high yield of product per unit time, whether it is in the terms of biomass or metabolic product. 6. Downstream Processing: Easy downstream processing i.e. product recovery and purification of the product from the culture and medium is very important because in certain cases downstream processing plays a substantial role in determining the overall cost of a fermentation process. 7. Toxicity: The organism or its products should not be toxic.

SELECTION OF MICROORGAMISMS The ideal isolation procedure commences with targeting an environmental source which has high probability of supporting a particular kind of organism. Generally, soil is considered to be a rich source of myriad kinds of microorganisms. Thus, soil holds a huge potential for acting as the reservoir for commercially important organisms which need an exhaustive search of a range of this natural environment. For this, the isolation procedure should be designed in a way so as to favor the growth of organisms possessing industrially important characteristics. Generally, a selective pressure of some kind, for example, certain chemical compounds, toxins, dyes etc. can be incorporated into the culture medium, so as to select for the taxon showing the desired characteristics. Alternately, selective killing of the undesired microorganisms can be undertaken in order to encourage only the novel or desired microorganisms. The following paragraphs describe some of the selective methods which are constantly used for selection and isolation of industrially important microorganisms.

1. Chemical Methods A number of chemical methods can be utilized, which make use of either presence or lack of a specific chemical component in the culture media as an essential criterion of selection.

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(a) Enrichment Culture Method This technique was designed by a soil microbiologist, Beijerinck, to isolate the desired microorganisms from a heterogeneous microbial population present in soil. Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to other species present in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium or by modifying the physical conditions of the incubation. The medium of known composition and specific condition of incubation favors the growth of desired microorganisms but is unsuitable for the growth of other types of microorganisms. Specifically, the use of a particular carbon or nitrogen source in the medium can help to enrich for a particular microorganism which is the only organism capable of utilizing that specific component of the culture medium. In order to understand this, let us consider a simple nutritional medium like nutrient agar. This medium is a very rich medium which can support the growth of numerous organisms including those which are capable of producing an inducible enzyme cellulase for the degradation of cellulose. In order to enrich the medium for cellulase producers, we need to prepare an enrichment medium in which cellulose is the sole source of carbon. Under ideal conditions of temperature, pH and humidity, the enrichment medium will allow only those organisms to grow, multiply and form colonies which are capable of utilizing cellulose by producing cellulase. Similarly, only those microorganisms can grow on a nitrogen free medium, which are capable of fixing atmospheric nitrogen and utilizing it for their growth, thus selecting for N-fixers only. b) Use of minimal nutrient levels There are many microorganisms which require very dilute medium components for thriving and thus are cultivated in the presence of minimal nutrient levels. The cultivation of these microorganisms is discussed ahead under the subheading of culturing the unculturable bacteria. c) Use of Inhibitors Certain dyes, bile salts, salts of heavy metals and antibiotics can be used for the isolation of select groups of bacteria depending upon their sensitivity to these chemicals (Table 2.1). For example, low concentration of dyes can inhibit the growth of Gram positive bacteria, thus selecting for gram negative bacteria. A very good example in this regard is the use of MacConkey Agar Medium. This medium is designed to select for few gram negative enteric bacteria like E. coli, Shigella and Salmonella. It contains crystal violet dye and sodium deoxycholate (bile salt). Crystal violet inhibits all Gram positive bacteria, except the enteric ones, which survive the presence of bile salts. MacConkey is thus a selective medium. Similarly, the use of antibiotics/antifungal agents in the medium can allow for the selective growth of antibiotic resistant bacteria or fungi.

2. Physical Methods In addition to chemical methods, microorganisms can be isolated on the basis of certain physical methods which are outlined below:

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Table 2.1. Certain antibacterial compounds used in the selective media for the isolation of actinomycetes. (Adapted from Goodfellow and O’Donnell 1989) Sr. No. 1 2 3 4 5 6 7 8

Antimicrobial agent Bruneomycin Dihydromethylfuratriazone Gentamycin Kanamycin Nitrofurazone Novobiocin Tellurite Tunicamycin

Microorganism Micromonospora Microtetraspora Micromonospora Actinomadura Streptomyces Micromonospora Actinoplanes Micromonospora

Reference Preobrazhenskayai et al. (1975) Tomita et al. (1980) Bibikova et al. (1981) Chormonova (1975) Yoshiokova (1952) Sveshnikova et al. (1976) Willoughbey (1971) Wakisaka et al. (1982)

(a) Heat In order to isolate the endospore forming bacteria, a sample of mixed culture of microorganisms, such as soil, is heated to a high temperature (80°C) and then inoculated in culture medium. Heating to this temperature kills the vegetative cells of microorganisms in the mixed culture. However, since the endospores are heat resistant, they are able to survive at this temperature. On inoculating them in the culture media, the endospores germinate to form vegetative cells of the endospore formers, thus favoring their selection. (b) Incubation Temperature Readers will be aware that the microorganisms can be differentiated into three broad categories on the basis of their optimum temperature of growth as thermophiles, mesophiles and psychrophiles. All the microorganisms show best growth at their optimum temperature of growth. Thus, incubation at particular temperature may favor the selective growth of the corresponding group of microorganisms. Accordingly, thermophiles can be selected if high temperature of incubation (say 55°C and above) is provided to the mixed culture. Alternately, to select for psychrophiles, mixed cultures need to be incubated at low temperatures such as 0-5°C. (c) pH Similarly, the microorganisms prefer to live in a particular pH range and thrive best at their optimum pH. High pH can be used for many alkali tolerant bacteria. In order to select for acidophiles (microorganisms showing optimal growth at acidic pH), a low pH of the medium needs to be maintained with the help of buffers.

3. Biological Methods Sometimes living organisms may serve as the selective medium for the isolation and purification of a particular microorganism. An excellent example is the routine use of specific regions of chick embryo for growth and purification of selective groups of viruses. Bacteriophages can be isolated from sewage sample by providing suitable bacterial host, where they would form plaques over bacterial lawns. A sputum sample may contain a large number of bacterial species including Streptococcus pneumoniae. These bacteria can be

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isolated from such a mixed culture of bacteria in sputum by injecting the sputum sample into laboratory mice. Pathogenic bacteria will multiply rapidly in the mammalian host while nonpathogenic bacteria will be killed and eliminated by the immune system of the mice. Here the mice serve as the selective medium. Whatever method of isolation of microorganisms might be followed, the aim of all these methods is to isolate pure cultures of microorganisms.

PURE CULTURE Pure culture is defined as a culture containing a growth of a single kind of organism free from other organisms. Pure culture involves not only isolation of individual microorganisms from a mixed population, but also the maintenance of such individuals and their progenies in contaminant free artificial media. However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions. For this, great deal of laboratory manipulation is required. If inoculum from any natural habitat is taken and allowed to grow in a culture medium, a large number of diverse colonies may develop that, due to crowdedness, may run together and, thereby, may lose individuality. Therefore, it is necessary to make the colonies well-isolated from each other so that each appears distinct, large and shows characteristic growth forms. Such colonies may be picked up easily and grown separately for detailed study. Several methods for obtaining pure cultures are in use (See Table 2.2). Some common methods are in everyday use by the majority of microbiologists, while the others are methods used for special purposes. Pure culture of microorganisms that form discrete colonies on solid media, e.g., many bacteria, yeasts, some microfungi, and certain unicellular microalgae, may be most commonly obtained by plating methods. These methods include spread plate method, pour plate method and streak plate method. Table 2.2. Methods of isolation and study of pure cultures of microorganisms Sr. No. I

II

Methods Conventional Methods Spread Plate Method Pour Plate Method Streak Plate Method Special Methods Single Cell Isolation Methods  Capillary Pipette Method  Use of Micromanipulator Culture Independent Molecular Techniques  Partial Community DNA Analysis  Whole community DNA Analysis Novel Methods for Isolating Microorganisms  Use of Dilute Nutrient Medium  Extended Incubation  Simulated Environments  Co-culture

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Techniques for Isolation of Pure Culture 1. Spread Plate Method In this method the mixed culture of microorganisms are diluted in a series of tubes containing sterile liquid, usually, water or physiological saline (Figure 2.1). A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent glass- rod and incubated at ambient temperature required for the growth of microorganisms. After incubation, well isolated colonies of microorganisms can be found on the surface of agar plate due to the separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate. 2. Pour Plate Method This method involves plating of diluted samples mixed with molten agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. The mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The bacteria and the melted medium are mixed well and the contents of each tube are poured into Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, isolated colonies are found to develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another petri plate to ensure purity. Pour plate method has certain disadvantages as follows: a) Subsurface colonies are produced which need to be dug up from within the agar thus interfering with other colonies. b) This technique is unsuitable for psychrophiles since the microbes being isolated must be able to withstand temporary exposure to 42-45° temperature of the liquid agar medium. However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.

3. Streak Plate Method This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is taken on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. These plates are incubated to allow the growth of isolated colonies. The key principle of this method is that by streaking, a dilution gradient is established across the surface of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited thereby giving isolated colonies. Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/needle and re-streaked onto fresh media to ensure purity. The modern streak plate method has progressed from the efforts by Robert Koch and other microbiologists to obtain

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microbiological cultures of bacteria in order to study them. The dilution or isolation by streaking method was first developed by Loeffler and Gaffky in Koch's laboratory. Many variations of the method have evolved with time. The most important variants are zig-zag streak, T-streak and Quadrant streak (Figure 2.2).

Figure 2.1. Serial dilution technique.

Figure 2.2. Streak Plate method. The petriplate is rotated by 45°C after each streak and a new streak is made.

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4. Serial Dilution Method This method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution (See Figure 2.1). The objective here is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. The higher the dilution showing growth of microorganism, the higher is the probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.

Special Methods of Isolation of Pure Culture In addition to the above mentioned methods, various special methods can also be employed for pure culture isolation.

1. Single Cell Isolation Methods An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow. The following two methods are in use: (i) Filter method Several small drops of a suitably diluted culture medium are put on a sterile glasscoverslip by a sterile pipette drawn to a capillary. Then each drop is examined under the microscope until a drop containing only one microorganism is found. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture. (ii) Micromanipulator method Micromanipulators allow the biotechnologists to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette. A hanging drop containing a single microorganism is searched, drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism. This technique ensures that the cultures come from a single cell and one can obtain strains within the species. However, the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. Accordingly, this method is reserved for use in highly specialized studies.

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2.Culture Independent Molecular Techniques for Monitoring Complex Microbial Communities Over the years, the scientific community has realized that only a small minority of microorganisms are readily cultivated in vitro, with the majority of all bacteria remaining ‗unculturable‘ using standard methods. There are currently estimated to be 61 distinct bacterial phyla, of which 31 have no cultivable representatives (Hugenholtz et al. 2009). It is well established that only approximately 1% of bacteria on Earth can be readily cultivated in vitro. This is attributed to the so called ‗Great Plate Count Anomaly‘ which indicates that the microorganisms obtained in culture are much less than those observed microscopically (Staley and Konopka 1985; Amann et al.1995; Hugenholtz et al.1998). The most common reasons attributed to this anomaly is that if the specific requirements for the growth of a bacterium are not met by the artificial medium and incubation conditions, or if there is competition for nutrients among mixtures of organisms cultured together, some bacteria may not grow. Moreover, growth may also be inhibited by bacteriocins released from other bacteria in a mixed culture or by antibacterial substances present within the medium (Tamaki et al. 2005). Yet it is only through the isolation of bacterial species in pure culture that they may be fully characterized, both for their physiological as well as pathological properties. Hence, the endeavor to devise novel cultivation methods for microorganisms that appear to be inherently resistant to artificial culture is the most important one. Molecular ecology methods are now well established for the culture-independent characterization of complex bacterial communities associated with various environmental and animal habitats and are revealing the extent of their diversity (Vartouikian et al. 2010). Advances in the field of molecular biology have made it possible to develop techniques which no longer require the isolation and culture of bacteria. These methods involve lysis of the bacterial cells directly in the soil followed by direct extraction of nucleic acids from the matrix. Thereafter, the targeted sequences or the entire genetic information is analysed. Ranjard and coworkers (2000) have divided the available molecular techniques to study bacterial communities using DNA directly extracted from the soil into two groups as shown in Figure 2.3. (i) Partial community DNA analysis: includes molecular approaches which usually investigate parts of the information by focusing on genome sequences which are targeted and amplified by PCR. Techniques employed involve PCR fragment cloning followed by restriction and/or sequencing analysis and genetic fingerprinting. Genetic fingerprinting techniques are also based on PCR amplification, but do not require a clone library. They are based on the principle of resolving the diversity of the amplified sequences simply by differential electrophoretic migration on agarose or polyacrylamide gels, which depend on their size, including amplified ribosomal DNA restriction analysis (ARDRA), terminal RFLP (t-RFLP), ribosomal intergenic spacer analysis (RISA), Randomly Amplified Polymorphic DNA (RAPD); or upon sequence such as Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE). Of special interest to the reader are two electrophoretic techniques which use denaturing gradient for the separation of DNA sequences that are of same size but vary in their sequence. Both these techniques involve the separation of amplicons on polyacrylamide gels containing a linear gradient of a DNA denaturing agent. This denaturing agent may be chemical (urea or

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Loveleen Kaur and Robinka Khajuria formamide), in which case the method is known as DGGE (denaturing gradient gel electrophoresis) or it may be a physical factor such as the temperature, in which case the name TGGE (temperature gradient gel electrophoresis) is used (Felske et al.1998; Muyzer et al.1993). (ii) Whole community DNA analysis: involves molecular approaches which try to investigate all the genetic information in the extracted DNA.

3. Novel Methods for Isolating Microorganisms Many microorganisms are considered unculturable. Unculturable does not indicate that such microorganisms can never be cultured, instead that current laboratory culturing techniques are unable to grow a given bacterium in the laboratory, probably because we lack critical information on their biology. Culturing such microorganisms may open up vast avenues both in terms of development of novel microbiological techniques and also providing knowledge about previously hidden metabolic diversity. The basic reason that we have not been able to culture microorganisms, even though we know that they exist is that the microbiologists are failing to replicate essential aspects of the environment of these bacteria. Attempt to vary all of the growth conditions at once results in a multidimensional matrix of possibilities which cannot be tested at once under lab conditions.

Figure 2.3. Some molecular techniques to study bacterial communities using DNA directly extracted from the soil.

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In such cases, different methods can be applied in order to increase the chances of culturing such microorganisms. Following are some of the methods which are catching attention of the scientific community. (i) Use of Dilute Nutrient Media: The majority of culture media used to date have been nutrient-rich. These conditions favor the growth of faster-growing bacteria at the expense of slow-growing species, some of which thrive in nutrient poor environments (Koch 1997; Connon and Giovannoni 2002). The use of dilute nutrient media in such cases has led to the successful cultivation of previously unculturable bacteria from various aquatic and terrestrial habitats. Consequently, the use of dilute nutrient media has led to the successful cultivation of previously unculturable bacteria from various aquatic and terrestrial habitats (Watve et al.2000; Connon and Giovannoni 2002; Rappe et al. 2002; Zengler et al. 2002). Certain bacteria, for example, those belonging to the genera Caulobacter can be isolated by inoculating a mixed culture into a medium containing very low concentration of nutrients. This limits the growth of other organisms which are unable to grow at low nutritional levels while encouraging the growth of desired organism. (ii) Extended incubation: Many oligotrophic bacteria are very slow-growing. Extended incubation times are a prerequisite for the cultivation of such bacteria. An added benefit is that faster-growing members within the mixed populations progressively die off over time, reducing the bacterial competition and creating conditions favorable for the growth of slow-growers. The culture of soil bacteria for up to 12 weeks has revealed increasing colony counts and an increased recovery of rarely isolated strains with time (Davis et al. 2005). Long-term incubation for up to 24 weeks has led to isolation of strains from the SAR11 clade (Song et al. 2009). (iii) Simulated Environments: This technique is becoming increasingly important and has yielded many fascinating results. This is because it is difficult to replicate a natural environment if it is not known which of the parameters are important for the growth of a given bacterial taxon from that setting. An alternative is to take the bacteria back to the environment to grow them, by moving a portion of the environment into the laboratory. For example, designing diffusion chamber by enclosing the very dilute suspensions of bacteria within a semipermeable chamber (Epstein and Lewis 2004) or encapsulating single bacterial cells in microdroplets of solidified agarose (Keller et al. 2004) which are kept in simulated environmental conditions will yield pure bacterial cultures. In one such study, Graf and coworkers (2011) used highthroughput sequencing of RNA transcripts to determine that an uncultured Rikenellalike bacterium in the leech gut was utilizing mucin as a carbon and energy source. Using this insight, they were able to culture this isolate on medium containing mucin. According to the authors, the RNA sequence information indicated which genes were actually being expressed in the growing bacterium and the information could be used to design the culture conditions. (iv) Co-culture: The use of co-culture technique is not new. A classic example is the dependence of Haemophilus influenzae on Staphylococcus aureus. The reason for this is that H. influenzae needs an exogenous source of both heme and NAD, for aerobic growth. The heme is released from blood added to the medium, and NAD is released by S. aureus growing in co-culture. While blood normally contains NAD,

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Loveleen Kaur and Robinka Khajuria blood from sheep and other animals commonly used as a source of blood for culture media also contains enzymes that can destroy this factor. The dependence of H. influenzae on S. aureus can be overcome by preheating the blood used in the petri plates (called chocolate agar); this heat treatment both releases heme and inactivates the enzyme that breaks down NAD.

It has been observed that co-culturing or conditioning the media with cell free extracts from helper cells has helped in isolation of species such as Symbiobacterium spp. It is believed that signalling molecules may be responsible for growth promotion. Figure 2.4 highlights some of the mechanisms by which helper microorganisms promote the growth of other microorganisms in co-culture. Zinser et al. (2008) separated a dependent strain of Prochlorococcus (MIT9215) from its heterotrophic helpers. They selected for streptomycinresistant mutants and killed the helper population by treatment with streptomycin, resulting in a pure Prochlorococcus culture. By maintaining this culture at high density (105 cells/ml), they were able to propagate it in pure culture. However, at low densities, MIT9215 could not form isolated colonies on petri plates without adding back the helper bacteria. Further studies suggested that helper bacteria provided catalase to Prochlorococcus for reducing oxidative stress. Supplementing the media with catalase helped in pure culture isolation of Prochlorococcus.

Figure 2.4. Mode of action of helper microorganisms in co-cultures.

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Thus, combining traditional culturing methods with new ways of making the medium more similar to the environment can be a key to culturing the unculturable bacteria. For more information, readers can refer to articles by Vartoukianet al. (2010); Pham and Kim (2012) and Harwani et al.(2013) The isolated microorganisms may have variable capabilities of producing an industrially important metabolite. Accordingly, a filtration/screening strategy needs to be applied in order to screen only the desirable, high yielders from a vast population.

SCREENING OF MICROORGANISMS Screening may be defined as the use of highly selective procedures to allow the detection and isolation of microorganisms of interest from among a large microbial population. Thus to be effective, screening must, in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population. The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs. During screening programs, generally a natural source such as soil is diluted to provide a cell concentration such that aliquots applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300). However, crowded plate technique, which will be described ahead, is an exception to this general rule. As such, primary screening is as a rule succeeded by secondary screening in order to zero in onto a prospective commercially important isolate. Readers will be introduced to a number of examples pertaining to the primary and secondary screening of microorganisms producing industrially important compounds.

Primary Screening Primary screening allows for selection of a large number of microorganisms that exhibit the potential of producing an industrially important compound irrespective of the fact whether the strain could be used at the commercial level of not. Following paragraphs discuss some examples for the primary screening of microorganisms.

1. Primary Screening for Organic Acid/ Amine Producers For primary screening of organic acid or organic amines producers, serially diluted soil sample is plated onto a poorly buffered agar nutrient medium with a pH indicating dye such as Neutral red (Pink to yellow) or Bromothymol blue (Yellow to blue). The production of these compounds is indicated by a change in the color of the indicating dye in the close vicinity of the colony to a color representing an acidic or alkaline reaction. The approach can be improved by using a media of greater buffer capacity so that only those microorganisms that produce considerable quantities of the acid or amine can induce changes in the color of the dye. Alternately 1-2 % calcium carbonate can be incorporated into the medium so that organic acid production is indicated by a cleared zone of dissolved calcium carbonate around

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the colony. However, these procedures have a limitation that inorganic acids or bases also are potential products of microbial growth. For instance, if the nitrogen source of the medium is the nitrogen of ammonium sulfate the organism may utilize the ammonium ion, leaving behind the sulfate ion as sulfuric acid, a condition indistinguishable from organic acid production. Thus, cultures yielding positive reactions require further testing to be sure that an organic acid or base actually has been produced.

2. Primary Screening of Antibiotic Producers The crowded plate technique is the simplest screening technique employed in detection and isolation of antibiotic producers. It consists of preparing a series of dilution of the source material for the antibiotic producing microorganisms, followed by spreading the dilution on the agar plates. The agar plates having 300-400 or more colonies per plate after incubation for 2-4 days are observed since they are helpful in locating the colonies producing antibiotic activity which is indicated by the presence of a zone of inhibition surrounding the colony. It is necessary to carry on further testing to confirm the antibiotic activity associated with a microorganism since zone of inhibition surrounding the colony may sometimes be due to other causes. Notable among these are a marked change in the pH value of the medium resulting from the metabolism of the colony, or rapid utilization of critical nutrients in the immediate vicinity of the colony. Antibiotic screening can be improved, by the incorporation of a ―Test organism‖ that acts as an indicator for the presence of specific antibiotic activity. Dilutions of microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition, a rough approximation of the relative amount of antibiotic produced by various colonies can be gained by measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing colonies again must be isolated and purified before further testing. (iii) Primary Screening of Growth Factor The technique that is most commonly employed for the primary screening of microorganisms that produce growth factors such as vitamins or amino acids is referred to as Auxanography. An important consideration in this regard is that the medium at makeup must be totally lacking in the metabolite under consideration. The microbial source is diluted and plated to provide well-isolated colonies and the test organism is applied to the plates before further incubation. The choice of the particular test organism to be used is critical since the test organism must possess a definite growth requirement for the particular metabolite and for that metabolite only, so that production of this compound will be indicated by zones of growth or at least increased growth of the test organism adjacent to colonies that have produced the metabolite.

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Secondary Screening Secondary screening is strictly essential in any systematic screening program intended to isolate industrially useful microorganisms, since primary screening merely allows the detection and isolation of microbes that possess potentially interesting industrial applications. Moreover, primary screening does not provide much information needed in setting up a new fermentation process. Secondary screening helps in detecting really useful microorganisms in fermentation processes. This can be realized by a careful understanding of the following points associated with secondary screening: It is important for sorting out commercially important microorganisms from a variety of microorganisms obtained during primary screening. In case of a new product, comparative studies with already known product can help to infer whether it will provide more cost effective fermentation process as compared to the known process. Moreover, it can help us to analyze whether a particular product produced by a microorganism is a novel product or not. This may be accomplished by paper, thin layer or other chromatographic techniques. It gives an idea about the economic position of the fermentation process involving the use of a newly discovered culture. Secondary screening also determines the optimum conditions for growth or accumulation of a product associated with a particular culture. It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration is taken into account. It detects gross genetic instability in microbial cultures. This type of information is very important, since microorganisms which tend to undergo mutation or alteration is some way may lose their capability for maximum accumulation of the fermentation products. It gives information about the number of products produced in a single fermentation. Additional major or minor products are of distinct value, since their recovery and sale as by-products can markedly improve the economic status of the prime fermentation. Chemical, physical and biological properties of a product are also determined during secondary screening. Moreover, it reveals whether a product produced in the culture broth occurs in more than one chemical forms. Secondary screening gives answers to many questions that arise during final sorting out of industrially useful microorganisms. This is accomplished by performing experiments on agar plates, in flasks or small bioreactors containing liquid media, or a combination of these approaches. A specific example of antibiotic producing Streptomyces species may be taken for an understanding of the sequence of events during a screening program. Those streptomycetes able to produce antibiotics are detected and isolated in a primary screening program. These streptomycetes exhibiting antimicrobial activity are subjected to an initial secondary screening where their inhibition spectra are determined. A simple ―Giant Colony technique‖ is used to do this. Each of the streptomycetes isolates is streaked in a narrow band across the centers of the nutritional medium containing plates. Then, these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are streaked from the edges of the plates upto but not touching the streptomycete growth. Again, the plates are incubated. At the end of incubation, growth inhibitory zones for each test organism are measured in millimeters. Thus, the microbial inhibition spectrum study extensively helps in discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting microbial inhibition spectra need further testing. With streptomycetes suspected to produce antibiotics with poor solubility in water, the initial secondary screening is done in a different

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manner. Samples are withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory.

MAINTENANCE AND PRESERVATION OF PURE CULTURES Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium, a procedure known as subculturing which allows continuous growth and viability of microorganisms. Subculturing favors the growth of microorganism by providing fresh nutrients and diluting the toxic compounds produced during growth. This is important; otherwise the culture might be lost over a period of time due to degeneration or death. However, since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination at each subculture. Therefore, other methods such as refrigeration, storage in paraffin wax, cryopreservation, and lyophilization (freeze drying) etc. are employed for both short term and long term preservation of microorganisms. These methods have been briefly outlined below.

Refrigeration Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus, their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.

Paraffin Method/ Mineral Oil Storage This is a simple and the most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant of culture which is stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.

Cryopreservation Cryopreservation (i.e., freezing in cultures for long storage times. In this frozen in liquid nitrogen at -196°C. cryoprotectant. Cryoprotectants are the

liquid nitrogen at -196°C) helps survival of pure method, the microorganisms of culture are rapidly The process invariably relies on the use of a chemical compounds, which protect the biological

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material from damage due to formation of ice crystals during the freezing or thawing of the cryopreserved cultures. Cryoprotectants such as glycerol, dimethyl sulfoxide (DMSO), and polyethylene glycol etc. have been used in the preservation of bacterial, fungal, plant and animal cultures. Cryoprotectants are found to eliminate most of the multiple destructive factors during freezing of biological structures (Uzunova and Donev 2005). According to Meryman (1971a, b), the cryoprotective agents prevent the formation of intracellular ice crystals and reduce electrolyte concentration and cell dehydration during cooling process. The major part of World Culture Collection recommends 5 to 10 per cent protecting concentration of DMSO (Sidyakina 1985) and 10 to 15 per cent protecting concentration of glycerol (Emtzeva et al. 1991; Panoff et al. 2000). For freezing at the American Type Culture Collection (ATCC), glycerol and DMSO are routinely employed at 10 per cent and 5 per cent (v/v) concentration, respectively in distilled water (Butterfield et al. 1974; Hwang and Howelle 1968; Davis 1965). Glycerol solutions are sterilized by autoclaving in the manner of ordinary culture media and the DMSO by filtration. Trials carried out have shown that DMSO is an effective cryopreservant for tropical mushroom cultures such as mycelia of Calocybe indica and Volvariella volvacea (Kaur et al.2008) while glycerol is preferred for temperate cultures such as Pleurotus florida and Agaricus bisporus (Kaur et al.2011)

Lyophilization Lyophilization, also known as freeze drying, is a method of preservation where the culture is rapidly frozen at a very low temperature (-30 to -70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures are then sealed and stored in the dark at 4°C in refrigerators as described in Figure 2.5. Lyophilization is associated with a number of advantages such as requirement for minimal storage space and hundreds of lyophilized cultures can be stored in a small area, easy packaging and transportation. Lyophilized cultures are revived by opening the vials, adding liquid medium, and transferring the rehydrated culture to a suitable growth medium. Freeze-drying method is the most frequently used technique by culture collection centers. Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.

Preservation Using Silica Gel Another method of choice for preservation of microorganisms, especially fungi is the use of silica gel as an inert substrate. Neurospora has been successfully preserved over silica gel. Screw cap tubes, half filled with desiccant activated silica gel (6-12 mesh, grade 40) are oven sterilized. After the tubes have cooled, a skim milk (10% v/v) suspension of conidia or mycelium is dispersed (0.5 ml) into each tube. The tubes are quickly cooled to reduce heat generated as the liquid is absorbed and then vortexed to break up clumps. After being dried at 25°C, they are stored in closed containers with desiccants.

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Figure 2.5. Steps for freeze drying of microbial cultures.

Preservation on Paper Spore forming fungi, actinomycetes, and unicellular bacteria can be preserved by drying the spores on some inert substrates. Fruiting bodies of the Myxobacteria, containing myxospores, may be preserved on pieces of sterile filter paper and stored at room temperature

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or at 6°C for 5 to 15 years. Pieces of agar containing fruiting bodies are placed on sterile filter paper in a Petri dish, dried in desiccators under vacuum, and stored at room temperature. Alternatively, vegetative cells are transferred from the growth medium to small pieces of sterile filter paper on water agar and incubated until fruiting bodies develop. After the development, they are allowed to mature for 8 days. The filter papers are then placed into sterile containers, such as screw cap tubes, and dried over silica gel in a desiccator. After few days the containers are tightly closed and stored.

Preservation on Beads The method was developed by Lederberg and is successful for many bacteria. Porcelain beads are autoclaved in screw cap glass vials. Cell suspensions are prepared from 24 to 48 hours culture slants with a 20% (w/v) sucrose solution. The sterile beads are transferred to a sterile Petri dish and inoculated (0.2 - 0.3 ml per bead) with the cell suspension. The beads are returned to the vial with sterile forceps, and the vial is loosely capped and dried in a vacuum desiccator for 72 - 96 hours.

Preservation on Soil For many soil borne species, survival in soil is necessary. Survival for up to 20 years in soil has been reported for many fungi such as Pythium, Fusarium, Verticillium spp etc. Survival for more than 1 year in soil is relatively common for other plant pathogens. Soil free of lumps, rocks, insects etc. is passed through sieves, filled in glass vials and autoclaved. Tyndallization may be carried out in order to sterilize the soil. Thereafter, the sterile soil is inoculated with pure culture of microorganisms and refrigerated.

CULTURE COLLECTION BANKS Microorganisms play an important role in a large number of processes. However, conservation of microbial strains has still not been given its due share. Astronomical numbers of microorganism have been isolated, yet only a very small fraction of microorganisms have been preserved and a large number have been left unattended and lost, after the termination of the project. It is in this context that a global initiative towards conservation of biological resources was established during the Earth Summit in Rio de Janeiro in 1992. During the meeting, the convention on Biological Diversity was drafted with an aim to conserve the natural biological resources. In 1991 (revised in 1999), World Federation for Culture Collections (WFCC) prepared a document that outlines all the activities to be performed by culture collection banks. The WFCC guidelines for the establishment and operation of Collections of Culture of Microorganisms focus on the following aspects: organization, funding, objectives, the kind and number of strains to be maintained, staff services, safety and quality standards. Culture collection banks (see Table 2.3 for a list of some culture collection banks) are encouraged to

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join WFCC and register with the World Data Centre on Microorganisms. Culture Collections are divided into three different categories based on their functions and specialties. Private Culture Collection: This type of culture collection bank is maintained by individuals, hospitals, institutes, laboratories and commercial firms. The cultures with private collections are not open to public. There may be a few to hundreds of cultures in the Private Culture Collection Table 2.3. Major culture collection banks Culture collection American Type culture collection, Maryland America (ATCC)

National Collection of Type Cultures (NCTC), London, UK The National Collection of Yeast Cultures (NCYC), London, UK Japan Collection of Microorganisms (JCM), Japan Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany Belgian Co-ordinated Collections of Microorganisms (BCCM), Belgium Culture Collection, University of Gothenburg (CCUG), Sweden European Culture Collections' Organisation (ECCO), Denmark European Collection of Cell Cultures, Porton Down, UK Microbial Culture Collection, Pune, India SERI microalgae culture collection, USA Microbial type culture Collection Collection of International Mycological institutes, Surrey, UK National Collections of Industrial, Food and Marine bacteria, Aberdeen, UK Collection Nationale de Cultures de Microorganismes (CNCM), France

Cultures Standard reference microorganisms, cell lines and other materials for research in the life science; more than 4,000 human, animal and plant cell lines and an additional 1,200 hybridomas. Over 5000 bacterial cultures, over 100 mycoplasmas and more than 500 plasmids, host strains, bacteriophages and transposons. Over 4,000 yeast cultures Approximately 8,300 strains of bacteria, 370 strains of archaea and 4,800 strains of fungi including yeasts About 30,000 cultures, such as 19,000 different cultures of microorganisms, 750 plant cell cultures, 550 plant viruses, 740 human and animal cell lines, and 6700 patent and safe deposits 47,500 strains of bacteria, filamentous and yeast fungi and 950 plasmids. Bacterial, filamentous fungal and yeasts cultures except extremophiles and biosafety level 3 cultures. Various microbial cultures Cell lines. Various microbial cultures. Algal cultures Actinomycetes, Bacteria, Fungi, Yeasts and Plasmids Mycological cultures 8,000 strains of bacteria, plasmids and bacteriophages Bacteria, Recombinant bacteria, hybridomas, animal and human cell lines, fungi, yeasts, phages and viruses

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Public culture collection: Public collections consist of large number of strains which are for public service. The number of cultures present varies amongst different collection centers. Specialized collection: These collections deal with a particular group of microorganisms having specialized functions, for example fungal cultures, microorganisms used in brewing, plant pathology etc.

STRAIN IMPROVEMENT Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal (Adrio and Demain 2006). However, depending on the system, it may be desirable to isolate strains which require shorter fermentation times, which do not produce undesirable pigments or other by-products, which have reduced oxygen needs, which exhibit decreased foaming during fermentation, or which are able to metabolize inexpensive substrates. Additionally, wild strains frequently produce a mixture of chemically closely related substances which is generally undesirable for a commercially important system. Thus, mutants that synthesize one component as the main product are preferable, since they make possible a simplified process for product recovery. Studies have shown that the changes in the genotype of microorganisms can lead to the biosynthesis of new metabolites. Thus, mutants which synthesize modified antibiotics may be selected.

Mutation Microorganisms can generate new genetic characters chiefly by two means: mutation and genetic recombination. In mutation, a gene is modified either unintentially (leading to spontaneous mutation) or intentially (referred to as induced mutation). Although the change is usually detrimental and eliminated by selection, some mutations are beneficial to the microorganism. Even if it is not beneficial to the organism, but beneficial to humans, the mutation can be detected by screening and can be preserved indefinitely. This is indeed what the fermentation microbiologists did in the strain development programs that led to the great expansion of the fermentation industry in the second half of the twentieth century. The early studies in basic genetics concentrated on the production of mutants and their properties. The ease with which ‗permanent‘ characteristics of microorganisms could be changed by mutation and the simplicity of the mutation techniques had tremendous appeal to microbiologists. The most common method used to obtain high yielding mutants is to treat a population with a mutagenic agent until a certain ‗desired‘ kill is obtained, plate out the survivors and test each resulting colony or a randomly selected group of colonies for product formation in flasks. The most useful mutagens include nitrosoguanidine (NTG), 4-nitroquinolone-1-oxide, methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), hydroxylamine (HA) and ultraviolet light (UV). The optimum level of kill for increased production of antibiotics is

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thought to be in the range 70–95% (Simpson and Caten 1979), although some industrial programs use much higher levels, e.g. up to 99.99%. Although single cells or spores are preferred for mutagenesis, non-spore-forming filamentous organisms have been mutated successfully by mutagenizing mycelia, preparing protoplasts and regenerating on solid medium (Keller, 1983). The following paragraphs deal with brief discussion on different types of industrially important mutants.

(i) Mutants Producing Increased Quantities of Metabolites Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decrease in costs can be attributed to a large extent to mutagenesis and selection of higher quantities producing microbial strains. At least five different classes of genes control metabolite production (Malik 1979): (i) structural genes coding for product synthases, (ii) regulatory genes determining the onset and expression of structural genes, (iii) resistance genes determining the resistance of the producer to its own antibiotic, (iv) permeability genes regulating entry, exclusion and excretion of the product, and (v) regulatory genes controlling pathways providing precursors and cofactors. Microorganism can be developed to overproduce microbial metabolites by (i) increasing precursor pools, (ii) adding, modifying or deleting regulatory genes, (iii) altering promoter, terminator and/or regulatory sequences, (iv) increasing copy number of genes encoding enzymes catalyzing bottleneck reactions, and (v) removing competing unnecessary pathways (Strohl 2001). Morphological mutants have been very important in strain improvement. Although almost nothing is known about the mechanisms causing higher production in superior random or morphological mutants, it is likely that many of these mutations involve regulatory genes, especially as regulatory mutants obtained in basic genetic studies are sometimes found to be altered in colonial morphology. These include mutants affected in mycelia formation, which produce colonies with a modified appearance or a new color. Color changes have also been important for pigment producers. This increased yield may be several times that formed by the parent culture. Haggag and Mohamed (2002) studied the influence of gamma radiation on improving anti-fungal metabolities like phenolic compounds and antibiotics including gliotoxin, trichodermine and viridine in Trichoderma species against Sclerotium cepivorum and found a significant increase in metabolite production over the wild type strains. (ii) Auxotrophic Mutants Very early in the development of the concepts of regulation, geneticists realized that the end product of a biosynthetic pathway to a primary metabolite exercises strict control over the amount of an intermediate accumulated by an auxotrophic mutant of that pathway. The accumulation of end product of the substrate of the deficient enzyme generally occurs at a growth limiting concentration of the end product in order to bypass feedback inhibition or repression. The production of secondary products such as antibiotics is also markedly affected by auxotrophic mutation, even when auxotrophs are grown in nutritionally complete and even complex media. Examples include the overproduction of phenylalanine by tyrosine auxotrophs and vice versa, and the overproduction of lysine by auxotrophs requiring threonine and methionine. The levels of primary metabolities in micro-organisms are regulated by feedback control systems including feedback inhibition and feedback repression.

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In feedback inhibition, the end product of biochemical pathway inhibits the activity of an enzyme that catalyses one of the reactions of the pathway whereas in feedback repression, the end product of a biochemical reaction prevents the synthesis of an enzyme catalyzing some reaction of the pathway. Figure 2.6 gives the control of biosynthesis of arginine in C. glutamicum. In the case of branched pathways leading to a primary metabolite and a secondary metabolite, auxotrophic mutants requiring the primary metabolite sometimes overproduce the secondary metabolite. Auxotrophic mutants of Corynebacterium glutamicum have been used for the synthesis of lysine by feedback inhibition of Aspartokinase enzyme by lysine and threonine as shown in Figure 2.7.

Figure 2.6. Control of the biosynthesis of arginine by C. glutamicum.

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Figure 2.7. The control of Aspartate family of amino acids by C. glutamicum.

Moreover, reversion of an auxotroph to prototrophy sometimes leads to new prototrophs possessing higher enzyme activity than present in the original ‗grandparent‘ prototroph. Such increased enzyme activity is probably the result of a structural gene mutation producing a more active enzyme or an enzyme less subject to feedback inhibition.

(iii) Antimetabolite-Resistant Mutants Basic studies on regulation have shown that it is possible to select regulatory mutants, which overproduce the end products of primary pathways and using toxic metabolite analogues.

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Such antimetabolite-resistant mutants often possess enzymes that are insensitive to feedback inhibition, or enzyme-forming systems resistant to feedback repression. A variation of the antimetabolite selection techniques is possible when a precursor is toxic to the producing organism. The principle here is that the mutant most capable of detoxifying the precursor by incorporating it into the antibiotic will be the best grower in the presence of the precursor. When the secondary metabolite produced is itself a growth inhibitor of the producing culture, as in the case of certain antibiotics, the metabolite can sometimes be used to select resistant mutants that are improved producers. Scherr and Rafelson (1968) described a method whereby mutants having a directed increased yield of metabolites such as pyridoxine, nicotinic acid and thiamine may readily be isolated. In this method, a culture of bacterial cells is exposed to a gradient concentration of an antimetabolite that is inhibitory to the growth of the organism and mutants are isolated which survive because of an increased yield of the metabolite.

(iv) Mutants Resistant to Nutritional Repression Nutritional repression can also be decreased by mutation to antimetabolite resistance. Examples of selection agents are 2-deoxyglucose (2-DOG) for enzymes and pathways controlled by carbon source regulation, methyl ammonium for those regulated by nitrogen source repression, and arsenate for phosphate regulation. Qiu et al. (2011) were able to screen Phanerochaete chrysosporium mutants resisting nutritional repression by using the guaiacol nitrogen sufficient differential medium and also characterized laccase produced by the mutants. Moreover, they characterized enzyme production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions. Mutants that use phosphate less efficiently for growth sometimes show improved antibiotic production. Thus, screening for small colonies on phosphate-limiting media could be a useful strain improvement technique for phosphate-regulated products. (v) Permeability Mutants Product excretion in overproducing strains often occurs when uptake and/or catabolism is impaired. Thus, genetic lesions eliminating active uptake can be used to specifically enhance excretion of metabolites. It is often of benefit to isolate mutants unable to grow on the desired product as sole carbon or energy source. Such mutants are often impaired in their ability to take up the product and they contain lower intracellular levels of the product, thus lessening feedback regulation. In certain improved mutants, there is an increase in sensitivity to deoxycholate and lysozyme, indicating a change in permeability. A classic example in this regard is the isolation of a mutant of Corynebacterium glutamicum which was deficient in the production of biotin as well as in the synthesis of enzyme α-ketoglutarate dehydrogenase by Kinoshita et al. (1957). Biotin deficient strains do not produce proper membrane and are thus deficient in selective permeability. Moreover, defect in production of the enzyme αketoglutarate dehydrogenase does not allow succinic acid formation from α-ketoglutaric acid. The α-ketoglutaric acid is diverted to glutamic acid synthesis. Oxaloacetate is regenerated by the activity of the glyoxalate pathway as shown in Figure 2.8.

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Figure 2.8. Biosynthesis of glutamate by C. glutamicum.

(vi) Mutants Producing New Antibiotics Mutant methodology has been used to produce new molecules. The medically useful products dimethyltetracycline and doxorubicin were discovered by simple mutation of the cultures producing tetracycline and daunorubicin, respectively. Smirnov et al. (2000) were able to successfully generate insertion mutants of Pseudomonas which were capable of producing a new antibiotic batumin by transposon Tn5. An analysis of over 7000 clones was done in order to select the mutant clones with increased and decreased levels of batumin synthesis and the mutants that lost the ability to synthesize this antibiotic.

Genetic Recombination Genetic material derived from one species can be transferred to another species by means of recombinant DNA Technology. In contrast to the extensive use of mutation in industry, genetic recombination was not much used at first, despite early claims of success (Jarai 1961; Mindlin 1969), mainly due to the absence or the extremely low frequency of genetic recombination in industrial microorganisms (in Streptomycetes, it was usually 10−6 or even less). Other problems were evident with the β-lactam-producing fungi. Although Aspergillus

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exhibited sexual and parasexual reproduction, the most commercially interesting genera, Cephalosporium and Penicillium, were the most difficult to work with as they only reproduced parasexually, which rarely resulted in recombination. After 1980, there was a heightened interest in the application of genetic recombination to the production of important microbial products such as antibiotics. Recombination is especially useful when combined with conventional mutation programs to solve the problem of ‗sickly‘ organisms produced as a result of accumulated genetic damage over a series of mutagenized generations.

Protoplast Fusion Protoplasts are cells devoid of their cell walls. Protoplast fusion has been widely used as a method of choice for exchange of genetic material across species barrier in a wide range of microorganisms in order to improve their characteristics. The process of protoplast fusion is depicted in Figure 2.9. For example, a cross via protoplast fusion was carried out with strains of Cephalosporium acremonium for a commercial strain improvement program. A low-titer, rapidly-growing, spore-forming strain which required methionine to optimally produce cephalosporin C was crossed with a high-titer, slow-growing, asporogenous strain which could use the less expensive inorganic sulfate. The progeny included a recombinant which grew rapidly, sporulated, produced cephalosporin C from sulfate and made 40% more antibiotic than the high-titer parent (Hamlyn and Ball 1979). Recently, mild electric stimulation is being used to fuse protoplast in preference to Polyethylene glycol mediated fusion (Kaur et al. 2014). Avram et al.(1992) employed electrofusion for hybrid construction in ergosterol-producing yeast strains. Some fusion products proved to be hybrid with respect to ergosterol content and to remain stable over several generations. Liu et al. (1996) were able to prepare an L-tryptophan auxotroph and milky mutants from an inducible cholesterol oxidase-producing bacterium, Arthrobacter simplex USA18 and a constitutive cholesterol oxidase producer strain US3011. Upon PEG induced protoplast fusion, the fusion frequency was about 1.5–1.7×10−3. The cholesterol oxidase activity of four fusants in a cholesterolcontaining medium was 20–60% higher than that of parental strains. Similarly, Tokdar et al.(2014) fused protoplasts isolated from induced mutants of Paracoccus denitrificans ATCC 19367 having antibiotic resistant markers. Among the generated fusants, one fusant namely PF-P1 showed 1.73 folds enhancements in specific CoQ10 content than wild type strain.

Improvement of Microbial Processes by Genetic Engineering New processes for the production of amino acids and vitamins have been developed by recombinant DNA technology. Escherichia coli strains were constructed with plasmids bearing amino acid biosynthetic operons. Plasmid transformation was accomplished in Corynebacterium, Brevibacterium and Serratia and as a result, recombinant DNA technology has been used routinely to improve such commercial amino acid-producing strains (Sahm et al. 2000). Genetic Engineering has been able to improve many characteristics of baking, brewing and wine making yeasts including improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate

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utilization (Dequin 2001). A recombinant strain of E. coli (made by mutating to isoleucine auxotrophy, cloning in extra copies of the thrABC operon, inactivating the threoninedegrading gene tdh, and mutating to resistance to high concentrations of L-threonine and Lhomoserine) produced 80 g L−1 L-threonine in 1.5 days at a yield of 50% (Eggeling and Sahm 1999). Cloning extra copies of threonine export genes into E. coli led to increased threonine production (Kruse et al. 2002). A process for riboflavin production in Corynebacterium ammoniagenes (previously Brevibacterium ammoniagenes) was developed by cloning and overexpressing the organism's own riboflavin biosynthesis genes (Koizumi et al. 2000) and its own promoter sequences. The resulting culture produced 15.3 g L−1 riboflavin in 3 days. A recombinant E. coli strain was constructed that produced optically active pure D-lactic acid from glucose at virtually the theoretical maximum yield, i.e. two molecules from one molecule of glucose (Zhou et al. 2003). The organism was engineered by eliminating genes of competing pathways encoding fumarate reductase, alcohol/aldehyde dehydrogenase and pyruvate formate lyase and by a mutation in the acetate kinase gene. The application of recombinant DNA technology to the production of secondary metabolites has been of great interest (Baltz and Hosted 1996; Diez et al.1997). The tools of the recombinant geneticist for increasing the titers of secondary metabolites have included: (i) transposition mutagenesis, (ii) targeted deletions and duplications by genetic engineering and (iii) genetic recombination by protoplast fusion (Baltz 2003). Recent additions to these techniques include genomics, transcriptome analysis, proteomics, metabolic engineering, and whole genome shuffling.

Figure 2.9. Process of protoplast fusion.

Isolation and Screening of Industrially Important Microorganisms

45

Genes encoding many microbial enzymes have been cloned and the enzymes expressed at levels hundreds of times higher than those naturally produced. Recombinant DNA technology has been used (Falch 1991): (i) to produce industrial enzymes obtained from microbes that are difficult to grow or handle genetically; (ii) to increase enzyme productivity by use of multiple gene copies, strong promoters, and efficient signal sequences; (iii) to produce useful enzymes obtained from a pathogenic or toxin-producing microorganism; in a safe host and (iv) to improve the stability, activity or specificity of an enzyme by protein engineering. The industrial enzyme business adopted rDNA methods to increase production levels and to produce enzymes from industrially-unknown microorganisms in industrial organisms such as species of Aspergillus and Trichoderma, as well as Kluyveromyces lactis, S. cerevisiae, Yarrowia lipolytica and Bacillus licheniformis. The properties of many enzymes have been altered by genetic means. ‗Brute force‘ mutagenesis and random screening of microorganisms over the years led to changes in pH optimum, thermostability, feedback inhibition, carbon source inhibition, substrate specificity, Vmax, Km and Ki. This information was later exploited by the more rational techniques of protein engineering. Single changes in amino acid sequences have yielded similar types of changes in a large variety of enzymes. Today, it is no longer necessary to settle for the natural properties of an enzyme; these can be altered to suit the needs of the investigator or the process. Dashtban and Qin (2012) successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of Trichoderma reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The recombinant β-glucosidase was thermotolerant and remained fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintained about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0.

Novel Genetic Technologies New technologies that have proven to be very useful for increasing production of primary metabolites include genome-based strain reconstruction, metabolic engineering, and whole genome shuffling. A genomic technique called ‗genome-based strain reconstruction‘ allows one to construct a strain superior to the production strain because it only contains mutations crucial to hyperproduction, but not other unknown mutations which accumulate by brute-force mutagenesis and screening (Ohnishi et al. 2002). The production of amino acids shows many examples of this approach. A useful review of metabolic engineering in C. glutamicum, especially in relation to L-lysine production, was published by Sahm and colleagues (2000). Metabolic flux studies of wild-type C. glutamicum and four improved lysine-producing mutants available from the ATCC showed that yield increased from 1.2% to 24.9% relative to the glucose flux. Other recent examples are on overproduction of aromatic amino acids and derivatives (Bongaerts et al. 2001), L-lysine (Wittmann and Heinzle 2002) and glutamate (Kimura 2003). There are many other successful applications of metabolic engineering for products such as 1,3-propanediol (Nakamura and Whited 2003), carotenoids (Rohlin et al. 2001; Visser

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Loveleen Kaur and Robinka Khajuria

et al. 2003; Wang and Keasling 2003), organic acids (Kramer et al. 2003), ethanol (Nissen et al. 2000), vitamins (Zamboni et al. 2003; Sybesma et al. 2004) and complex polyketides in bacteria (Pfeifer et al. 2001; Khosla and Keasling 2003). During the last few years, an expanded view of the cell has been possible due to impressive advances in all the ‗omics‘ techniques (genomics, proteomics, metabolomics, etc.) and high-throughput technologies for measuring different classes of key intracellular molecules. ‗Systems biology‘ has emerged as a term to describe an approach that considers genome-scale and cell-wide measurements in elucidating processes and mechanisms (Stephanopoulos et al. 2004). However, progress in strain development will depend, not only on all the technologies mentioned above, but also on the development of mathematical methods that facilitate the elucidation of mechanisms and identification of genetic targets for modification. A genome-wide transcript expression analysis called ‗massive parallel signature sequencing‘ (Brenner et al. 2000) was used successfully to discover new targets for further improvement of riboflavin production by the fungus A. gossypii (Karos et al. 2004). The authors identified 53 genes of known function, some of which could clearly be related to riboflavin production. This approach also allowed the finding of sites within the genome with high transcriptional activity during riboflavin biosynthesis that are suitable integration loci for the target genes found. These recent technologies and mathematical approaches have been contributing to the generation and characterization of microorganisms able to synthesize large quantities of commercially important metabolites. The ongoing sequencing projects involving hundreds of genomes, the availability of sequences corresponding to model organisms, new DNA microarray and proteomics tools, as well as the new techniques for mutagenesis and recombination described above will accelerate strain improvement programs. ‗Directed evolution‘ is a rapid and inexpensive way of finding variants of existing enzymes that work better than naturally occurring enzymes under specific conditions (Arnold 1998). The process involves evolutionary design methods using random mutagenesis, gene recombination and high-throughput screening (Arnold 2001). ‗Molecular breeding techniques‘ (DNA shuffling, Molecular Breeding™) not only recombine DNA fragments, but also introduce point mutations at a very low controlled rate (Stemmer 1994; Zhao and Arnold 1997). ‗Whole genome shuffling (WGS)‘ is a novel technique for strain improvement combining the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes. This method was applied successfully to improve acid-tolerance of a commercial lactic acid-producing Lactobacillus sp. (Patnaik et al. 2002). ‗Combinatorial biosynthesis‘ is being used for the discovery of new and modified drugs (Reeves 2003). In this technique, recombinant DNA techniques are utilized to introduce genes coding for antibiotic synthases into producers of other antibiotics or into non-producing strains to obtain modified or hybrid antibiotics.

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Koizumi, S., Yonetani, Y., Maruyama, A. & Teshiba, S. (2000). Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes. Appl Microbiol Biotechnol,51, 674-679. Kramer, M., Bongaerts, J., Bovenberg, R., Kremer, S., Müller, U., Orf, S., Wubbolts, M.&Raeven, L. (2003). Metabolic engineering for microbial production of shikimic acid. Metab Eng,5, 277-283. Kruse, D., Kraemer, R., Eggeling, L., Rieping, M., Pfefferle, W., Tchieu, J. H., Chung, Y. J., Saier, M. H. Jr. &Burkorski, A. (2002). Influence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol,59, 205-210. Liu, W. H., Chow, L. W. & Lo, C. K. (1996). Strain improvement of Arthrobacter simplex by protoplast fusion. Journal of Industrial Microbiology,16(4)., 257-260. Malik, V. S. (1979). Genetics of applied microbiology. Adv Genet,29, 37-126. Meryman, H. T. (1971a). Cryoprotective agents. Cryobiology,8, 173-83. Meryman, H. T. (1971b). Osmotic stress as a mechanism of freezing injury. Cryobiology,8, 489-500. Mindlin, S. Z (1969). Genetic recombination in the actinomycete breeding. Genetics and Breeding of Streptomyces (Sermonti G, Alacevic M, eds.)., Yugoslav Academy of Sciences and Arts, Zagreb, 147-159. Muyzer, G., De Waal, E. C. & Uitterlinden, A. G (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol,59, 695-700. Nakamura, C. E. &Whited, G. M. (2003). Metabolic engineering for the microbial production of 1,3-propanediol.Curr Opin Biotechnol,14, 1-6. Nissen, T. L., Kielland-Brandt, M. C., Nielsen, J. & Villadsen, J. (2000). Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng,2, 69-77. Ohnishi, J., Mitsuhashi, S., Hayashi, M., Ando, S., Yokoi, H., Ochiai, K. &Ikeda, M. (2002). A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl Microbiol Biotechnol,58, 217-223. Panoff, J. M., Thammavongs, B. & Gueguen, M. (2000). Cryoprotectants lead to phenotypic adaptations to freeze-thaw stress in Lactobacillus delbruekii sp. bulgaricus. Cryobiology,40, 264-69. Patnaik, R., Louie, S., Gavrilovic, V., Perry, K., Stemmer, W. P. C., Ryan, C. M. &del Cardayre, S. (2002). Genome shuffling of Lactobacillus for improved acid tolerance. Nature Biotechnol,20, 707-712. Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E. &Khosla, C. (2001). Biosynthesis of complex polyketides in a metabolically engineered strain of Escherichia coli.Science,291, 1790-1792. Pham, V. H. T. & Kim, J. (2012). Cultivation of unculturable soil bacteria. Trends in Biotechnology,30(9), 475-484. Preobrazhenskayasi, T. P., Lavrova, N. V. M., Ukholina, R. S. & Nechaeva, N. P. (1975). Isolation of new species of Actinomadura on selective media with streptomycin and bruneomycin. Antibiotiki,30, 404-408. Qiu, A., Li, W., Zheng, Y., Fan, X., Ye, Y.& Meng, Y. (2011). Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression. Wei Sheng Wu Xue Bao, 51(3)., 352-359.

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Chapter 3

MEDIA DESIGN Once the microorganisms have been isolated and screened based upon their commercially important properties, the microorganisms need to undergo extensive studies for their characterization in order to be used at a large scale. In order to study industrially important microorganisms such as bacteria, it is important that they be obtained as pure cultures. Thus, successful cultivation of microorganisms requires a sound knowledge about the physical as well as nutritional characteristics affecting their growth. However, this is not an easy task since microorganisms are extraordinarily diverse in their requirements for growth. Microorganisms are greatly affected by environmental conditions and will grow in accordance to how these environmental niches support their individual needs. Factors that affect microbial growth include but are not limited to, pH, osmolarity, water activity, temperature and oxygen levels. There is a great deal of nutritional diversity among microorganisms; therefore, microbial growth is greatly affected by the nutrients that are available in their environment. All life forms ranging from microorganisms to plants to animals including humans share certain common nutritional requirements for growth and maintenance of cellular structure. Table 3.1 outlines the various elements required for growth by the microorganisms. Like all living organisms, the bacteria require certain essential nutritional components which will be discussed in this chapter. This will be followed by a detailed account of growth media, its formulation, preparation and sterilization. Finally certain aspects related to the economics of the fermentation media will be discussed.

NUTRITIONAL REQUIREMENTS OF MICROORGANISMS Source of energy: Like all living organisms, microorganisms require a source of energy. Light energy from the sun is converted to chemical energy stored temporarily in the bonds of organic compounds such as sugars or starches via photosynthesis. This energy is released, as the bonds of compounds are broken down by the organisms such as microbes and is utilized by them for their metabolic functions. The organisms that rely on radiant energy (light) as their energy source are known as phototrophs.eg. Rhodospirullum rubrum is a phototrophic bacterium. On the other hand, chemotrophs such as Escherichia coli obtain energy from the oxidation of organic or inorganic chemical compounds.

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Loveleen Kaur and Robinka Khajuria Table 3.1. Major elements, their sources and functions in bacterial cells

Carbon

% of dry weight 50

Oxygen

20

Nitrogen

14

Hydrogen

8

Phosphorus

3

Sulfur

1

Potassium

1

Organic compounds or CO2 H2O, organic compounds, CO2, and O2 NH3, NO3, nitrogen containing organic compounds, N2 H2O, organic compounds, H2 Inorganic phosphates (PO4) SO4, H2S, S°, organic sulfur compounds Potassium salts

Magnesium

0.5

Magnesium salts

Calcium

0.5

Calcium salts

Iron

0.2

Iron salts

Element

Source

Function Main constituent of cellular material Constituent of cell material and cell water; O2 is electron acceptor in aerobic respiration Constituent of amino acids, nucleic acids nucleotides and coenzymes Main constituent of organic compounds and cell water Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoic acids Constituent of cysteine, methionine, glutathione, several coenzymes Main cellular inorganic cation and cofactor for certain enzymes Inorganic cellular cation, cofactor for certain enzymatic reactions Inorganic cellular cation, cofactor for certain enzymes and a component of endospores Component of cytochromes and certain non-heme iron-proteins and a cofactor for some enzymatic reactions

Source of electrons: All organisms require a source of electrons for carrying out metabolic reactions. Depending on the source of electrons, microorganisms can be either Lithotrophs or Organotrophs. The microorganisms that rely on reduction of inorganic chemical compounds as electron donors are designated as lithotrophs while the ones which use organic compounds as electron donors are called organotrophs. Bacillus, Clostridium and members of Enterobacteriaceae are examples of organotrophs while Thiobacillus and Beggiatoa are lithotrophs which gain energy by oxidizing hydrogen sulfide to sulfur. Source of Carbon: In order to synthesize their cellular components, all organisms require a source of carbon. Accordingly, they may be classified as autotrophs or heterotrophs. The organisms that can make use of CO2 as their sole source of carbon are known as autotrophs while those that make use of organic compounds as their carbon source are assigned to the heterotroph group. The common colon bacterium E. coli is a heterotroph while Nitromonas europa is an autotrophic bacterium. Depending upon the above characteristics, most microorganisms can be categorized as belonging to one of four major nutritional types depending on their sources of carbon, energy, and electrons: Photolithotrophic autotrophs, Photoorganotrophic heterotrophs, Chemolithotrophic autotrophs or Chemoorganotrophic heterotrophs. Apart from these,

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another group called mixotrophs is also found that combines autotrophic and heterotrophic metabolic processes, relying on inorganic energy sources and organic carbon sources. Nitrogen: Nitrogen is one of the chief components of all cellular systems. Some bacteria, such as nitrogen fixing eubacteria and some cyanobacteria can fix atmospheric nitrogen. Some microorganisms depend upon inorganic nitrogen compounds while still others make use of organic compounds such as amino acids as their nitrogen source. Oxygen, Phosphorus and Sulfur: Obligate aerobes must grow in the presence of oxygen. At the same time obligate anaerobes do not carry out oxidative phosphorylation. Furthermore, they are killed by oxygen since they lack certain enzymes such as catalase (which breaks down hydrogen peroxide to water and oxygen), peroxidase (by which NADH + H2O2 are converted to NAD+ and O2) and superoxide dismutase (by which superoxide, O2., is converted to H2O2). These enzymes detoxify peroxide and oxygen free radicals produced during metabolism in the presence of oxygen. Aerotolerant anaerobes are bacteria that respire anaerobically, but can survive in the presence of oxygen. Facultative anaerobes can perform both fermentation and aerobic respiration. In the presence of oxygen, anaerobic respiration is generally shut down and these organisms respire aerobically. Another group of bacteria called microaerophilic bacteria grow well in low concentrations of oxygen, but are killed by higher concentrations. Phosphorous is needed for the synthesis of nucleic acids, nucleotides, cofactors, etc. Almost all microbes use inorganic phosphate as a source of phosphorous. However, some microbes utilize organophosphates. Apart from oxygen and phosphorous, sulfur is needed for the synthesis of certain amino acids and vitamins. Most microbes reduce SO42- to sulfur while some reduce the amino acids cysteine or methionine to acquire sulfur. Major and minor elements: Macroelements are required in significant amounts, the lack of which can limit growth of the microorganisms. C, O, H, N, S, P are the major elements which serve as the components of proteins, carbohydrates, lipids, and nucleic acids. K, Ca, Mg, Fe are needed as ions since they play important roles in microbial physiology. Table 1 outlines all the important major elements required by all the life forms including bacteria. Microelements (trace elements) are required in minute amounts and are usually not a growth limiting factor due to their ubiquitous nature. Since they are required in such small amounts, they are present as "contaminants" of the water or other media components. As metal ions, the trace elements usually act as cofactors for essential enzymatic reactions in the cell. One organism's trace element may be another's required element and vice-versa, but the usual cations that qualify as trace elements in bacterial nutrition are Mn, Co, Zn, Ni, Cu, and Mo. It has been seen that some microbes have special requirements that reflect their morphology or environment, e.g., diatoms need silicon as in silicic acid. Vitamins and growth factors: Growth factors are organic compounds that are essential cellular components that cannot be made by a microbe. Three types of growth factor have been identified which determine the growth of microorganisms in laboratory conditions. These include amino acids, purines and pyrimidines and vitamins which are small organic compounds that make up all or part of enzyme cofactors. Some microorganisms produce vitamins themselves and can thus support the growth of other organisms. For instance Lactobacilli produce both extracellular and intracellular folate. Water: All organisms need water for their survival. Water acts as a solvent and is required for all the metabolic reactions of the cell.

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All these components need to be supplied to the bacteria in order to support their growth in laboratory conditions. The nutritional medium which is primarily used to support the growth of microbes in laboratory scale vessels constitutes the growth or culture medium while the one which is used to obtain larger volumes of products at pilot plant or industrial scale constitutes the production medium.

GROWTH MEDIA In order to cultivate an industrially important bacterium for any purpose such as biomass or metabolite production, it is necessary to provide the appropriate biochemical and biophysical environment. The culture medium is responsible for the biochemical (nutritional) environment. A culture medium is a liquid or gel designed to support the growth of microorganisms or cells. Depending upon the special needs of particular bacteria, different types of culture media have been developed with different purposes and uses. Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties. Microbiological culture media are defined according to DIN EN 1659 (1996) and contain a large variety of ingredients to foster microbial growth including nutrients (extracts, digests of proteins, etc.). Some components include:     

Enrichments (essential ingredients for auxotrophic or fastidious microorganisms) Antioxidants and neutralizing agents against toxic substances like disinfectants (e. g. blood, coal, letheen) Source of energy (e. g. Glucose) Salts for maintaining adequate osmolarity (e.g. NaCl) Buffer for pH-regulation (e. g. potassium salts, sodium salts)

Types of Media 1. Based on Consistency: Solid, Semisolid and Liquid Media The manner in which bacteria are cultivated, and the purpose of culture media, varies widely. Liquid medium also known as broth is used for growth of pure batch cultures, while solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. The most common and frequently used gelling agent for solid or semisolid medium is agar, a hydrocolloid derived from red algae from genera Gelidium and Gracilaria. Agar is a unique compound due to certain physical properties that it possesses. For instance, it melts at 100°C and remains liquid until cooled to 40°C, (the temperature at which it gels). It is an inert component of the medium which cannot be metabolized by most bacteria. 2. Based on Composition: Defined and Undefined Media Culture media may be classified into several categories depending on their composition or use. A chemically-defined medium, also known as synthetic medium is the one in which

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the exact chemical composition is known. A defined medium is a minimal medium if it provides only the exact nutrients (including any growth factors) needed by the organism for growth. The use of defined minimal media requires the investigator to know the exact nutritional requirements of the organisms in question. Chemically defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. On the contrary, a complex (undefined) medium is one in which the exact chemical constitution of the medium is not known. Defined media are usually composed of pure biochemicals whereas complex media usually contain complex materials of biological origin such as blood, milk, yeast extract or beef extract, the exact chemical composition of which is obviously undetermined. Complex media usually provide all the growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria with complex nutritional requirement (i.e., organisms that require a lot of growth factors, known or unknown). Such organisms are known as fastidious. Hence, complex media are usually used for cultivation of bacterial pathogens and other fastidious bacteria.

3. Special Purpose Media A number of special purpose media have been devised depending upon the requirement. Following sections give a brief outline of a few of these media. Basal media are used for the growth or culture of microorganisms without any enrichment of the media. Examples include nutrient broth, nutrient agar, peptone water etc. which support the growth of numerous microorganisms. Selective media are those media that allow the growth of certain type of organisms, while inhibiting the growth of other organisms. This selectivity is achieved in several ways. For instance, organisms that have the ability to utilize a given sugar are screened easily by making that particular sugar the only carbon source in the medium for the growth of the microorganism. Like-wise, the growth of certain microorganisms can be achieved by the selective inhibition of some other types of microorganisms. This can be done by adding certain dyes, antibiotics, salts or specific inhibitors that will affect the metabolism or enzymatic systems of the organisms. For example, tellurite agar medium containing potassium tellurite, sodium azide or thallium acetate at different concentrations which inhibit the growth of all Gram-negative bacteria. On the other hand, media supplemented with the antibiotic penicillin at concentration of 5-50 units/ml or crystal violet @ 2 mg/l inhibit the growth of Gram-positive bacteria. Thus, Tellurite agar is used to select for Gram-positive organisms, and nutrient agar supplemented with the antibiotics such as penicillin can be used to select for the growth of Gram negative organisms. Other examples include Mannitol salt agar, Hektoen enteric agar (HE), Phenylethyl alcohol agar etc. Differential media also known as indicator media are widely used for differentiating closely related organisms or groups of organisms. Because of the presence of certain dyes or chemicals in the media, the organisms will produce certain characteristic changes or growth patterns that are used for identification or differentiation of microorganism. Examples are MacConkey agar, Eosin Methylene Blue (EMB) agar and blood agar. Enrichment medium is a medium with specific and known qualities that favor the growth of a particular microorganism. The enrichment culture's environment will support the growth of a selected microorganism, while inhibiting the growth of others. Selenite broth is used to selectively isolate Salmonella species. Alkaline Peptone Water is used for the cultivation of

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Vibrio. Both these examples are clinically relevant for clinical microbiology relating to stool samples. Enriched Media are enriched usually by adding blood, serum, egg etc. Examples include blood agar media for Streptococci and Lowenstein Jensen medium for selective cultivation of Mycobacterium. Transport Media are used when the specimen cannot be cultured immediately after collection and needs to be transported to a distant site for study. Examples are Cary-Blair medium, Amies medium and Stuart medium. Storage Media are used for preserving the microorganisms for a long period of time. These media donot contain a readily metabolizable carbon source, thereby reducing the growth for long term storage. Examples of storage media are egg saline medium, chalk cooked meat broth etc.

ROLE OF MEDIA IN FERMENTATION Media are required in several stages of most industrial fermentation processes. They may include inoculum propagation (which is known as starter culture), pilot-scale fermentations and the main production fermentation. The technical objectives of inoculum propagation and the main fermentation are often very different, which may be reflected in differences in their media formulations. Where biomass or primary metabolites are the target product, a production medium is designed so as to allow optimal growth of the microorganism for maximum biomass production. Conversely, for secondary metabolite production, such as antibiotics, biosynthesis is not growth related. Consequently, for this purpose, media are designed to provide an initial period of cell growth, followed by conditions optimized for secondary metabolite production. In many cases, the supply of one or more nutrients (carbon, phosphorus or nitrogen source) may be limited and rapid growth ceases. Some considerations made when formulating media for fermentation:    

 

An optimally balanced culture medium containing all critical elements is mandatory for maximal production. The composition of culture media must be adapted to the fermentation process. In addition to product yield, product recovery must be examined in trial fermentations. If catabolite repression or phosphate repression cannot be eliminated by optimization of the nutrient medium or suitable fermentation management, deregulated mutants must be used as production strains. Besides material cost and product yield, it must be considered whether materials used are readily available in sufficient supply without high transportation costs. It should also be considered whether impurities will hinder product recovery or increase the cost of product recovery.

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Media Preparation Medium formulation is a very important step in fermentation process. Most fermentation processes require liquid media. However, many solid-substrate fermentations are also known, such as enzyme fermentations. It is essential that the fermentation media satisfies all the nutritional requirements of the microorganisms and fulfill the technical objectives of the industrial process (Waites and Morgan 2001). Since media are required in a number of stages during the fermentation process, it becomes imperative on a large scale to select those nutrients for formulation of media which meet most of the set criteria in a fermentation industry. Table 3.2 gives some industrially important media for carrying out various fermentations. Stanbury et al. (2011) have outlined certain criteria for an ideal medium: i. ii. iii. iv. v. vi. vii.

It should produce the maximum yield of product or biomass per gm of substrate used. It should produce maximum concentration of product or biomass. It should permit the maximum rate of product formation. There should be the minimum yield of undesired products. It should be of a consistent quality and be readily available throughout the year. It should cause minimal problems during media preparation and sterilization. It should cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment.

In order to successfully formulate a fermentation medium, it is of utmost importance that one should have a sound knowledge of the overall process including the stoichiometry for growth and product formation. For this, consideration of the input of the carbon and nitrogen sources, minerals and oxygen and their conversion to cell biomass, metabolic products, carbon-dioxide, water and heat is very important as such information can help one to calculate the minimum quantities of each element required to produce a certain quantity of biomass or metabolite. The elemental formula of microbial cells can be represented roughly as C4H7O2N, which on the basis of dry weight is 48% C, 7% H, 32%O and 14% N. Ideally, a knowledge of the complete elemental composition of the specific industrial microorganism allows further medium refinement. This ensures that no element is limiting, unless this is desired for a specific purpose. An aerobic fermentation process is generally represented as: Carbon and energy source + Nitrogen source + O2 + other requirements → Biomass + products + CO2 + H2O + heat On the basis of this information, the minimum quantities of each element including carbon source, energy source, nitrogen source, aeration etc. required for producing a particular quantity of biomass and metabolic end product may be calculated. Once the amounts of nutrients to be incorporated have been detected, then suitable nutrient sources can be added into the media (Stanbury et al. 2011).

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Loveleen Kaur and Robinka Khajuria Table 3.2. Composition of some industrially important media Citric Acid (Jernejc et al. 1982)

Amylase (Hartman and Tetrault 1955)

Deionized sugar140 gl-1 NH4NO32,500 mgl-1 MgSO4.7H2O250 mgl-1 KH2PO41,000 mgl-1 CuSO4.5H2O40 mgl-1 pH2.5 Citric Acid (Kumari et al. (2008)

Tripticase2% N-Z-Case0.4% KNO30.5-0.75% Malt syrup/Dimalt1% Soluble starch0.1%

KH2PO47 gl-1 Na2HPO47 gl-1 MgSO4.7H2O1.5 gl-1 CaCl20.15 gl-1 ZnSO4.7H2O0.02 gl-1 MnSO4.H2O0.06 gl-1 FeCl3.6H2O0.15 gl-1 Glycerol38.77 gl-1 (NH4)2SO40.401 gl-1

Glucose3% Casein1% (NH4)2SO40.2% NaCl0.1% KH2PO40.04% CaCO30.5% K2SO40.6% MgSO4.7H2O0.02% FeSO4. 7H2O0.001% ZnSO4. 7H2O0.005% Endotoxin from Bacillus thuringiensis (Holmberg et al. 1980) Molasses0.4% Soy flour2-6% KH2PO40.5% K2HPO40.5% MgSO4.7H2O0.005% MnSO4.4H2O0.003% FeSO4. 7H2O0.001% CaCl20.005% Na(NH4)2PO4.4H2O0.15% Lysine Cane Blackstrap molasses Soybean meal hydrolysate20% CaCO3 or MgSO4 (as buffer) Antifoam agent

Penicillin (Gordon et al. 1947) CSL30 ml/L Lactose40 g/L CaCO310g/L Phenylacetamide0.25 g/L Antifoam1500 ml/L Itaconic Acid ( Nubel and Ratajak 1962) Cane molasses150 g dm-3 ZnSO41 g dm-3 ZnSO4.7H2O3 g dm-3 CuSO4. 5H2O0.01 g dm-3

Streptomycin (Shirato and Motoyama 1966)

Another consideration during media formulation is the scale of the fermentation. It is desirable that for small scale laboratory fermentations pure chemicals be used in well defined media. Pure chemicals are also desirable when fermentation is carried out for many purified end products of high value since crude media components can cause a problem in recovery of the product. However, this is not possible for most industrial-scale fermentation processes, simply due to cost, as media components may account for up to 60-80% of process expenditure. However, many industrial scale fermentation processes use cost effective complex substrates, where many carbon and nitrogen sources are almost indefinable (Waites and Morgan 2001). Most are derived from natural plant and animal materials, often by-products of other industries, with varied and variable composition. For example, for preparing a

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medium for culturing yeast for animal consumption, vegetable waste matter can be used as a starting material, although, such a waste matter is not suitable for direct feeding of animals. Corn-cob and/or stalks, reeds, sunflower stalks, sewage, wheat and paddy straw etc. are other crude substrates of interest.

Properties of Media Due to the inconsistency of the raw substrate, small-scale trials are usually performed with each new batch of substrate, particularly to examine the impact on product yield and product recovery. The main factors that affect the final choice of individual raw materials are as follows: 1. Cost and availability: ideally, materials should be inexpensive, of consistent quality and should have year round availability. 2. Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g. requirements for temperature control. 3. Sterilization requirements and any potential denaturation problems. 4. Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages. 5. The concentration of target product attained, its rate of formation and yield per gram of substrate utilized. 6. The levels and range of impurities, and the potential for generating further undesired products during the process. 7. Overall health and safety implications. A medium that is easily sterilized with minimum thermal damage is vitally important. Thermal damage not only reduces the level of specific ingredients, but can also produce potentially inhibitory by-products that may also interfere with downstream processing. Let us now discuss all the desired components of a fermentation medium.

Medium Components Carbon sources A carbon source is required for all biosynthesis leading to multiplication of cells, cell maintenance and product formation. Additionally, it serves as the energy source. Carbon requirements may be determined from the biomass yield coefficient (Y), which is an index of the efficiency of conversion of a substrate into the cellular material: Ycarbon (g/g) = biomass produced (g)/Carbon substrate utilized (g) Carbohydrates are traditional carbon and energy sources for microbial fermentation, although other sources are increasingly gathering importance, such as alcohols, alkanes and

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organic acids. In addition to main carbon source, animal fats and plant oils may be incorporated into some media as supplements (Crueger and Crueger 1990, Stanbury et al. 1995). A large number of raw materials can act as a carbon source in the medium. Following are some of the commercially used C sources: Simple sugars such as pure glucose or sucrose can seldom be used as the sole carbon source. Since these substrates are relatively expensive, so they are used primarily for the production of high cost, low volume products. Molasses, a byproduct of sugar production, is one of the cheapest sources of carbohydrates. Besides a large amount of sugar, molasses contains nitrogenous substances, vitamins, and trace elements. However, the composition of molasses varies depending on the raw material used for sugar production. Additionally, it requires removal of certain metal ions which can reduce the yield of the fermentation product. Malt extract, an aqueous extract of malted barley, is an excellent substrate for many fungi, yeasts, and actinomycetes. Dry malt extract consists of about 90-92% carbohydrates, and is composed of hexoses (glucose, fructose), disaccharides (maltose, sucrose), trisaccharides (maltotriose), and dextrins. In addition to carbohydrates, malt extracts serve as a rich source of nitrogen. Nitrogenous substances present in malt extract include proteins, peptides, amino acid, purines, pyrimidines, and vitamins. The amino acids composition of different malt extracts varies according to the grain used, but proline always makes up about 50% of the total amino acids present. However, sterilization of medium containing malt extract needs special consideration and should be done carefully. This is because when overheating occurs, the Maillard reaction results, due to the low pH value and the high proportion of reducing sugar. In this conversion, the amino groups of amines, amino acid (especially lysine), or proteins react with the carbonyl groups of reducing sugars, aldehydes or ketones, which results in the formation of brown condensation products. These reaction products are not suitable substrates for microorganisms and may inhibit their growth. The Maillard reaction is one of the main causes of damage to culture media during heat sterilization, resulting in considerably reduced yields. Starch and dextrins can be directly metabolized as carbon sources by microorganisms that are capable of producing amylase. In addition to glucose syrup, which is frequently used as a fermentation substrate, starch is finding more importance as a substrate for ethanol fermentation. Sulfite waste liquors are sugar-containing waste products of the paper industry. Sulphite waste liquors have a dry weight of 9-13% and are primarily used in the cultivation of yeasts. Sulfite liquors from coniferous trees have a total sugar content of 2-3%, and 80% of the sugars are hexoses (glucose, mannose, galactose), the others being pentoses (xylose, arabinose). Sulfite liquors from deciduous trees contain mainly pentose sugars. Cellulose is being extensively studied as a substrate for conversion to sugar or alcohol due to its wide availability and low cost. It is usually not possible to use cellulose directly as a carbon source, so it must first be hydrolyzed chemically or enzymatically. The sugar syrup formed from cellulose hydrolysis has been used for ethanol fermentation, and the fermentative production of butanol, acetone, and isopropanol is also being considered. Research groups are working on developing one-step processes for direct conversion of cellulose to ethanol, using fermentative organisms which produce cellulases. Whey, a byproduct of the dairy industry, is produced annually on a world-wide basis to the amount of 74 million tons which might contain 1.2 million tons of lactose and 0.2 million tons of milk protein. Only about 56% of this product is used for human or animal feed. The

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lactose is used primarily for the production of ethanol or single-cell protein, but is also used in the production of xanthan gum, vitamin B12, 2,3-butandiol, lactic acid, and gibberellic acid. However, because of storage and transportation costs, whey is often not economical as a substrate. Animal fats such as lard, animal and plant oils are readily utilized by some microorganisms, but are generally added as supplemental substrates rather than as the sole fermentable carbon source. For instance, in certain antibiotic fermentations, soy, palm, and olive oils are used. Oils contain approximately 2.4 times the energy of glucose on per weight basis and also occupy less volume, thereby, giving a better fermenter working volume. Methanol is the cheapest fermentation substrate with respect to its carbon content, but it can be metabolized by only a few bacteria and yeasts. Methanol has commonly been used as a substrate for single cell protein production. Research has been carried out on processes for the production of glutamic acid, serine, and vitamin B12 using methanol as the sole carbon source or as a co-substrate. Ethanol is available in ample supply from the fermentation of either saccharified starch or cellulose, and can be metabolized by many microorganisms as the sole carbon source or as a co-substrate. Acetic acid, for instance, is presently made by the oxidation of ethanol. However, at the present time, the cost of ethanol is too high to make it utilizable as a general industrial carbon source. Hydrocarbons such as n-alkanes with a chain length of C12 to C18 are readily metabolized by many microorganisms. The use of alkanes as an alternative to carbohydrates depends on the price of petroleum. On a dry weight basis, n-alkanes have approximately twice the carbon and thrice the energy content of the same weight of sugar. It was believed that hydrocarbons and their derivatives might be used as feedstocks in fermentation of high value products such as pharmaceuticals, fine chemicals and agricultural chemicals (Drozd 1987).

Nitrogen Sources Most of the industrially important microorganisms can utilize both inorganic and organic nitrogen sources. Many fermentations such as antibiotic fermentations are influenced by type and concentration of nitrogen source in the culture medium (Aharonowitz 1980).Table 3.3 deals with the different kinds of nitrogen sources used in various fermentation processes. 1.

Inorganic nitrogen may be supplied as ammonium salts, often ammonium sulfate and diammonium hydrogen phosphate, or ammonia. These are readily available to the microorganisms. Ammonia can also be use to adjust the pH of the fermentation medium. Many large-scale processes utilize ammonium salts, urea, or gaseous ammonia as nitrogen source. However, ammonium salts such as ammonium sulfate usually lead to decrease in pH of the fermentation broth when ammonium ions are utilized, thereby generating free acid. On the other hand, ammonium nitrate leads to alkaline conditions. Initially, a drop in pH is observed as ammonium ions are utilized and acid is produced. At this stage nitrate assimilation is repressed since in the presence of ammonium ions, the activity of nitrate reductase (an enzyme that converts nitrate to ammonium ions) is repressed. Once the ammonium ions are exhausted, nitrate is used as an alternative source (Morton and MacMillan 1954).

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Loveleen Kaur and Robinka Khajuria 2. Organic nitrogen sources include amino acids, proteins and urea. In many fermentations, nitrogen is supplied in crude forms that are essentially by-products of other industries, such as corn steep liquor, yeast extracts, peptones and soya meal. Growth rate is increased with a supply of organic nitrogen. A few microorganisms have an absolute requirement of amino acids. Purified amino acids act as precursors for specific products and are thus added accordingly (Waites and Morgan 2001).

A nitrogen source, which is efficiently metabolized, is corn steep liquor, which is formed during starch production from corn. The concentrated extract (about 4% nitrogen) contains numerous amino acids, such as alanine, arginine, glutamic acid, isoleucine, threonine, valine, phenylalanine, methionine and cystine. The sugar present in corn steep liquor is largely converted to lactic acid (9-20%) by lactic acid bacteria. Cornsteep Liquor, in addition to acting as a carbon and nitrogen source, also acts as a precursor in penicillin production. Complex nitrogen sources when used in antibiotic fermentation help in creating favourable physiological conditions in exponential phase of growth (trophophase) that lead to antibiotic production in stationary phase (idiophase, See Figure 1.2). Yeast extracts are other excellent substrates for many microorganisms. They are produced from baker‘s yeast through autolysis/plasmolysis in the presence of high concentrations of NaCl. Yeast extract contains amino acids and peptides, water-soluble vitamins and carbohydrates. The glycogen and trehalose of yeast cells are hydrolyzed to glucose during yeast extract production. The composition of yeast extract varies partly because the substrates used for yeast cultivation affect the quality of the yeast extract. Peptones are protein hydrolysates which can be utilized by many microorganisms but they are relatively expensive for industrial application. Sources of peptones include meat, casein, gelatin, keratin, peanut seeds, soy meal, cotton seeds, and sunflower seeds. Peptone composition varies depending upon its origin. For instance, peptone from gelatin is rich in proline and hydroxyproline, but has almost no sulfur-containing amino acids. On the other hand, peptone from keratin has a large proportion of proline and cysteine, but lacks lysine. Peptones of plant origin such as soy peptone and cottonseed peptone have large proportions of carbohydrates. The end product is also influenced by the type of hydrolysis, whether acid or enzymatic, especially in regard to its tryptophan content. Table 3.3. Best nitrogen sources for some secondary metabolites (Adapted from Stanbury et al. 2011) Secondary Metabolite Penicillin Bacitracin Riboflavin Novobiocin Rifomycin Gibberellins Butirosin Polyenes

Nitrogen source in fermentation medium Corn steep liquor Peanut granules Pancreatic digest of gelatin Distiller‘s soluble Soybean meal, (NH4)2SO4 Ammonium salts and natural plant nitrogen source Dried beef blood or hemoglobin with (NH4)2SO4 Soybean meal

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Soy meal, the residue from soybeans after the extraction of soybean oil, is a complex substrate which contains a protein content of 50%, a carbohydrate content of 30% (sucrose, stachyose, raffinose, arabinoglucan, arabinan, and acidic polysaccharides), 1% residual fat, and 1.8% lecithin. Soy meal is frequently used in, antibiotic fermentations; catabolite regulation does not occur because of the slow catabolism of this complex mixture.

Water Most fermentation, except solid substrate fermentation, require large quantities of water in which medium is formulated. Water is required in heating, cooling, cleaning and rinsing. Many early industrial establishments were located close to a river or stream which provided ample, clean water for use. It also provides trace mineral elements. The mineral content of water is of utmost importance in breweries and decides the type of beer produced. For instance, hard water containing CaSO4 is good for producing English bitter beer while water rich in carbonate content is better for stout beer. Prior to use, removal of suspended solids, colloids and microorganisms and removal of hardness is usually required. In order to minimize water costs, recycle/ reusage of water is practiced, which also reduces the volume requiring waste water treatment (Stanbury et al. 1995). Readers can refer to topic by Levi et al. (1979) for water reusage.

Minerals Usually sufficient quantities of cobalt, copper, iron, manganese, molybdenum and zinc are also present in water supplies. Moreover, certain trace elements also occur as impurities in other media ingredients. For example, corn steep liquor satisfies the requirements of minor and trace mineral needs. Specific salts of calcium, magnesium, phosphorus, potassium, sulfur and chloride ions have to be added in the medium as inorganic salts fulfill the requirements (Waites and Morgan 2001). For more details on concentration of various minerals required in fermentation media, refer to table 3.4. Table 3.4. Typical forms and concentrations of mineral components in fermentation medium (Adapted from Stanbury et al. 2011) Component KH2PO4 MgSO4.7H2O KCl CaCO3 FeSO4.4H2O ZnSO4. 8H2O MnSO4. H2O CuSO4.5H2O Na2MoO4.2H2O

Concentration 1.0-4.0 0.25-3.0 0.5-12.0 5.0-17.0 0.01-0.1 0.1-1.0 0.01-0.1 0.003-0.01 0.01-0.1

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Vitamins and Growth Factors Some bacteria, filamentous fungi and yeasts cannot synthesize all necessary vitamins from basic elements; they must be added as supplements to the fermentation medium. Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants. Many vitamins such as B-complex vitamins are rapidly destroyed at elevated temperatures. Garrett (1956) studied vitamin stability at elevated temperature in liquid preparations. When a single vitamin is required by a culture, it is better to use it in pure form as it will be more economical than using a larger amount of cheaper source. Other necessary growth factors, amino acids, nucleotides, fatty acids and sterols are added either in pure form, or as plant and animal extracts (Stanbury et al. 1995).

Precursors Specific precursors have to be added in some fermentations, notably for secondary metabolite production. Precursors are the chemicals which when added to the fermentation media are directly incorporated into the desired product. They are often added in controlled quantities and in a relatively pure form. For example, D-threonine is used as a precursor in Lisoleucine production by Serratia marsescens (Kisumi et al. 1973). Earliest evidences of use of precursors was an increase in the yield of penicillin from 20 units cm-3 to 100 units cm-3 on addition of corn steep liquor in the medium which can be attributed to the presence of phenylethylamine. It has now become standard practice to add phenyl acetic acid to the medium in order to yield benzyl penicillin (penicillin G, Smith and Bide 1948). Some other examples of precursors has been outlined in the Table 3.5.

Inducers and Elicitors Majority of enzymes which are of industrial use, being inducible, require a specific inducer or a structural analogue, which must be incorporated into the culture medium or added at some specific stage. Inducers are often necessary in fermentation by genetically modified microorganisms (GMMs) (Waites et al. 2009). Common examples include use of substrates such as starch for amylase production, pectin for pectinase production and cellulase for cellulose production. In certain cases analogues of common substrates might be used as inducers (Table 3.5). While deciding on inducers it is important that cost consideration be taken into the account. For example: Fatty acids induce the production of lipase, however their inclusion in fermentation medium might not be financially feasible (Crueger and Demain 1990). A classical example is the use of yeast mannan in streptomycin production (Inamine et al. 1969). During streptomycin fermentation both streptomycin and mannosidostreptomycin are produced. Mannosidostreptomycin has only about 20% of the biological efficiency of streptomycin, making it an undesirable product. Addition of yeast mannan induces the production of β- mannosidase by Streptomyces griseus which converts mannosidostreptomycin into streptomycin.

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Table 3.5. Examples of industrially important precursors and metabolic regulators in fermentation industry Compound Used Precursors used Phenylacetic acid and related compounds Phenoxyacetic acid Chloride Cyanides β-Iononones α- amino butyric acid D-Threonine Inhibitors Sodium bisulphite Bromide Penicillin Alkali metal/phosphate, ph4% chromium), mild steel (coated with glass or epoxy material), wood, plastic or concrete may be

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used as vessel construction material depending upon the requirement of the fermentation process. One of the essential features of the used construction material is that it should be non-toxic and corrosion proof. Glass vessel: is generally made up of borosilicate glass. Glass vessels generally are of two types: Type I – glass vessels with round or flat bottom with top plate which can be sterilized by autoclaving. The largest type I glass vessels have a diameter of 60cm. Type II – glass vessel with flat bottom and with top and bottom stainless steel plate. This type is used in in situ sterilization process and the largest vessel has a diameter of 30cm. Stainless steel: Pilot scale and industrial scale vessels are normally constructed of stainless steel or at least have stainless steel cladding to limit corrosion. According to American Iron and Steel Institute, steels containing less than 4% chromium are considered as alloys and those containing more than 4% chromium are classified as stainless steels. Stainless steel is corrosion resistant due to the presence of a thin hydrous oxide film on the surface of the metal. This film is stabilized by chromium and is considered to be non-porous, continuous and self-healing. Increasing chromium concentration enhances the resistance to corrosion, but only steels with 10-13% chromium are able to develop effective films. Inclusion of nickel in to the stainless steel increases carrion resistance and improves engineering. Presence of molybdenum provides resistance to halogen salts, brine, and sea water.

Figure.4.1. Components of a typical Fermenter (Stanbury et al., 1995).

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Sealing An important factor to be considered while designing a fermenter is to ensure proper sealing between the top plate and the vessel to maintain airtight and aseptic conditions. Sealing needs to be carried out between three types of surfaces viz. between glass-glass, glassmetal and metal-metal. There are three main types of sealing used in a fermenter. For glass and metal, a seal can be made with a compressible gasket, lipseal and ‗O‘ ring (Figure 4.2). For metal- metal sealings, only O ring is used. This sealing made up of fabric-nitryl or butyl rubbers ensures tight joint in spite of expansion of vessel material during fermentation. The seals should be changed after finite time.

Aeration and Agitation The purpose of aeration is to provide oxygen to microorganism submerged in the media for carrying out various metabolic operations and the medium must be suitably stirred (agitated) to keep the cells in suspension and to make the culture homogeneous. The objective of stirring is to achieve good mixing without causing damage to the cells. Efficient aeration is achieved by bubbling air through the medium (sparging), but this may damage animal cells due to the high surface energy of the bubble and on the cell membrane. The damage can be reduced by using larger bubbles, lower gassing rates and by adding non-nutritional supplements like Pluronic F-68 (polyglycol) and sodium carboxymcthyl cellulose (these protect cells from damage due to shear forces and bubbles, respectively). Aeration may be achieved by medium perfusion, in which medium is continuously taken from culture vessel, passed through an oxygenation chamber and returned to the culture. The cells are removed from the medium taken for perfusion so that the medium can be suitably altered, e.g., for pH control. Where considered safe and desirable, O2 supply in the culture vessel can be enhanced from the normal 21% to a higher value and the air pressure can be increased by 1 atmosphere. This increases the O2 solubility and diffusion rates in the medium, but there is a risk of O2 toxicity.

Figure 4.2. Joint Seals a) Gasket b) Lip seal c) O ring (Stanbury et al. 1995).

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The type of aeration-agitation system used in the fermenter is dictated by the characteristics of the fermentation process. The following components of the fermenter are a part of aeration and agitation system: a) Agitator or Impeller b) Stirrer glands and bearings c) Baffles and d) Sparger or the aeration system.

a) Agitator Agitators are required in a fermenter for achieving the following objectives: bulk fluid and gas-phase mixing, air dispersion, oxygen transfer, heat transfer, suspension of solid particles and maintenance of a uniform environment throughout the vessel. There are different types of agitators used in fermenters depending on the requirements of the fermentation process (Figure 4.3).These include: (i) Disc turbines: Disc turbine consists of a disc with a series of rectangular vanes set in a vertical plane around its circumference. Disc turbine prevents flooding by air bubbles. Flooding occurs when the air bubble is not properly dispersed and air pockets are formed. The medium is flooded at 120min/hour of air discharge when disc turbine is used as compared to open turbine and propeller where flooding is observed at 21min per hour of air discharge. (ii) Vaned discs: The vaned disc turbine has a series of rectangular vanes attached vertically to the underside of the disc. Air from the sparger hits the underside of the disc and is displaced towards the vanes where air bubbles are broken up into smaller bubbles. (iii) Open turbines with variable pitch: In case of variable pitch open turbine, the vanes are attached directly to a boss on the agitator shaft. In this case the air bubbles do not initially hit any surface before dispersion by the vanes or blades. (iv) Propellers: The marine propeller is similar to variable pitch open turbine, except that it has blades in the place of vanes. In this case also, air bubbles contact the vanes/blades directly and are broken up and dispersed by them. Since the 1940, Rushton disc turbine with 1/3 of fermenter diameter has been optimised for fermentation process. It has been experimentally proven that the disc turbines are the most suitable in a fermenter as it could break up a fast air stream without itself becoming flooded. With the advancements in fermentation process, new designs of agitator have been introduced. Scaba is one such agitator that can handle high flow rate before flooding and has radial flow. Prochem maxflow agitator has low power conception with high hydrodynamic thrust. This design has increased downward pumping capacity of blades. In this design agitator/vessel diameter ratio is 0.4. Appoximately 66% less power requirement is there along with improvement in oxygen transfer efficiency. These basic agitation devices have been variously modified so as to meet the needs of the fermentor. For example, the variable pitch open turbine scheme has been modified to develop four modern agitator types, viz., Lightning A315 and the Ekato Intermig. These new agitators are larger, require lower power input (they do not lose as much power as the Rushton turbines when aerated), are able to handle higher air volumes without flooding, and give better bulk blending and heat transfer in more viscous media. But they can cause mechanical problems mostly of vibrational nature. Good mixing and aeration in high viscosity broths may also be achieved by a dual impeller combination in

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which the lower impeller primarily dispenses the air, while the upper impeller primarily enhances mixing of the broth.

b) Stirrer Glands and Bearings Maintenance of aseptic conditions over long periods is one of the most critical aspects of fermentation process which requires a satisfactory sealing of the stirrer shaft assembly. The entry point of stirrer into fermenter may be from top to bottom or sides. Mostly used entry point is from the bottom as it leaves more space for entry ports on top. There are three types of stirrer glands and bearings: (i) Stuffing box: Comprises of several layers of packing rings of asbestos or cotton yarnpressed against the shaft by a gland follower. The drawback of this type of seal is that at high speeds, the packing wears. Also the packing is difficult to sterilize due to unsatisfactory heat penetration. (ii) Mechanical seal: comprises of two parts: stationary part in the bearing housing and the other rotates on the shaft. The two parts are pressed together by springs or expanding bellows. Steam condensate is used to lubricate and cool the seals during operation and servers as a contaminant barrier. Most modern fermenters use mechanical seals; these seals are more expensive, but they are more durable and less prone to leakage or contaminant entry. (iii) Magnetic drives: comprise of two magnets one driving and the other driven. The driving magnet is held in the bearing in the housing on outside of head plate and connected to drive shaft. The driven one is placed on one end of impeller shaft held in bearing in suitable housing. Magnetic drives, although quite expensive, are being used in some animal cell culture vessels.

3. Baffles Baffles are metal strips roughly one-tenth of the vessel diameter and attached radially to the fermenter wall. They are normally used in fermenters having agitators to prevent vortex formation and to improve aeration efficiency. Usually, four baffles are used, but larger fermenters may have 6 or 8 baffles. Extra cooling coils may be attached to baffles to improve cooling. Further, the baffles may be installed in such a way that a gap exists between the baffles and the fermenter wall. This would lead to a scouring action around and behind the baffles, which would minimise microbial growth on the baffles and the fermenter wall. 4. Aeration System (Sparger) Spargers are used for introducing air into the fermenter. Fine bubble aerators must be used for introducing air which will facilitate oxygen transfer to a greater extent as large bubbles will have less surface area than smaller bubbles. Agitation is not required when aeration provides enough agitation which is the case in Air lift fermenter. But this is possible with medium having low viscosity and low total solids. For aeration to provide agitation, the vessel height/diameter ratio (aspect ratio) should be 5:1.There are three types of sparger viz. porous sparger, orifice sparger and nozzle sparger.

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Figure 4.3. Types of agitators: a) Disc turbines b) Vaned discs c) Open turbines with variable Pitch d) Propellers (Stanbury et al. 1995).

(i) Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non agitated vessel. The size of the bubble formed is 10-100 times larger than pore size. There is a pressure drop across the sparger and the holes tend to be blocked by growth which is the limitation of porous sparger. (ii) Orifice sparger: used in small stirred fermenters. It is a perforated pipe kept below the impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air holes are drilled on the under surfaces of the tubes and the holes are at least 6mm in diameter. This type of sparger is used mostly with agitation. It is also used without agitation in some cases like yeast manufacture, effluent treatment and production of SCP. (iii) Nozzle sparger: Mostly used in large scale fermenters. It is single open/partially closed pipe positioned centrally below the impeller. When air is passed through this pipe there is lower pressure loss and it does not get blocked. In small fermenters, a combined sparger-agitator may be used. In this case, the air is introduced via a hollow agitator shaft, and it comes out through holes drilled in the disc between the blades and connected to the base of the main shaft. This design gives a good aeration in baffled vessels over a range of agitator speeds.

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Temperature Regulation The fermenter must have an adequate provision for temperature control. Metabolic activities of the microorganism and agitation lead to generation of heat. Now, if this heat generates a temperature that is optimum for the fermentation process, then heat removal or addition may not be required. But in most cases, this is not true; in all such cases, either addition of heating or removal of the excess heat is required. Temperature control may be considered at laboratory scale, and pilot and production scales. 1. In laboratory scale fermentations, normally little heat is generated. Therefore, heat has to be added to the system; this can be achieved in the following ways: (a) the fermenter may be placed in thermostatically controlled bath, (b) internal heating coils may be used, (c) water may be circulated through a heating jacket, or (d) a silicone healing jacket may be used. The silicone jacket consists of two silicone rubber mats, and heating wires between these mats. This jacket is wrapped around the fermenter and is held in place by Velcro strips. 2. In case of larger fermenters beyond a certain size, excess heat is generated, and the fermenter surface becomes inadequate for heat removal. The size at which fermenter surface becomes inadequate for heat removal will depend on the fermentation process and the ambient temperature at which fermentation is being carried out. In such cases, internal coils have to be used to circulate cold water through them for removing the excess heat. The cooling surface area necessary for temperature control will depend mainly on the following factors: (i) temperature of cooling water, (ii) the culture temperature, (iii) the type of microorganism, and (iv) the energy provided by stirring. The cooling water consumed during bacterial fermentation in a vessel of a size around 55,000 L would range between 500 to 2,000 Lh-1. Fungal fermentation, however, may need 2,000 to 10,000 L cooling water per hour as they have a lower optimum temperature for growth.

Maintenance of Aseptic Conditions Once parameters like aeration, agitation and temperature regulation are optimised, the next step is to ensure that the fermenter design meets the requirements of the degree of asepsis and containment required of a fermentation process. Maintenance of aseptic conditions requires a three way approach: 1. Sterilization of the Fermenter: The fermenter should be designed in such a manner that it can be sterilised under pressure. The media may be sterilised in the fermenter or separately. In case of in situ sterilization of the medium, its temperature should be raised before injection of the live steam to prevent formation of large amount of condensate. This can be achieved by introducing steam in fermenter coils or steam jackets. Also every point of entry and exit in a fermenter is a potential source of contamination; therefore steam should be introduced through every entry and exit point. All the pipes should be constructed so as to ensure that steam reaches each and every part of the fermenter.

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2. Sterilization of the air supply: Aerobic fermentations require a very large volume of sterile air. There are two main ways of sterilizing air: by heat which is not economically viable at industrial scale, and filtration. Previously, glass wool, mineral slag wool or glass fibre were used for filtration of air but now most of the fermenters use cartridge-type filters with a membrane pore size of 0.2 to 0.3 µm for sterilization. 3. Sterilization of exhaust gases: is normally carried out by using a 0.2µm filter on the outlet pipe. One of the problems encountered in this method is plugging of the filter due to the moisture and solid matter released during fermentation in the form of an aerosol. To avoid this, cyclone separators for solids and coalesce for liquids are included in the upstream of the filters in series.

Addition of Inoculum, Nutrients and Other Supplements To maintain aseptic conditions when operating a fermenter, it is essential that the fermenter and the addition vessel are maintained at positive pressure and the addition ports are sterilised with steam supply prior to release of inoculum or nutrients into the fermenter. While introducing inoculum into the fermenter, care should be taken that the microorganisms are not released into the external environment. This is normally achieved by aseptic piercing of the membrane or connections with steam locks. Addition of nutrients, acid or alkali in small fermenters is normally done with the help of silicone tubes which are sterilised separately and pumped by a peristaltic pump. In large fermenters, the feed reservoirs and associated piping is an integrated part of the fermenter and is sterilised along with the vessel by using steam.

Foam Control Foam is produced during most microbial fermentations. Foaming may occur either due to a medium component, e.g., protein present in the medium, or due to some compound produced by the microorganism. Foaming can cause removal of cells from the medium; such cells undergo autolysis and release more proteins into the medium which in turn, further stabilizes the foam. Five different patterns of foaming that are recognized are listed below. 1. Foaming remains at a constant level throughout the fermentation. Initial foaming is due to the medium, but later microbial activity contributes to it. 2. Foaming declines steadily in the initial stages, but remains constant thereafter. This type of foaming is due to the medium. 3. The foaming increases after a slight initial fall‘, in this case, microbial activity is the major cause of foaming. 4. The foaming level increases with fermentation duration; such foaming pattern is solely due to microbial activity. 5. A complex foaming pattern that combines features of two or more of the above patterns.

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Foaming can lead to several physical and biological problems such as a decrease in the working volume of the fermenter caused by circulation of oxygen-depleted gas bubbles in the system, a decline in the heat and mass transfer rates. Foaming may interfere with the functioning of sensing electrodes resulting in invalid process data, and incorrect monitoring and control of pH, temperature, etc. The biological problems of foaming include deposition of cells in the upper parts of the fermenter, interference in sterile operation as the air filter exits of the fermenter becomes wet, and increased risk of contamination. In addition, there may be product loss due to siphoning of the culture broth. The problem of excessive foam formation can be resolved by adopting the following strategies: (1) A defined medium may be used to avoid foam formation. This may be combined with modifications in physical parameters like pH, temperature, aeration and agitation. This approach will be successful in such cases where medium is the main culprit, but will fail whenever microbial activity is the main contributor. (2) Often the foam may be unavoidable; in such case, antifoam should be used. This is the most standard approach to combat foaming. Antifoams are surface active agents; they reduce surface tension in the foams and destabilize protein film by the following effects: (a) hydrophobic bridges between two surfaces, (b) displacement of the absorbed protein, and (c) rapid spreading of the surface film. A number of compounds have been found to be suitable antifoam agents for different fermentation processes; these include: alcohols (stearyl and octyldecanol), esters, fatty acids and their derivatives (especially, triglycerides like cottonseed oil, linseed oil, soybean oil, sunflower oil, etc.), silicones, sulfonates, and miscellaneous compounds like oxaline, Alkaterge C, and polypropylene glycol. Many of the antifoams are of low solubility; therefore, they are added with a carrier like lard oil, liquid paraffin and castor oil. Their carriers, however, may be metabolized, and they may affect the fermentation process. Further, many antifoams would reduce oxygen transfer by up to 50% when used at effective concentrations. Antifoams are generally added when foaming occurs during fermentation. But foam control in fermentation industry is still an empirical art. Therefore, the best method of foam control for a particular process in one factory is not necessarily the best for the same process in other factories. Further, the design and operating parameters of the fermenters may affect the properties and the foams produced during the fermentation process. (3) A mechanical foam breaker may also be used.

Valves and Steam traps Valves: attached to fermenters and additional vessel are required to control the flow of liquids and gases. There are four types of addition valves: (a) Simple ON and OFF: which are either fully open or closed (b) For coarse control of flow rates (c) Accurate adjustment valves for precise adjustment of flow rates

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(d) Safety valve: which is constructed in such a way that they allow flow of liquids and gases in one direction only. There are different models of valves. 1. Opening and closing, raising or lowering of blocking unit a. Gate valve - a sliding disc moves in/out of flow path by a turn of the stem b. Globe valve - horizontal disc/plug – raised/lowered c. Piston valve - similar to globe valve except a piston controls flow d. Needle valve - similar to globe valve except disc replaced with tapered plug/needle 2. Drilled sphere/plug a. Plug valve - parallel/tapered plug with orifice – on 90 turn closes/open the flow path b. Ball valve - similar to plug valve – except a ball (s) with orifice replaces the plug 3. Disc rotating between bearings: Butterfly valve - a disc rotates about a shaft – closes against seal to stop flow 4. Rubber diaphragm/tube pinching a. Diaphragm valve - similar to pinch valve – except not pinching, but pushing from one side against a diaphragm b. Pinch valve - flexible sleeve closed by a pair of pinch bars (rubber, neoprene etc.) Check valves: Valves used to prevent accidental reversal flow of liquid or gas due to break down. Pressure control valves: These types of valves are used for two purposes. a) Pressure reduction b) Pressure retaining Safety valve: These are types of safety valves by which the increase in pressure is released. They are; a) A spindle lifted from its seating against the pressure – releases pressure b) Bursting/rupturing of discs to release pressure. In case of releasing the gas, the escaping gas must be treated before release. Steam traps: are important for the removal of steam condensates. They consist of two components viz. valve and seat assembly and open/close device. The operation of the component is based on, i) density of fluid: A float (ball / bucket) float in water, sink in steam. When it floats it closes and when it sinks it opens the valve ii) Temperature of fluid: It has water/alcohol mixture which senses the change in temperature. This mixture expands in hot steam and closes the valve. As temperature drops, it contracts and opens the valve. iii) Kinetic effect of fluid in motion. The conversion of pressure energy into kinetic energy controls the opening and closing.

Control and Monitoring of Fermentation System The integral part of a high-quality bioreactor is a process controller. Such a controller is specially formed for a definite bioreactor brand. This is rather connected with the fact that microorganism cultivation processes have relatively high requirements in respect to precision

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and sophistication. All this is despite the fact that almost all bioreactors monitor and regulate the same values invariably. There are three types of sensors used in fermenter. In-line sensors form integral part of fermenter. The directly measured value controls the process. Eg. Antifoam probe On-line sensors form integral part of fermenter. The measured value must be entered into control system to control process. Eg. Ion specific sensors, mass spectrophotometer. Off-line sensors do not form integral part of fermenter. The measured value must be entered into control system for data collection.

1. (a) Temperature Measuring Devices Temperature is an important parameter of fermentation, since, in the cultivation of many microorganisms, the temperature deviation by a couple of degrees can diminish dramatically the growth and biosynthetic productivity. Among the various types of temperature measuring devices available, devices such Mercury-in-glass, Electrical resistance and Thermistors are used widely in fermentation process. Mercury-in-glass thermometers: Mercury enclosed in bulb expands with increase in temperature. Expansion is read as measure of temperature and is used only as indication. Electrical resistance: The property of some metals whose resistance changes with change in temperature are used to measure temperature. Bulb with mica is used for accurate measurement and ceramic for less accurate measurement. This is covered by temperature sensing platinum. Thermistors: These are semi-conductors of pure oxides of iron, nickel or other metals. They exhibit large change in resistance with a small temperature change and hence are highly sensitive even with little temperature change.

(b) Temperature Controlling Device The temperature in laboratory bioreactors is controlled by one of the following ways: 1. A heater is located inside the bioreactor vessel, and cooling is ensured by thin-wall pipes located in the upper cover, which are connected with an electromagnetic valve with the cooling water. 2. Heating and cooling proceed in a thermostat, and this water, with the help of a pump, circulates through the bioreactor jacket. Variant 1 above is less complicated, and it ensures a more economic constructive solution. This variant works very well for small bioreactors with the volume up to about 5 litres. Variant 2 ensures a more even distribution of heat throughout the bioreactor volume, which is essential in microorganisms' cultivation.

2. (a) Gas Flow Rate Measuring Device Flow rate can be measured by simple variable area meter. Rotameter: is a vertically mounted glass tube with an increasing bore size and enclosing a free moving float. The position of float indicates the flow rate. This is less accurate at low

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flow rate. Since air coming out of it is non-sterilized, it is placed between inlet and filter. Metal tubes can replace glass tubes. Float position is determined by magnetic or electrical techniques. This can be used to measure gas and liquid flow rates (Figure 4.4). Thermal mass flowmeter: In this 2 thermistors are placed upstream and downstream of the heat source which may be inside or outside the piping. The mass flow rate Q can be calculated from the equation, H = QCp(T1-T2) Where, Cp– specific flow rate of the gas, T1temperature of gas before heat transfer and T2 – temperature of gas after heat transfer. This change could induce voltage signal which could be used in data logging.

(b)Gas Flow Rate Controlling Device Needle valve is used to control the gas flow rate. Piston movement of the valve is controlled by fluctuations in pressure in flow measuring device. This should be placed upstream of supply when regulated air flow rate is required and downstream when fluctuations and back pressure is constant. 3. (a) Liquid Flow Rate Measuring Device Electric flow transducer: is used for measuring liquid flow rate. In this, magnetic coils on the sides of liquid flowing tube supplied with current create magnetic field. Relative velocity of fluid and magnetic field are proportional to voltage induced. Potential difference in fluid is measured by a pair of electrodes. Rotameter Load cells: are also used for this purpose. The compressive strain by axial load is measured by electrical resistance strain gauge fixed to surface of cylinder. This is already calibrated by measuring compressive strain with various loads. The change of resistance is proportional to load which can be determined. This is fitted to either reservoir or fermenter vessel.

Figure 4.4. Rotameter.

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(b) Liquid Flow Rate Controlling Device Syringe pump: is useful in case of fed batch for controlling the liquid flow rate. A syringe filled with liquid is secured onto the pump. Syringe plunger is linked to a movable piston and secured firmly to the main body of pump. Regulated piston movement controls the flow rate. This is ideal for low flow rates. Peristaltic pump: is also used to control flow rate by squeezing and releasing pulse flow. The tubing through which the liquid flows is housed on roller. The circular motion (speed) pinches and released the tubing (Figure 4.5). Diaphragm pump: The liquid is allowed to flow through a flexible tube which utilizes a piston and pump controls the flow. The ball valves present on the way of flow prevent leakages and control direction of flow (Figure 4.6).

Figure 4.5. Peristaltic Pump.

Figure 4.6. Diaphragm Pump (Stanbury et al. 1995).

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4. Pressure Measuring and Controlling Devices Bourbon tube pressure gauge: is used to measure the pressure changes (Figure 4.7). This is a partial coil which has an elliptical cross section. This becomes circular with increasing pressure and the motion straightens out the coil. The process pressure is fixed to one end of the coil and the other end fixed to geared sector and pinion movement which actuates an indicator pointer. The pointer moves on a calibrated meter which is used to indicate the pressure. Diaphragm gauge: is another device used to measure the pressure under aseptic condition. The change in pressure cause movements in diaphragm fixed to a mechanically levered pointer. Pressure bellows: connected to core of transformer. Movement of core generates voltage output which can be measured. Strain gauge: a wire subjected to strain results in change of electrical resistance which can be measured. Piezo electric transducer: a solid crystal (quartz), has asymmetrical charge distribution. Any change in shape due to pressure produce equal external electric charge on the opposite face of crystal (piezo electric effect). This charge be can measured by means of electrodes on both surfaces. Pressure can be controlled by regulatory valves and safety valves. 5. Agitation Measuring and Controlling Device Agitation speed can be measured by power consumed by agitator shaft. Wattmeter is usually used in large scale process. It is a measure of power consumed for rotation of agitator shaft. This measure is less accurate because power required to rotate against friction in the bearing is taken into consideration. Torsion dynamometer is used in small scale. This has to be placed outside the vessel and is less accurate due to friction. Strain gauges can be mounted on shaft within fermenter from which electric signal is picked up through lead wires passing out of fermenter via an axial hole. Tachometer can be used to control the agitation speed. The rate of rotation is monitored either by electromagnetic induction or voltage generation or light sensing or magnetic force. Final choice is made by the type of signal required to record or monitor the signal. The agitator speed is also controlled by gear box usage, modifying the size of wheels and drive belts and changing the drive motor. 6. Foam Sensing For elimination of foam, 2 methods or their combinations are commonly used: Additional metering of antifoam: based on the information provided by the foam sensor. The given impulses are relatively low, with long pauses and a limited metering time. This additional control is necessary to avoid the possible overdose, since, in this case, the mass exchange parameters can decrease dramatically. Mechanical metering of foam: For this purpose, an upper drive with a special disktype or other type of the mechanical foam breaking mixer is installed in the bioreactor's upper cover. If an intensive foaming begins, then the mechanical breaking of foam will not help any more. An optimal solution is the combination of both the parameters. Foam formation can be sensed by a probe which is a stainless steel rod insulated except at tip and set at a defined level. When foam touches the probe tip, current passes through, with foam as electrolyte and vessel

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as earth. This current actuates a vessel/pump to release antifoam into fermenter. Process timers are also included which ensure time gap for antifoam mixing in the medium and reducing foam before next sensing occurs.

7. Dissolved Oxygen Monitoring One of the most specific aspects of the fermentation monitoring is pO2 measurement and control. pO2 control is characteristic only for fermentation processes. There are different pO2 control principles: 1. Varying the mixer's rotational speed n, assuming that pO2 ~ n. 2. Combining the change of the mixer's rotation speed n and the amount of the inlet compressed air Q. It is assumed that pO2 ~ n, pO2 ~ Q. First of all, n is usually regulated until it reaches one of the limiting values - nmin or nmax, and its regulation is realised by varying Q. 3. If n and Q have reached the limiting values, but pO2 is not within the necessary limits, then the regulating effect does not occur. 4. Feeding up the substrate or its any component. It is assumed that pO2 is proportional to the feeding up intensity. Feeding up is normally realised with controlled peristaltic pumps. Dissolved oxygen can be measured by: Galvanic electrodes: which consist of KCl or KOH or bicarbonate or acetate as an electrolyte. Lead is used as anode and silver acts as cathode. The electrodes measure the partial pressure of the dissolved oxygen concentration. The sensing tip of electrode is a membrane made up of Teflon, polyethylene or polystyrene that allows gas phase passage so that equilibrium is established between the gas phases inside and outside the electrode. Due to slow movement of oxygen the changes are read slowly. Polarographic electrode: consists of silver as anode, Pt or gold as cathode and KCl as the electrolyte. Fluorometric sterilizable oxygen sensors :are now being developed which are based on the fact that change in oxygen partial pressure quenches the fluorescence lifetime of a chromophore, tris (4,7-diphenyl – 1,10 – phenanthroline ruthenium II) complex. This is reliable only at low oxygen tension. Tubing method: is also used to measure dissolved oxygen concentrations. It comprises of a probe made up of a coil of permeable membrane tubing which is placed inside the fermenter. Through this membrane helium or nitrogen is passed. Oxygen that diffuses into tubing from the medium is then measured by Paramagnetic gas analyser. The paramagnetic oxygen analyzer is based on the scientific principle that oxygen is a paramagnetic material, which means that it can be attracted into a magnetic field. Magnetic susceptibility is a measure of the intensity of the magnetization of a substance when it is placed in a magnetic field. By measuring magnetic susceptibility, concentration of dissolved oxygen can be estimated.

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Figure 4.7. Bourbon tube pressure gauge (Stanbury et al. 1995).

Figure 4.8. Glass electrode and Combined electrode.

8. pH Monitoring Devices pH measurement is the determination of the activity of hydrogen ions in an aqueous solution. Many important properties of a solution can be determined from an accurate measurement of pH, including the acidity of a solution and the extent of a reaction in the

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solution. pH electrode used for estimating the pH of the media consists of two parts: a sensing electrode and a reference electrode. The sensing electrode consists of a thin hydrogen permeable membrane containing a solution and an electrode. The membrane of the sensing electrode allows hydrogen ions to slowly pass, creating a positive voltage across the membrane. The voltage created in this electrode is then compared to the voltage in the reference electrode. The voltage difference between the two electrodes is then used to determine the pH of the unknown solution using the Nernst equation. Most modern pH electrodes consist of a single combination reference and sensing electrode instead of separate electrodes. This type of pH electrode is much easier to use and less expensive than the electrode pair. A combination electrode is functionally the same as an electrode pair (Figure 4.8). Figure 4.9 shows the arrangement of different Control and monitoring systems used in a typical fermenter.

Figure 4.9. Schematic diagram of Control and monitoring fermentation systems (Huang et al. 2001).

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TYPES OF FERMENTERS 1. Stirred Tank Fermenter The Stirred tank reactor comprises of baffles and a rotating stirrer attached either at the top or at the bottom of the bioreactor (Figure 4.10). The typical decision variables are: type, size, location and the number of impellers; sparger size and location which determine the hydrodynamic pattern in the reactor, thus influencing mixing times, mass and heat transfer coefficients, shear rates etc. The conventional fermentation is carried out in a batch mode. Since stirred tank reactors are commonly used for batch processes with slight modifications, these reactors are simple in design and easier to operate. Many of the industrial bioprocesses even today are carried out in batch reactors though significant developments have taken place in the recent years in reactor design, the industry, still prefers stirred tanks because in case of contamination or any other substandard product formation, the loss is minimal. The batch stirred tanks generally suffer due to their low volumetric productivity. The downtimes are quite large and unsteady state fermentation imposes stress to the microbial cultures due to nutritional limitations. The Stirred tank reactors offer excellent mixing and reasonably good mass transfer rates. The cost of operation is lower and the reactors can be used with a variety of microbial species.

Figure 4.10. Stirred Tank Reactor.

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2. Airlift Fermenter Airlift fermenter (ALF) is generally classified as pneumatic reactor without any mechanical stirring arrangements for mixing. The turbulence caused by the fluid flow ensures adequate mixing of the liquid. The draft tube is provided in the central section of the reactor. The introduction of the fluid (air/liquid) causes upward motion and results in circulatory flow in the entire reactor (Figure 4.11). The air/liquid velocities will be low and hence the energy consumption is also low. ALFs can be used for both free and immobilized cells. There are very few reports on ALFs for metabolite production. The advantages of Airlift reactors are the elimination of attrition effects generally encountered in mechanical agitated reactors. It is ideally suited for aerobic cultures since oxygen mass transfer coefficient is quite high in comparison to stirred tank reactors. This is ideal for SCP production from methanol as carbon substrate. This is used mainly to avoid excess heat produced during mechanical agitation. Modifications of airlift fermenters include various modifications of draught (riser) tubes (Figure 4.12).

3. Tower Fermenter A tower fermenter has been defined by Green-shields and co-workers as an elongated non-mechanically stirred fermenter that has an aspect ratio (height to diameter ratio) of at least 6:1 for the tubular section and 10:1 overall, and there is a unidirectional flow of gases through the fermenter. There are several different types of tower fermenters, which are grouped on the basis of their design:

Figure 4.11. Air-lift Fermenter.

Fermenter Design

Figure 4.12. Air-lift fermenter with outer loop and inner loop (Stanbury et al. 1995).

Figure 4.13. Bubble Column Tower Fermenters (Carrington et al.1992).

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(i) Bubble Column Tower Fermenters These are the simplest type of tower fermenters; they consist of glass or metal tubes into which air is introduced at the base. Fermenter volumes from 3 l to up to 950 l have been used and the aspect ratio may be up to 16:1. These tower fermenters have been used for citric acid and tetracycline production, and for a range of other fermentations based on mycelial fungi (Figure 4.13). (ii) Vertical-Tower Beer Fermenters These fermenters were designed for beer production and to maximise yeast biomass yields. A series of perforated plates are placed at intervals to maximise yeast yields. It has a settling zone free of gas; in this zone, yeast cells settle down to the bottom and return to the main body of the tower fermenter, and clear beer could be removed from the fermenter. Tower of up to 20,000 l capacity and capable of producing up to 90,000 L beer per day have been installed. (iii) Multistage Tower Fermenters In these fermenters, a column forms the body of vessel, which is divided into compartments by placing perforated plates across the fermenter. About 10% of the horizontal area of plates is perforated. In a variant of this type of fermenter (down-flow tower fermenter), the substrate is fed in at the top and overflowed through down spouts to the next section, and the air is supplied from the base. These fermenters have been used for continuous culture of E. coli, S. cerevisiae (baker‘s yeast), and activated sludge.

4. Fluidised Bed Bioreactor Fluidized bed bioreactors (FBB) have received increased attention in the recent years due to their advantages over other types of reactors. Most of the FBBs developed for biological systems involving cells as biocatalysts are three phase systems (solid, liquid and gas). The FBBs are generally operated in co-current upflow with liquid as continuous phase. Usually fluidization is obtained either by external liquid re-circulation or by gas fed to the reactor. In the case of immobilized enzymes, the usual situation is of two-phase systems involving solid and liquid but the use of aerobic biocatalyst necessitates introduction of gas (air) as the third phase. A differentiation between the three phase fluidized bed and the airlift bioreactor would be made on the basis that the latter have a physical internal arrangement (draft tube), which provides aerating and non-aerating zones. The circulatory motion of the liquid is induced due to the draft tube. Basically the particles used in FBBs can be of three different types: (i) inert core on which the biomass is created by cell attachment. (ii) Porous particles in which the biocatalyst is entrapped.(iii) Cell aggregates/ flocs (self-immobilization). In comparison to conventional mechanically stirred reactors, FBBs provide a much lower attrition of solid particles. The biocatalyst concentration can significantly be higher and washout limitations of free cell systems can be overcome. In comparison to packed bed reactors FBBs can be operated with smaller size particles without the drawbacks of clogging, high liquid pressure drop, channelling and bed compaction. The smaller particle size facilitates higher mass

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transfer rates and better mixing. The volumetric productivity attained in FBBs is usually higher than in stirred tank and packed bed bioreactors.

TYPES OF FERMENTATION Any fermentation process can be carried in either of the three configurations: 1. Batch Fermentation: is a closed culture system which contains initial, limited amount of medium that is not altered by further addition or removal. This form is simple and widely used in both laboratories and industries. As the growth of microorganism proceeds, the medium availability changes and the culture passes through a number of phases (Figure 4.14). After inoculation, there is a phase during which it appears that no growth occurs; this phase is called lag phase and is considered as the time of adaptation. This is followed by a phase when rate of cell growth gradually increases and this period is called log phase. This growth results in the consumption of nutrients and excretion of microbial products, leading to a decrease in the growth rate. This cessation of growth is called the stationary phase. 2. Continuous Fermentation: In this, the exponential growth phase of organism may be prolonged by the addition of fresh medium to the vessel. The vessel should be designed in such a way that the added volume displaces an equal volume of culture from the vessel. If medium is fed continuously to such vessel at a suitable rate, a steady state is achieved eventually. Steady state occurs when formation of new biomass in the vessel is equivalent to the loss of cells from the vessel. The medium flow into the vessel is related to the total volume of the medium in the vessel expressed as dilution rate, D, which can be expressed in the form of mathematical equation, D = F/V, where, F is the flow rate (dm3h-1) and V is the total volume. The net change in cell concentration over a time period may be expressed as, dx/dt = growth – output or µx - Dx Under steady state conditions the cell concentration remains constant, thus

dx/dt = 0.

and the equation becomes, µx = Dx and µ = D. Thus, under steady state conditions the specific growth rate is controlled by the dilution rate which is a controllable variable. An important objective of continuous culture operation is to control cell growth at a level at which productivity is optimum.

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Figure 4.14. Growth of a tyical microbial culture in batch conditions.

There are several ways in which this can be achieved. One is to maintain a constant fermenter volume and to use a flow rate that gives an appropriate productivity. In this mode of operation, the fermenter system is known as a chemostat. The chemostat is by far the simplest and most common mode of operation of a continuous culture. The cell growth in chemostat cultures is maintained steady by a constant inflow of fresh medium consisting of nutrients (nitrogen, phosphorous, glucose) at a concentration so as to be growth limiting. Other constituents of such a medium are present at concentrations higher than required. Increase or decrease in the concentration of the growth limiting factor is correspondingly expressed by increase or decrease in the growth rate of cells. The volume of the chemostat can be controlled either by using a pump. Where contamination can be a significant problem, a pump based control system is preferred. This setup is commonly used in laboratory investigations and animal cell culture systems. An overflow system has the advantage in that only one pump is required. However, as the effluent flow rate is determined by gravity alone, there is a greater possibility of contaminants moving move up the effluent tube into the reactor. Overflow systems are however widely used in wastewater treatment and have been used in the large scale continuous culture of bacteria. In other techniques, a fermenter variable, eg. turbidity or pH, will be monitored using an appropriate detector and the liquid flow rate will be automatically adjusted so as to maintain the variable at a constant level. Examples of these types of continuous fermenters are the pH-stat, turbidostat and nutristat. Apart from the pH-stat, these reactors are however rarely used as the necessary measurementcontrol systems are generally unreliable over long period of time. A turbidostat is a continuous culturing method where the turbidity of the culture is held constant by manipulating the rate at which medium is fed. If the turbidity tends to increase, the feed rate is increased to dilute the turbidity back to its set point. When the turbidity tends to fall, the feed rate is lowered so that growth can restore the turbidity to its set point. The most widespread large scale application of continuous culture reactors is in wastewater treatment.

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3. Fed-Batch Fermentation: Two basic approaches to the fed-batch fermentation can be used: the constant volume fed-batch culture or Fixed Volume Fed-Batch - and the Variable Volume Fed-Batch. Fixed volume fed-batch: In this type of fed-batch, the limiting substrate is fed without diluting the culture. The culture volume can also be maintained practically constant by feeding the growth limiting substrate in undiluted form, for example, as a very concentrated liquid or gas (ex. oxygen). Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiation can be the growth limiting factor without affecting the culture volume. A certain type of extended fed-batch - the cyclic fed-batch culture for fixed volume systems - refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the starting point for a further fed-batch process. Basically, once the fermentation reaches a certain stage (for example, when aerobic conditions cannot be maintained anymore), the culture is removed and the biomass is diluted to the original volume with sterile water or medium containing the feed substrate. The dilution decreases the biomass concentration and result in an increase in the specific growth rate. Subsequently, as feeding continues, the growth rate will decline gradually as biomass increases and approaches the maximum sustainable in the vessel once more, at which point the culture may be diluted again. Variable volume fed-batch: As the name implies, a variable volume fed-batch is one in which the volume changes with the fermentation time due to the substrate feed. The way this volume changes it is dependent on the requirements, limitations and objectives of the operator. The feed can be provided according to one of the following options: (i) the same medium used in the batch mode is added; (ii) a solution of the limiting substrate at the same concentration as that in the initial medium is added; and (iii) a very concentrated solution of the limiting substrate is added at a rate less than (i), (ii) and (iii). This type of fed-batch can still be further classified as repeated fed-batch process or cyclic fed-batch culture, and single fed-batch process. The former means that once the fermentation reached a certain stage after which is not effective anymore, a quantity of culture is removed from the vessel and replaced by fresh nutrient medium. The decrease in volume results in an increase in the specific growth rate, followed by a gradual decrease as the quasi-steady state is established. The latter type refers to a type of fed-batch in which supplementary growth medium is added during the fermentation, but no culture is removed until the end of the batch. This system presents a disadvantage over the fixed volume fed-batch and the repeated fed-batch process: much of the fermenter volume is not utilized until the end of the batch and consequently, the duration of the batch is limited by the fermenter volume.

REFERENCES Bailey, J. E. & Ollis, D. F. (1986).Biochemical Engineering.2nded, McGraw-Hill Book Co, New York. Carrington, R., Dixon, K., Harrop, A. J. & Macloney, G. (1992). Oxygen transfer in industrial air agitated fermentations. In, Harnessing Biotechnology for the 21st century. Ladisch MR, Bose A(Eds.) American Chemical Society, Washington DC, 183-189 .

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Chen, N. Y. (1990). The design of Air-lift fermenter for use in biotechnology. Biotechnology and Genetic Engineering Reviews, 18, 379-396. Doran, P. M. & Bioprocess, (1995).Engineering Principle. Academic Press, New York Hastings, J. J. H. (1978). Acetone- butanol fermentation. In, Economic microbiology.,2, 31-45 (Editor Rose AH) Academic Press London. Huang, T. K., Wang, P. M. & Wu, W. T. (2001).Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes. Biochemical Engineering Journal.,7, 35-39. Karimi, A., Farideh, G., Momammad, R. M., Masoud, N., Kazem, M., Ahmad, N. & Mohammad, R. P. (2013).Oxygen mass transfer in a stirred tank bioreactor using different impeller configureurations forenvironmental purposes. Iranian Journal ofEnvironmental Health Sciences and Engineering, 10,6. Shuler, M. L. & Kargı, F. (1992). Bioprocess engineering, basic concepts. Prentice Hall, New Jersey Stanbury, P. F., Whitaker, S. J. & Hall, A. (1995). Principles of Fermentation Technology 2nd ed. Pergamon Press Oxford

Chapter 5

DOWNSTREAM PROCESSING Downstream processing refers to the recovery and purification of fermentation products from natural sources such as animal or plant tissue or fermentation broth.The products formed may be secreted into the broth or may be retained within the cell introducing complexity in the recovery of the product. In view of the complexity, downstream processing involves various techniques and methodologies. Bioproducts differ greatly in their nature, hence different separation principles and mechanisms depending on molecular mass, charge distribution, hydrophobicity, distribution coefficient, structure and immunogenic structure and specific affinity towards other biomolecules becomes necessary for their isolation and purification. The choice of the separation methodology depends to a large extent on the nature of the product, its quantity and the extent of purity required. The entire downstream processing operation can be divided into following 4 stages: 1. Removal of insolubles: This step involves capture of the product as a solute in a particulate-free liquid, for example the separation of cells, cell debris or other particulate matter from fermentation broth. Typical operations to achieve this include filtration, centrifugation, coagulation and flocculation. Additional operations such as grinding, homogenization, or leaching, required for recovering products from solid sources such as plant and animal tissues are usually included in this group. 2. Product isolation: This step involves separation of the desired product from other components whose properties vary markedly from that of the product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved. 3. Product purification: Involves separation of products from contaminants which resemble the product very closely in terms of their physical and chemical properties. This stage contributes a significant fraction of the entire downstream processing expenditure. Examples of operations include affinity, size exclusion, reversed phase chromatography, crystallization and fractional precipitation. 4. Product polishing: is the final processing step which ends with packaging of the product in a form that is stable, easily transportable and convenient. Crystallization, desiccation, lyophilization and spray drying are typical unit operations. Depending on the product and its intended use, polishing may also include operations to sterilize the product and remove or deactivate trace contaminants which might compromise

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Loveleen Kaur and Robinka Khajuria product safety. Such operations might include the removal of viruses or depyrogenation. This chapter will discuss some of the most extensively used methods in downstream processing:

SEPARATION OF INSOLUBLE PRODUCTS The first step of product recovery involves separation of solids such as biomass and insoluble particles from the fermentation broth. Depending on the nature of the final product, different protocols may be used for the initial recovery of the product; for example if the product is the biomass, then separation of the solid is the major step in product recovery resulting in significant volume reduction. On the other hand if the product is soluble in fermentation broth, then insoluble solids in the broth need to be removed first before the broth is given special treatments for product recovery. For intracellular products, the first step involves cell disruption followed by removal of insoluble products. The major methods used for separation of insoluble products are: Filtration, Centrifugation, Coagulation and Flocculation.

Filtration It is defined as the separation of solid in a slurry consisting of the solid and fluid by passing the slurry through a septum. Filtration is one of the most commonly use separation process at all scales of separation. It comprises of allowing the fermentation broth to pass through a filter medium leading to the formation of a filter cake. As the filter cake increases in thickness, the resistance to flow of broth will gradually increase. If constant pressure is applied to the surface of the slurry, the rate of flow will gradually diminish. Thus in order to keep the flow rate constant the pressure will gradually have to be increased. Flow through a uniform and constant depth porous bed can be represented by the Darcy equation: Rate of flow = Where µ= liquid viscosity L= depth of filter bed P= pressure differential across the filter bed A=area of the filter exposed to the liquid K=constant of the system Filtration can be carried out under a variety of conditions; the selection of specific filtration equipment depends upon a number of factors such as: 1. 2. 3. 4.

The size and shape of the solid particles to be filtered Solid: Liquid ratio Scale of operation Viscosity and Density of Filtrate

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5. Type of operation: Batch or continuous 6. The need for ascetic conditions 7. The need for pressure or vacuum suction

Types of Filters Batch Filters 1. Plate and Frame filter: is a type of pressure filter which comprises of alternately arranged plates and frames. The plates are covered with filter cloth or filter pads. The plates and frames are arranged on a horizontal framework in such a manner to ensure that no leakage of fermentation broth can take place (Figure 5.1). The framework comprises of continuous channels formed by holes in the corners of press and frames, through which the broth in introduced into the press. The broth passes through the filter pads and the filtrate is discharged through the grooves in the filter plates. The solids are retained within the frames and the filtration is stopped when the frames are completely loaded with filter cakes. These filters are most suitable for filtering broths with low solid content and low resistance to filtration. The pros and cons of Plate and Frame filters are discussed in table 5.1. Table 5.1. Advantages and Disadvantages of Plate and Frame filters Advantages On industrial scale, it is one of the cheapest filters per unit of filtering space. Requires least floor space.

Disadvantages Considerable tear in the filter pads due to mounting and dismounting. Labor intensive and time consuming

Figure. 5.1. Plate and Frame filters.

2. Pressure Leaf Filters: These filters comprise of a number of metals frameworks of grooved plates which are covered with fine wire mesh or filter cloth. These

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Loveleen Kaur and Robinka Khajuria frameworks are known as leaves. The fermentation broth is fed into the filter which is normally operated under pressure or by suction provided by a vacuum pump. The broth passes through the filter mesh or cloth leading to accumulation of filtration cake on the leaves. Leaf filters include Vertical leaf filters and Horizontal leaf filters. The former includes a number of vertical porous leaves mounted on a hollow shaft in a cylindrical pressure vessel. The solids gradually deposit on the surface of the leaves and filtrate is removed from the bottom of the vessel. Solids are normally removed at the end of a cycle by blowing air through the shaft into the leaves. The latter on the other hand includes leaves (often the upper face is porous) mounted on a vertical hollow shafts within a pressure vessel (Figure 5.2). The cake is deposited in the space between the disc shaped leaves and is discharged by releasing the pressure and spinning the shaft with a drive motor.

Continuous Filters 1. Rotary vacuum filter drum: consists of a drum covered with a fabric or metal filter which is partially immersed in a trough of liquid to be filtered (Figure 5.3). The drum is pre-coated with a filter aid, typically of diatomaceous earth (DE) or Perlite. After pre-coat has been applied, the liquid to be filtered is sent to the tub below the drum. The drum rotates through the liquid and the vacuum draws liquid and solids onto the drum‘s pre-coated surface, the liquid portion is passed through the filter media to the internal portion of the drum, while the solids adhere to the outside of the drum. Before the discharge of the filter cake, air pressure is applied internally to help ease off the filter cake from the drum. A number of spray jets are carefully aligned to rinse the cake. Cake can be discharged from the drum by using the following techniques:

Figure 5.2. Vertical leaf filters and Horizontal leaf filters.

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Figure 5.3. Rotary vacuum filter drum.

a) String Discharge: is normally used to discharge fibrous filter cake generally produced by fungal mycelia. Long lengths of string are threaded over the drum and two round rollers. The cake is lifted free from upper part of drum when vacuum pressure is released and passed on the small rollers where it falls free. b) Scraper Discharge: The filter cake which builds up on the drum is removed by a knife blade that is accurately positioned near the drum. This technique is generally used to collect yeast cells. One of the drawbacks of this technique is the gradual wearing of the filter cloth on the drum. 2. Tangential Filtration: One of the drawbacks of filtration methods discussed above is that the flow of broth is perpendicular to the membrane, eventually leading to blockage of the membrane and hence lower rate of productivity or use of filtration aids. One way of solving this issue is to allow the broth to flow tangentially across the surface of the filter, rather than into the filter. The principal advantage of this is that the filter cake is substantially washed away during the filtration process, increasing the length of time that a filter unit can be operational. Tangential filtration or cross filtration system consists of a media storage tank (can also be a fermenter containing the broth), a pump and a membranous pack. Membranous pack may contain either of the two types of membranes: Microporous membranes with specific pore sizes or ultrafiltration membranes with a specific molecular weight cut off. The selection of membrane will depend on the product to be harvested. Tangential flow filtration depends on two variables: 1. The transmembrane pressure (TMP): the force that drives fluid through the membrane, carrying along the permeable molecules. 2. The crossflow velocity (CF): the rate of the solution flow through the feed channel and across the membrane. It provides the force that sweeps away molecules that can foul the membrane and restrict filtrate flow. Fluid is pumped from the sample reservoir into the feed port, across the membrane surface (crossflow), out the retentate port and back into the sample reservoir. The crossflow sweeps away larger molecules and aggregates that are retained on the surface of the membrane, preventing the formation of a concentrated biomolecule layer on the membrane surface that can foul or plug the membrane. Liquid flowing through the narrow feed channel creates a pressure drop between the feed and retentate ports. This pressure, which is applied

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to the membrane, can be further increased by increasing the crossflow rate or by restricting the tubing at the retentate port. This transmembrane pressure (TMP) is the force that drives liquid through the membrane. Liquid that flows through the filter membrane carries molecules smaller than the membrane pores. The trick to using TFF effectively is to regulate both the TMP and crossflow rate to prevent membrane fouling, thus allowing a greater volume of product to be processed in the least possible time (Figure 5.4).

Centrifugation When it comes to removing microorganisms and other similar sized particles, filtration doesn‘t give satisfactory results. Although centrifugation is expensive as compared to filtration but it is preferred in cases where filtration is slow and difficult, the cells or suspended matter must be free from filter aids or continuous separation to a high standard of hygiene is required. Centrifugation employs centrifugal force to promote accelerated settling of particles in a solid-liquid mixture. Separation is achieved by means of accelerated gravitational force by rapid rotation. The particle size that can be separated by centrifugation lies in the range from 0.1µm to 100 µm. Separation of the particles is based on Stoke‘s law, which states that the rate of Newtonian viscosity characteristics is proportional to the square of the diameter of the particles and is expressed as: Vg

(1)

Where, Vg = rate of sedimentation (m/s) d = particle diameter (m) g = gravitational force (m/s2) ρP = liquid density (kg/m3) ρL = particle density (kg/m3) µ = viscosity (kg/m/s) This equation can then be modified for sedimentation in a centrifuge: Vc= d2w2r(ρP – ρL) / µ

(2)

Vc= rate of sedimentation in the centrifuge (m/s) w = angular velocity of the rotor (s-1) r = radial position of the particle (m) Dividing equation 2 with 1 Z= w2r/g This is a measure of the separating power of a centrifuge compared with the gravity settling. It is often referred to as the relative centrifugal force (Z).

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liquids

cake

centrate

Imperforate bowl

motor drive to rotate bowl

Figure 5.4. Tangential filtration or cross filtration system.

outer casting food suspension

(a)

(b)

Figure 5.5. (a)Tubular centrifuge and (b) Multi Chamber bowl centrifuge (Stanbury et al. 1995).

Types of Centrifuges The following section discusses the different types of centrifuges used for separation of microorganism or desired particles. The selection of centrifuge depends on the separation rate, energy requirements and man power requirements. 1. Tubular centrifuge: This is used to separate particle size of 0.1 - 200 µm and upto 10% solids in on-going slurry. The main component of the centrifuge comprises of a

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Loveleen Kaur and Robinka Khajuria tube rotating between bearings at each end. The broth enters at the bottom of the centrifuge via a nozzle and high centrifugal forces act to separate the solids and liquids. The bulk of solids will adhere to the walls of the bowl, while the liquids exit at the top of the centrifuge (Figure 5.5a). This centrifuge may be altered for different uses such as Light phase/Heavy-phase separation, Solids/liquid-liquid phase/heavyliquid phase separation or solids/liquid separation. The pros and cons of Tubular centrifuge are enlisted in table 5.2. 2. Multi Chamber bowl centrifuge: This is used for slurry of upto 5% solids (particle size of 0.1 - 200 µm). This centrifuge consists of a number of tubular bowls arranged coaxially within a rotor chamber. The main bowl contains cylindrical inserts that divide the volume of the bowl into a series of annular chambers, which operate in sequence. The feed enters the centre of the bowl and passes through each chamber. The solids settle on the outer walls of each chamber and the clarified liquid overflows from the largest diameter chamber. These vessels have greater solids capacity than tubular bowls and there is no loss of efficiency as the chambers are filled with solids. However, their mechanical strength and design limit their speed to a maximum of 6500rpm for a rotor of 46 cm diameter.(Figure 5.5b). Table 5.2. Advantages and Disadvantages of Tubular Centrifuge

Advantages High centrifugal forces can be achieved Good Dewatering Ease of cleaning

Disadvantages Limited solid capacity Difficulty in recovery of collected solids Loss in efficiency as the bowl fills

Figure 5.6. Disc-Bowl centrifuge (Stanbury et al. 1995).

3. Disc-Bowl centrifuge: This centrifuge comprises of a series of discs in the rotor or bowl. A central inlet pipe is surrounded by a tack of stainless-steel conical discs. Each disc has spacers so as to build a stack. The broth to be separated flows outwards

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from the central feed pipe and then upwards and inwards between the discs at an angle of 45oto the axis of rotation. The close packing of the discs assists rapid sedimentation and solids slide to the edge of the bowl, eventually accumulating on the inner wall of the bowl. These can also be used for continuous removal of solids through a number of nozzles along the circumference of the bowl. The feed rates range from 45 to 1800 dm3/min, with rotational speeds between 5000-10,000 rpm (Figure 5.6). 4. Decanter centrifuge: This type of centrifuge is used for continuous handling of fermentation broth. This sedimentation centrifuge is designed to handle significant solid concentration in feed suspension. It consists of horizontal cylindrical bowl rotating at a high speed, with a helical extraction screw placed co-axially. The screw perfectly fits the internal contour of the bowl, only allowing clearance between the bowl and the scroll. The differential speed between the screw and scroll provides the conveying motion to collect and remove solids that accumulate at the bowl wall (Figure 5.7). The speed of this type of centrifuge is limited to around 5000 rpm in larger bowls and 10000 rpm in smaller bowls. Bowl diameters are normally between 0.2 to 1.5 meters with feed rate ranging between 200 dm3/ h to 200 m3/h.

Coagulation and Flocculation These processes are generally carried out to form cell aggregates before centrifugation or filtration to enhance the performance of these separation processes. Coagulation is the formation of small flocs from dispersed colloids using coagulation agents (electrolytes). Flocculation is the agglomeration of these small flocs into larger particles that can settle down using flocculating agents such as calcium chloride. There are three main reasons for microorganism existing as discrete units in a fermentation broth: a. Their negatively charged surfaces – therefore they repel each other. b. Their hydrophilic cell walls support the formation of a shell of bound water around them which acts as a thermodynamic barriers to aggregation c. Stearic hindrance due to their irregular shapes Flocculation aims at negating the above mentioned mechanisms, thus leading to cell aggregation. This is achieved by neutralizing negative charges on cell surfaces and reduction in surface hydrophobicity by changing the pH or by presence of a range of compounds such as polyelectrolytes like Polystyrene sulfate, Polyacrylamide or Polyethylene imine. Flocculation involves mixing of the process fluid with a flocculating agent under conditions of high shear in a stirred tank. This stage is called coagulation which is followed by a period of gentle agitation where flocs grow in size.

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Figure 5.7. Decanter Centrifuge.

Cell Disruption Once the cells are separated from the fermentation broth, if the desired product is produced intracellularly, then an additional step is required which involves disruption of cell to release the intracellular products. The selection of disruption methods used will depend on the type of microorganism and the nature of desired product. The different methods of cell disruption can be divided into the following categories:

Chemical Methods i. Alkali Treatment: Alkali acts on the cell wall resulting in the saponification of membrane lipids. Sodium hydroxide on addition alters the pH and affects the integrity of the cell membrane. It is carried out at pH range of 11 to 12 for about 20 to 30 min. ii. Organic solvents: Cell wall absorbs the solvent resulting in its swelling and rupture. At low concentration the cell wall is not ruptured but the permeability is increased. Toluene, ethyl acetate, dimethyl sulfoxide, benzene, chlorobenzene, xylene etc are commonly used organic solvents for cell disruption. However, the final selection of the solvent will depend on the stability of the product. iii. Detergents: A number of detergents damage the lipoproteins of the microbial cell membranes leading to the release of intracellular components. Anionic detergents such as Sodium dodecyl sulfate (SDS), sodium sulphonate, Cationic detergents such as cetyltrimethyl ammonium bromide (CTAB) and non-ionic detergents such Triton X100 are used for disruption of cells. However, these detergents can also denature proteins and these should be removed before further processing. Biological Methods Enzymatic digestion: is involved in two stages: (i) cell wall disruption resulting in the release of cell wall proteins leaving the protoplast intact (ii) digestion of organelle membrane to release the organelle proteins. Digestion may be achieved by hydrolyzing cell walls by specific enzymes such as lysozyme or by combination of enzymes (β- 1,3 –glucanase, β - 1,6 – glucanase, mannanase, chitinase etc).

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Physical Methods i. Sonication: Ultrasound waves of frequencies greater than 20 kHz rupture the cell wall by a phenomenon known as cavitation. The passage of ultrasound waves creates alternating areas of compression and rarefaction which change rapidly. The cavities formed in the areas of rarefaction rapidly collapse as the area changes to one of compression. The bubbles produced in the cavities collapse creating shock waves which disrupt cell walls. ii. Grinding: with a ball mill or a waring blender. Waring blender is particularly effective with animal cells and tissues as well as with mycelial organisms. In industrial scale, cell disruption is carried out using a bead mill or high pressure homogenizer. Vertical or horizontal bead mill consists of a grinding cylinder with a central shaft fitted with a number of impellers and driven by motor. The cell suspension is pumped into the cylinder and cell disruption occurs due to shear forces produced between velocity gradients because of the rotary motion of cells and beads. iii. High pressure homogenization: involves a high pressure positive displacement pump coupled to an adjustable discharge valve with a restricted orifice. The cell suspension is pumped through the homogenizing valve at 200 – 1000 atmospheric pressure depending on microbes and cell concentration. Cell disruption occurs due to stress caused by impingement and pressure drop. iv. Osmotic shock: caused by a sudden change in the salt concentration which causes cell disruption. Freezing-thawing cycles cause loss of membrane integrity and cell wall is ruptured. In thermolysis, the heat inactivates the organism by disrupting the cell walls without affecting the products. The effect of heat shock depends on pH, ionic strength, and presence of chelating or sequestering agents such as EDTA.

SEPARATION OF SOLUBLE PRODUCTS Once the cells are separated from the microbial mass and other insolubles, the next step involves recovery of soluble products from the broth. Most of the microbial products such as organic acids, antibiotics, amino acids, enzymes are extracellular and soluble. Methods such as Extraction, Adsorption, Ultrafiltration, and Chromatography are used to recover these products.

Liquid-Liquid Extraction It is a classical method for recovery and concentration of various products. Solvent extraction has several advantages such as extraction of product directly from broth, reduction in product loss as the product is transferred to a second phase and easy scale up. Liquid extraction is commonly used to separate inhibitory fermentation products such as ethanol and acetone-butanol from the fermentation media. Solvent extraction involves extraction of compound in one phase to another phase based on the solubility difference of compounds in one phase relative to the other. When a compound is distributed between two

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immiscible liquids, the ratio of the concentrations in the two phases is known as the distribution coefficient: KD = YL/XH Where,YL= concentration of the solute in light phase XH = concentration of the solute in heavy phase One of the examples of the use of solvent extraction is recovery of antibiotics from the fermentation broth. Penicillin is more soluble in organic phase at low pH (2-3) and is highly soluble in aqueous phase at high pH (8-9). Therefore, penicillin is extracted between organic and aqueous phases by shifting the pH to improve the purity of the product. Sometimes products to be recovered from the broth can be weak acids or bases. Since non-ionizable compounds are soluble in organic phases, the pH conditions are selected such that the extracted compound is neutral and is soluble in an organic solvent. Therefore, weak acids are extracted at low pH and weak bases are extracted at high pH. Extraction systems are categorized into two types: Co-current system: In this system there are a number of mixer vessels in line and the rafinate is passed from one vessel to another vessel. Fresh solvent is added to each stage and the extracting solvent pass through the cascade in the same direction. At every stage the extract is recovered.A relatively large amount of solvent is used in this process but a high degree of extraction is achieved (Figure 5.8a) Counter –current system:This is the most efficient method of extraction, comprising of a number of mixtures attached in series. The extracted raffinate passes from vessel 1 to vessel n while the product-enriched solvent flows in the opposite direction. The feed and the extracting solvent pass through the cascade in the opposite directions (Figure 5.8b) Solvent Recovery: A major item of equipment in an extraction process is the solventrecovery plant which is usually a distillation unit. Distillation may be achieved in three stages: a) Evaporation: removal of solvent as vapor from a solution b) Vapor-liquid separation: in a column to separate the lower boiling more volatile component from other less volatile components. c) Condensation: of the vapor to recover the more volatile solvent fraction

Aqueous Two Phase Extraction One of the drawbacks of using organic solvents for extraction is that the final product may get affected by the solvent used in the extraction process. An alternative to using organic solvents is the use of aqueous two-phase systems. Phase separation occurs when hydrophilic polymers are added to an aqueous solution. At low concentration of polymers, homogenous solution is formed but at discrete concentration rise, two immiscible phases are formed using two aqueous phases with incompatible polymers such as PEG and dextran. For example, PEG water/dextran water and PEG water/K-phosphate water, PEG phosphates. Homogenates are

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prepared with the two incompatible polymers after which mixer-phase separation is done and the bottom phase and top phase are separated. Then the soluble and insoluble substances are separated by ultrafiltration and product recovered. The retentate may be recycled for further recovery. Two-phase aqueous systems are used extensively for the purification of proteins, enzymes, cells and in extractive bioconversions.

Supercritical Fluid Extraction This involves the dissolution power of super critical fluids ie. fluids above their critical temperature and pressure (Figure.5.9). Critical temperature is defined as the temperature above which a distinct liquid phase cannot exist regardless of pressure. The vapor pressure of the substance at its critical temperature is called the critical pressure. Alternately, pressure and temperature required to liquefy a gas are critical temperature and pressure. At temperature and pressure above but close to the critical point a substance exists as a supercritical fluid. For example: Carbon dioxide, NO, SO2 are used in extraction of βcarotene, vanilla and vegetable oil.

Figure 5.8(a). Co-current Flow Extraction.

Figure 5.8(b). Counter-current Flow Extraction.

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Figure 5.9. Supercritical fluid extraction process.

Adsorption Adsorption of solutes from liquid media onto solids is a common practice in separating soluble materials from fermentation broth. Solute is transferred from liquid to solid phase and equilibrium is reached after a while in a batch operation. The type of adsorbent used depends on the particular application. Adsorbents used are usually in the form of spherical pellets, rods, or monoliths with a hydrodynamic radius between 0.25 and 5 mm. They must have high abrasion resistance, high thermal stability and small pore diameters, which results in higher exposed surface area and hence high capacity for adsorption. The adsorbents must also have a distinct pore structure that enables fast transport of the gaseous vapors. Most industrial adsorbents fall into one of three classes: Oxygen-containing compounds – Are typically hydrophilic and polar, including materials such as silica gel and zeolites. ii. Carbon-based compounds – Are typically hydrophobic and non-polar, including materials such as activated carbon and graphite. iii. Polymer-based compounds – Are polar or non-polar functional groups in a porous polymer matrix. i.

Precipitation It involves separation of compounds by decreasing the solubility of the solutes in the broth. Solubility of the particle can be changed by a number of methods such as: i. ii. iii. iv. v.

Salting out – involves increasing ionic strength of the broth by adding salts as ammonium sulfate or disodium sulfate leading to precipitation of the product. Solubility reduction at low temperature – by adding organic solvents at low temperature. Solvent precipitation – adding salt, pH adjustment and low temperature. Isoelectric precipitation – by changing the pH to isoelectric pH (no charge in proteins). Use of electrolytes – ionic polymers (ionic polysaccharides), non ionic polymer (dextrans).

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Ultrafiltration It is a process in which solutes of high molecular weight are retained when the solvent and low molecular weight solutes are forced under hydraulic pressure through a membrane of a very fine pore size. Therefore, it is used for product concentration and purification. A range of membranes with different molecular weight cut-offs (500 to 500,00) are used for the separation of macro-molecules such as proteins, enzymes, hormones and viruses.

PRODUCT PURIFICATION This involves separation of product from contaminants that resemble the product very closely in terms of their physical and chemical properties. This stage contributes a significant fraction of the entire downstream processing expenditure. Some of the processes used for product purification are described below:

Chromatography In many fermentation processes, chromatographic techniques are used to isolate and purify relatively low concentrations of metabolic products. Chromatographic techniques are also used in the final stages of purification of a number of products. Different types of chromatographic techniques used for product purification include:

Ion Exchange Chromatography In ion exchange chromatography, separation and purification is done on the basis of charge. Basic principle involves reversible competitive binding of different ions of one kind to immobilized ion exchange groups of opposite charge which are bound to the chromatographic matrix called ion exchanger. The factors influencing the binding are net charge, anisotrophy (charge distribution on protein surface), ionic strength, pH of solvent, nature of ions and other additives.There are two types of ion exchangers: Cation exchangers having acidic groups with net negative charge and positively charged exchangeable ions. Anion exchangers having basic groups with net positive charge and negatively charged exchangeable ions. Charges on ion exchanger are based on pH of the solvent (Figure 5.10). In a charged column matrix, when oppositely charged components are loaded, more charged components bind strongly to the matrix whereas similarly charged and neutral components are eluted without retention.

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Figure 5.10. Ion exchange column.

Gel Permeation Chromatography In gel permeation chromatography, separation is based on the difference in the sizes of the particle. The gel column consists of polysaccharides such as dextrans (sephadex) and agarose (sepharose), polyacrylamide cross-linked to form a three dimensional network in the shape of bead. The extent of cross-linking during manufacture of gels controls the pore size within the gel beads. Small molecules can enter the pores of the gel but larger molecules are excluded. Elution of molecules is in decreasing order of size since eluting solvent has lesser access to the smaller molecules than the larger molecules. Hence, the larger molecules are eluted first followed by smaller ones (Figure 5.11). The factors affecting the resolution are void volume, size of the molecule, molecular weight of the molecule pore size of the gel bead, elution volume and number of theoretical plates.

Figure 5.11. Gel permeation chromatography.

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Affinity Column Chromatography This chromatography includes a group of closely related techniques such as bioaffinity, dye-ligand and immobilized metal ion affinity chromatographic techniques. The stationary phase matrix is specially prepared to isolate or purify biomolecules based on their specific interactions between the matrix immobilized functional groups and functional groups on the surface of the biomolecules. A ligand which has a functional group with affinity towards the biomolecules is usually attached covalently to the support matrix and packed in a column. When the biomolecules are loaded, only the ones which recognize the ligand are retained whereas the remaining molecules are eluted. The adsorbed component can then be eluted out in pure form by using a suitable solvent or eluent. Some specific ligands are lectins such as con A for glycoprotein isolation. The choice of the ligand depends mainly on two factors namely, (i) availability of chemically modified groups on the ligand to facilitate its attachment to the matrix while retaining binding capacity with the biomolecule (ii) affinity towards the counter-ligand with suitable K values to ensure maximum binding and desorption. Spacer arm usage helps in proper access of the binding site to the molecule. Commonly used spacer arms are linear aliphatic hydrocarbons with terminal functional groups to facilitate the binding of the spacer molecule to the matrix as well as the ligand. For coupling ligand to the matix the following steps have to be adopted. (i) chemical activation of the matrix, (ii) immobilization of the ligand via the chosen functional group and (iii) blocking order activating the residual active groups. After coupling and blocking, the matrix can be packed and used for separation. From the economic point of view, the column is regenerated and reused. Reverse Phase Chromatography This chromatographic method utilizes a solid phase which is modified so as to replace hydrophilic groups with hydrophobic alkyl chains thus allowing separation of proteins according to their hydrophobicity. More hydrophobic proteins bind strongly with the stationary phase and are therefore eluted after the less hydrophobic proteins. RPC can be combined with affinity techniques in the separation of, for example, proteins and peptides.

Crystallization Crystallization is the process of formation of solid crystals precipitating from a solution. It is an established method used in the initial recovery and purification of fermentation products such as amino acids. Crystallization is also a chemical solid–liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs. Crystallization can be achieved by: (a) Cooling the solution with negligible evaporation (b) Evaporation of the solvent with little or no cooling as in evaporator crystallizer, or (c) Combined cooling and evaporation as in adiabatic or vacuum crystallizers.

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PRODUCT POLISHING This is the final processing step which ends with packaging of the product in a form that is stable, easily transportable and convenient. Lyophilization and drying are typical unit operations that are a part of product polishing.

Drying Drying by Conduction Drying with evaporators removes the solvent, mainly water, from the desired product by heat conduction. A typical evaporator has two principal functions to exchange heat and to separate vapor that is formed from liquid. It has got three principal functional sections viz., the heat exchanger, the evaporating sections where the liquid boils and evaporates and the separator in which the vapor leaves the liquid and passes to the condenser. Types of Evaporator Direct contact drying involves bringing the material to be dried into contact with a heated surface and heat is supplied to the product mainly by conduction. 1. Open pans involve boiling of the liquid in open pans for evaporation. This is restricted to the heat stable product. 2. Horizontal tube evaporator is an improved open pan method in which pan is fixed in a vertical cylinder. 3. Long tube evaporator is tall slender, vertical tube with length to diameter ratio of 100:1in which the liquid passes down the heated tubes. 4. In Forced circulation evaporator, a pump circulation is included such that the heat exchange surface can be divorced from the boiling and vapour separating sections. 5. In Batch vacuum dryers the tray with materials is enclosed in a large cabinet which is evacuated. 6. Drum drying involves spreading the material over the surface of a heated drum.

Drying by Convection Hot air drying uses a moving stream of hot air in contact with the product to be dried. Heat is supplied to the product mainly by convection. Examples of such drying are Kiln driers, tray or compartment driers. 1. Tray dryers involve spreading out the materials as a thin layer on trays and applying heat by air current over the trays. 2. Tunnel dryers are developed tray dryers. The tray with the materials moves through a tunnel on trolleys where heat is applied and vapors removed. 3. In Pneumatic dryers or Conveyor dryers, the materials are conveyed rapidly in an airstream. Heated air sweeps away the heat. 4. In Fluidized bed dryers, the material is suspended against gravity in an upwardflowing air stream

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5. Rotary dryers are those in which the material is filled in a horizontally inclined cylinder and heated by air flow through the cylinder 6. Spray dryers involve spraying of small droplets of into a current of heated air.

Lyophilization Freeze drying technically known as Lyophilization is a process of sublimation in which the water molecules in a solid phase specimen are directly converted to free water molecules in vapor phase. It is the most complex and expensive form of drying; its use is restricted to delicate, heat sensitive materials. In the freeze drying process, material is frozen by exposure to cold air followed by sublimation of ice in vacuum from the frozen state to produce a dried product. In batch freeze dryers, a vacuum cabinet, a vacuum system and a heating system are present. Refrigerated condensers backed by a mechanical pumping system are commonly used commercially. The pumping system is essential to pump the frozen materials down the cabinet pressure initially in a short time to prevent melting of the frozen products. Heat may be supplied by conduction or radiation or from a microwave radiator. The removal of the water by sublimation results in a porous structured product which retains shape and size. There are four stages in the complete drying process: pretreatment, freezing, primary drying, and secondary drying.

Pretreatment Pretreatment includes treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent or increasing the surface area. In many instances, the decision to pretreat a product is based on theoretical knowledge of freeze-drying and its requirements, or is demanded by cycle time or product quality considerations. Methods of pretreatment include: freeze concentration, solution phase concentration, formulation to preserve product appearance, formulation to stabilize reactive products, formulation to increase the surface area, and decreasing high vapor pressure solvents. Freezing In a laboratory, this is often done by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature. This cycling process is called annealing. However, in the case of food, or objects with formerly-living cells, large ice crystals will break the cell walls (a problem discovered, and solved, by Clarence Birdseye), resulting in the destruction of more cells, which can result in increasingly poor texture and nutritive content. In this case, the freezing is done rapidly, in order to lower the material to below its eutectic

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point quickly, thus avoiding the formation of ice crystals. Usually, the freezing temperatures are between -50 °C and -80 °C. The freezing phase is the most critical in the whole freezedrying process, because the product can be spoiled if improperly done.

Primary Drying During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the material for the water to sublime. The amount of heat necessary can be calculated using the sublimating molecule‘s latent heat of sublimation. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be slow (can be several days in the industry), because, if too much heat is added, the material‘s structure could be altered. In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates provide a surface(s) for the water vapour to re-solidify on. This condenser plays no role in keeping the material frozen; rather, it prevents water vapor from reaching the vacuum pump, which could degrade the pump's performance. Condenser temperatures are typically below −50 °C. Secondary Drying The secondary drying phase aims to remove unfrozen water molecules, since the ice is removed in the primary drying phase. This part of the freeze-drying process is governed by the material‘s adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0 °C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually, the pressure is also lowered in this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal). However, there are products that benefit from increased pressure as well. After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed. At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%.

Diafiltration In diafiltration process a hydrophobic membrane is used. The solvent with solute fills the pores and the products do not pass across due to pressure on other side. The solute is immobilized on the membrane with phosphate that can be extracted afterwards.

REFERENCES Aiba, S., Humphrey, A. E. & Millis, N. F. (1973). Recovery of fermentation products. Biochemical Engineering 2nd ed. Academic Press, New York Bailey, J. E. & Ollis, D. F. (1986). Biochemical Engineering 2nded, McGraw-Hill Book Co, New York.

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Doran, P. M. & Bioprocess, (1995). Engineering Principles Academic Press, New York Roy, I., Gupta, M. N. Downstream processing of enzymes/Proteins (2002). Proc Indian Nat Sci Acad B68 (2), 175-204 Schügerl, K. (2000). Integrated processing of biotechnology products. Biotechnology Advances,18(7), 581-599. Shuler, M. L. & Kargı, F. (1992). Bioprocessengineering, basic concepts. Prentice Hall, New Jersey Stanbury, P. F., Whitaker, S. J. & Hall, A. (1995). Principles of Fermentation Technology 2nd ed. PergamonPress, Oxford

SECTION II: APPLICATIONS OF INDUSTRIAL BIOTECHNOLOGY

Chapter 6

PRODUCTION OF MICROBIAL METABOLITES I Microbial production of primary and secondary metabolites contributes significantly to the quality of life. Through fermentation, microorganisms growing on inexpensive carbon sources can produce valuable products such as amino acids, organic acids, antibiotics, vitamins etc. Microorganisms have the potential to provide many petroleum-derived products as well as the ethanol necessary for liquid fuel. Chapter 6 and Chapter 7 discuss the fermentation process used for commercial productions of some of these microbial metabolites.

BIOFUELS Fossil fuels like coal and oil have played a critical role in humanity‘s recent history, providing a vast energy source which has fuelled much of society‘s development and industrialization. These fuels are still the primary source of energy for the world and yet it is agreed that these traditional sources of energy cannot continue to power human growth into the future. The use of fossil fuels poses other problems as well, most notably that their consumption is environmentally unsustainable. Burning fossil fuels produces enormous quantities of the greenhouse gas carbon dioxide, which has a negative impact on the Earth‘s environment by contributing to global warming. For all of these reasons, there is great incentive to pursue the development of renewable energy sources, particularly microbial biofuels. Microbial biofuel production primarily involves sugar fermentation by yeast to produce ethanol. Although many microbes have been used in ethanol production, the yeast species Saccharomyces cerevisiae is primarily used in industry, which utilizes starch and sugars from plants as the starting material for the process. Ethanol fermentation by S. cerevisiae is primarily done via the standard glycolysis pathway. In the process, a single molecule of glucose is oxidized to two molecules of pyruvate. Anaerobic conditions are required so that molecular oxygen is not available for use as an electron acceptor, and instead pyruvate must be used as the terminal electron acceptor. This fermentative process involves the decarboxylation of pyruvate to form carbon dioxide and acetaldehyde, and the subsequent reduction of acetaldehyde to produce ethanol. The most common feed-stocks (carbon source utilized by the microbes) are agricultural products which can easily be processed to create the

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simple sugars needed for fermentation. Various substrates used for ethanol production and their processing is summarised below: 1. Sugar crops such as sugarcane, sugar beet, sorghum etc. provide a good substrate. Juices from these crops contain up to 12% fermentable sugars and can be used directly for fermentation. 2. By-product sugars from crop processing: such as molasses, sweet sorghum syrup and spent sulphite liquor are the most common substrates. Molasses obtained after sugar recovery contains 35% sucrose, 19% other reducing agents and 14% other organic substances; thus the total fermentable sugars are nearly 50-55%. Molasses is first suitably diluted, then treated with 0.5% by weight, concentrated sulfuric acid at 7095°C and used for fermentation. 3. Cereals such as maize, wheat sorghum contain 60-75% w/w starch, which on hydrolysis produces glucose in the ration 9:10. Conversion of starch into glucose is usually achieved by a cooking process aided by enzymes. Seeds are powdered and the resulting meal is mixed with water in the ratio of 1:2.5-3. α-amylase is then added and temperature is raised by steam injection to 105-110°C for 20 minutes. The suspension obtained after the treatment contains solubilised starch molecules and dextrins and is treated with glucoamylase to produce glucose from dextrins which is then used for fermentation (Figure 6.1). 4. Cellulosic substrates are the most abundant substrates such as wood, wastes like straw, stovers, bagasse, saw-dust etc. These wastes are first pre-treated by acid hydrolysis or enzymatic hydrolysis. A third approach involves direct conversion of cellulose into ethanol by using mixed culture of cellulolytic and fermenting bacteria. The substrates are subjected to fermentation. Both batch and continuous fermentation process are used and often yeast cells are recycled to save the substrate used up as cell matter. Ethanol recovery is based on distillation. The broth is distilled in a beer column to yield 85% v/v ethanol. This is followed by rectification to give 96.5% ethanol and then dehydrated to 99.4% using benzene or cyclohexane for ethanol to be used as fuel-blends. But many car engines can use 95% ethanol. A summary of the ethanol production from starch substrates is given in Figure 6.1.

Figure 6.1. A summary of the ethanol production process from starch substrates.

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ACETONE-BUTANOL PRODUCTION Acetone-Butanol production is a strictly anaerobic process carried out by Clostridium acetobutylicum. The process was industrialised for the production of acetone, used as solvent for cordite for explosive manufacture during World War I. Later, it was used for the production of butanol. Different strains of C. acetobutylicum utlize different substrates and the fermentation yields different products that include acetone, butanol and ethanol. During fermentation, starch is digested to yield glucose which is then metabolised to yield butanol and acetone (Figure 6.2). Corn is the most extensively used substrate for the production of acetone and butanol. Corn meal medium is prepared by grinding degermed corn to fine powder; 8-10% of this corn meal is heated to gelatinize the starch. The molasses medium contains 6% sucrose which is supplemented with ammonium sulfate, calcium carbonate, superphosphate and cornsteep liquor. Corn medium is used for fermentation by C. acetobutylicum while molasses medium is fermented by C. saccharoacetobutylicum. The fermentation passes through the following three phases: I.

Rapid bacterial growth, production of acetic and butyric acids and evolution of CO2 and H2 for 13-17 hours of incubation. II. Rapid conversion of the acids into acetone and butanol leading to a fast decline in the acidity of the broth. This is known as acid break. III. Marked decrease in gas evolution, and acetone and butanol production.

Figure 6.2. Biochemical Pathway for the production of n-butanol and acetone.

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The final broth contains about 2% acetone, ethanol and butanol which represent about 30% conversion of the carbohydrates. Typically, corn medium yields butanol, acetone and ethanol in the ratio of 6:3:1, while with molasses medium these values are 6.5:3:5. Recovery of the products is achieved by distillation process. Individual solvents are separated by fractional distillation.

BEER AND WINE PRODUCTION An alcoholic beverage is a drink containing ethanol, commonly known as alcohol, although in chemistry the definition of alcohol includes many other compounds. The process of culturing yeast under alcohol producing conditions is referred to as brewing and varies according to the end product. Beer involves a relatively short (incomplete) fermentation process and an equally short aging process (a week or two) resulting in an alcohol content generally between 3-8%, as well as natural carbonation. Wine on the other hand, involves a longer fermentation process, and a relatively long aging process (months or years, sometimes decades) resulting in an alcohol content between 7-18%.

BEER Beer is produced by fermentation of an extract of malted cereals, preferably barley. Aromatic herbs such as hops are generally added to give flavor to the beer. Barley malt is the most common grain used to make beer. Wheat, corn and rice are the secondary grains, used as an adjunct to the barley. The alcohol content of beer varies by local practice or beer style. Since the cereal grains contain very little fermentable sugar and the carbohydrates present is normally in the form of starch which a few strains of yeast are able to utilize, therefore, the grains are moistened to encourage germination prior to fermentation. The alcohol in beer comes primarily from the fermentation of sugars that are produced during mashing. In modern brewing, the malt is ground and extracted with hot water with the addition of ground unmalted cereals. Since sufficient enzyme activity remains in the malt after kilning, the starch of the adjunct grain is hydrolysed to fermentable sugar during mashing. Mashing temperature is generally in the range of 50 to 800C. The sugary extract called as wort drained from the mash is filtered and the mash is sparged with hot water to ensure maximum extraction of nutrients. The collected wort is then boiled with hops which also sterilizes the wort and inactivates enzyme activity. This is followed by filtration of wort and the hopped wort is inoculated with yeast. During the fermentation, the pH falls from the initial 5-5.2 to 3.8-4.0 and the yeast population grow about eight times. At the end of the fermentation, the yeast spontaneously flocculates into clumps and settles out rapidly. Further clarification can be carried out by filtration or centrifugation if required. The complete process of beer production has been summarised in Figure.6.3. Beers mainly are categorised as Ale and Lager. Ale is traditionally fermented by top stains of S. cerevisiae. It is called so because a portion of the yeast rises to form a thick yeast head on the surface of the fermenter. This yeast head is cooled and used as inoculum for next round of fermentations. In case of lager beer, malt is kilned at lower temperature and the yeast

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used S. carlsbergensis, does not form a yeast head and is harvested from the bottom of the fermentation vessels where it settles down at the end of the fermentation process. All beers require a period of conditioning after fermentation to improve the flavor of green beer drawn from the fermenters. This is followed by injection of carbon dioxide, filtration and pasteurization. Some brewers add one or more clarifying agents to beer. Common examples of these include isinglass finings, obtained from swimbladders of fish; kappa carrageenan, derived from seaweed; Irish moss, a type of red alga; polyclar (artificial), and gelatin. Clarifying agents typically precipitate out of the beer along with protein solids, and are found only in trace amounts in the finished product.

Figure 6.3. Flow diagram of beer production.

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WINE Wine is an alcoholic beverage made from the fermentation of unmodified grape juice. The natural chemical balance of grapes is such that they ferment without the addition of sugars, acids, enzymes or other nutrients. Fermentation normally begins with the crushing of the fruits. The juice or must is then treated with sulfur dioxide or sulphite to remove undesired non-wine yeast, mold and bacteria as fermentation yeasts are more resistant to sulfur dioxide and sulphite. During the fermentation, a succession of yeasts such as Saccharomyces, Kloeckera, Kluyveromyces, Zygosaccharomyces develop. Each yeast contributes its own flavor to the wine. The characteristics of wine are partly due to the variety of grapes and partly due to yeast strain used. Commercially, it is a standard practice to use pure yeast cultures or mixture of pure yeast cultures for fermentations. All microbial flora of the grapes is eliminated by the addition of higher amounts of sulfur dioxide or sulphite. The selected pure cultures are then added to the juice and incubated at 7 to 14°C. Red wines are produced from black grapes, the skins of which are left in contact with the fermenting juice. Grape skin pigments which are ethanol soluble are extracted from the skin as fermentation progresses. Tannins are also extracted which give bitter taste to young wines. As the wine ages, these tannins mellow down due to chemical reaction and fermentation. White wines on the other hand are produced from either green/yellow or black grapes whose skins have been removed before the beginning of the fermentation process. As a result white wines have lower tannin content than red wines. The progress of wine fermentation is essentially similar to the beer fermentation but normally lasts over longer period of time thus producing more alcohol. Ethanol production only requires few days but aging may continue for months or even years.

MICROBIAL ENZYMES Enzymes are commercially produced by two methods: Semisolid culture: The enzyme producing culture is grown on the surface of a suitable semi-solid substrate. The substrate usually consists of moistened wheat or rice bran, supplemented with nutrient salts. The desired pH of the media is adjusted with acid. Then the medium is steam sterilized in an autoclave while stirring. The sterilized media is then spread on metal trays up to a depth of 1-10 cm. Alternatively, the cultivation may be carried out in rotating drums. The fungal spores are inoculated; either in the autoclave after cooling or in the trays. Aeration is ensured by the circulation of suitably humidified air over the surface of the culture. It is necessary to keep temperature within narrow limits. Therefore the trays should be equipped with a cooling system. Submerged culture: Nowadays submerged culture methods are widely used in the production of enzymes. It is carried out in a cylindrical fermentation tank equipped with agitator, an aerating device, a cooling system and various ancillary equipments. The quantity of fermentation media taken in the fermentation tank varies from 1000-3000 gallons or more. Meida used in submerged fermentations is sterilized batch wise in the fermenter at 120°C for 1- 2hours. Alternatively the fermentation media can be sterilized by continuous sterilization.

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After the fermentation is completed, the fermented liquid is subjected to rapid cooling to about 50C to reduce deterioration. Separation of media is accomplished by filtration or centrifugation of the refrigerated media. The colloidal particles present in the filtrate are eliminated with coagulating or flocculating agents such as calcium phosphate. Removal of suspended solids is carried out by vacuum drum filtration or disc-type centrifuge. In order to obtain high degree of purity, the enzyme is precipitated with acetone, alcohols or inorganic salts. Extraction of endocellular enzymes involves the disintegration of the microbial cells. This can be accomplished by a homogenizer or a bead mill. Thereafter, purification methods being employed for exoenzymes are used for purification.

Amylases Amylases are one of the most extensively used enzymes in food industry. Therefore concentrations of α-amylses and β-amylases are prepared and used in a variety of ways. These enzyme preparations must be carefully standardized for activity, according to the purpose for which they are to be used. α-Amylases are produced by fungi such as Aspergillus niger, Aspergillus oryzae and bacteria such as Bacillus amyloliquefaciens, B. licheniformis. They are therefore classified as fungal amylases or bacterial amylases based on their source of origin. Fungal amylases are produced by growing the fungus in wheat bran (semi-solid culture). It is possible to produce fungal amylases by submerged culture but there is a problem encountered during agitation and aeration due to very high viscosity of the media caused by the fungal mycelia. Bacterial amylase on the other hand is produced by the two bacterial species mentioned above using submerged fermentation. α-Amylase production requires a temperature optimum of 30-40°C and pH of 7.0. It is essential to maintain the pH of the fermentation media as the amylases get denatured below a pH of 6. The production of α-Amylases begins when the bacterial count reaches 109-1010 cells per milliliter after about 10-20 hours and continues for another 100-150 hours. The most active preparations contain 2 per cent active α-amylase protein.

Proteases Complex mixtures of true proteinases and peptidases are usually called proteases. The concentration of peptidases in the production media is low, as they are endoenzymes. Proteases like amylases are produced by bacteria (Bacillus subtilis and B. licheniformis) as well as by fungi (Aspergillus niger and Aspergillus oryzae). Proteases are basically of two types: Alkaline serine proteases and Acidic proteases. Alkaline serine proteases: Subtilisin Carlsberg is the most widely used detergent protease obtained from B. licheniformis by submerged fermentation in a media supplemented with starch hydrolyzate, soyabean meal and casein. The temperature of the fermentation is kept in the range of 30-40°C and pH of 7.0.The production of the enzyme begins when maximum cell growth is reached after 10-20 hours and this continues at a constant rate till the end of the fermentation. The enzyme is available in markets in the form of dust-free granules that contain 1.5-2 per cent enzyme protein.

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Acid Proteases: are mostly produced by fungi such as Mucor pusillus, M. miehei, A. oryzae, A. phoenicis, A niger var. macropus. Acid proteases may be produced by either semisolid-culture or submerged-culture depending on the fungal species employed. For example, Mucor pusillus is cultivated on semi-solid medium. The medium consists of 60% wheat bran with water. The optimum temperature of the fermentation is 30°C and requires 3 days for completion. Finally, the enzyme is extracted with water. On the other hand, M. miehei is grown by submerged culture method. The optimum temperature of the fermentation is 30°C and requires 7 days for completion. These preparations contain 0.2-0.3 per cent of active enzyme.

Pectinases Pectinases are produced by species of Aspergillus and Penicillium. These fungi are grown on beet molasses because it contains pectins. Semisolid cultures can be used to produce pectinases employing A. niger. At the end of the fermentation, drying and pulverization of the mycelia is carried out. Subsequently a mixture of pectin esterase and polygalaturonase, polymethylgalacturonase and pectin transeliminase is extracted with the help of cold water. After the extraction is completed, the crude enzyme mixture is subjected to concentration and refrigeration without solidifying.

REFERENCES Gottschalk, G. & Peinemann, S. (1992). The anaerobic way of life.In, The Prokaryotes. Balows A et al. (eds), 300-311. Berlin, SpringerVerlag. Muller, V. & Bacterial Fermentation, (2001). Encyclopedia of Life Sciences. Nature Publishing Group Rao, D., Swamy, A. & Siva Rama Krishna, G. (2006). Bioprocess technology Strategies, Production and Purification of Amylases, An overview. The Internet Journal of Genomics and Proteomics.,2(2), Shuler, M. L. & Kargi, F. (1992). Bioprocess engineering, basic concepts. Prentice Hall, New Jersey Stanbury, P. F., Whitaker, S. J. & Hall, A. (1995). Principles of Fermentation Technology. 2nd ed. Pergamon Press, Oxford Vidyalakshmi, R., Paranthaman, R. & Indhumath, J. (2009). Amylase Production on Submerged Fermentation by Bacillus spp. World Journal of Chemistry,4(1), 89-91

Chapter 7

PRODUCTION OF MICROBIAL METABOLITES II ORGANIC ACIDS An organic acid is an organic compound with acidic properties. The most common type of this would be the carboxylic acid group. Organic acids possess a long chain of carbons attached to a carboxyl group. This category of acid is used widely in the food industry as both a food additive and also in chemical feedstock. Fermentation processes play a major role in the production of most organic acids. Broadly, microbial fermentations involve the use of microorganisms to metabolize (either aerobically or anaerobically) a nutrient molecule to yield a specific product. Table 7.1 shows some of the organic acids produced by microbial fermentation. All acids of the tricarboxylic acid (TCA) cycle can be produced microbially in high yields, other acids can be derived indirectly from the Krebs cycle (pseudonym for the TCA cycle) such as itaconic acid, or can be derived directly from glucose (gluconic acid). Some acids are formed as the end products from pyruvate or ethanol (lactic and acetic acid). Organic acid production can be stimulated and in a number of cases, conditions have been optimized that result in almost quantitative conversion of carbon substrate into acid. This is exploited in large-scale commercial production of a number of organic acids like citric-, gluconic- and itaconic acid. Other organic acids produced in lower scale are Lactic acid, Malic acid, Gibberellic acid, and Kojic acid.

1. Citric Acid Citric acid (2-hydroxy-propane-1, 2, 3-tricarboxlic acid) is the most widely used acidulant in the food and beverage industries. It has many uses such as a blood anticoagulant, a metal cleaning agent and a descaler. Until 1923, citric acid was chemically prepared from the juices of lemons and limes (Berovic and Legisa 2007). A fermentation system was then developed which utilized Aspergillus niger. Different fungal strains such as A.niger, A.clavatus, Penicillium luteum, P.citrinium, Mucor piriformis, Ustulina vulgaris etc. have been reported to produce citric acid (Max et al. 2010). However A.niger is the most extensively used fungus due to the following reasons: i) Gives higher yield ii) Possesses

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uniform biochemical properties iii) Easily cultivated, and iv) Produces small amounts of oxalic acid. There are two main strategies for the production of citric acid: a) Surface Fermentation: This was the first technique developed for citric acid fermentation and is still practiced today. The process involves inoculating the culture across the surface of the production media under stationary conditions. A. niger forms a mat on the surface of the media (Majumdar et al. 2010). Different process parameters to be maintained during the fermentation process are discussed below: 1. Inoculum: A. niger is inoculated in shallow pans on a suitable solid sporulation media at 25oC for 5-14 days. After suitable incubation period, the spores are collected and spore suspension is prepared by suspending them in suitable diluent such as water. This suspension is then used for the inoculation of the production media. 2. Carbon source: Among various sugars, sucrose up to a concentration of 15% has been reported as the best carbon source for citric acid production. However, due to its high cost, sucrose is not preferred for the commercial production processes. For commercial production, beet molasses is the most extensively used carbon source. Since beet molasses contains high amounts of trace elements, therefore it is pretreated with ferrocyanide or ferricyanide which react with metals present in beet molasses leading to their precipitation. 3. Inorganic Salts: In addition to carbon, hydrogen and oxygen, trace elements such as nitrogen, potassium, phosphorus, sulfur and magnesium are also need in the fermentation media for the production of citric acid. It is very critical to maintain the concentrations of nitrogen, phosphorus and magnesium as higher concentration of these salts have an adverse effect on the citric acid yield. The actual concentrations of different metal salts required will eventually depend on the fungal strain being used in the fermentation process. For example, A. niger62 requires 0.1mg of iron per litre of media for optimum production while A. niger-59 has a requirement of 10mg per litre. 4. pH: A low pH of around 3.4-3.5 has been reported to support maximum citric acid yield. Also the pH of the media is adjusted using HCl as other acids such as nitric acid, sulfuric acid are known to inhibit the production. Low pH fermentation is desirable for citric acid production as it suppresses the production of oxalic acid thus giving higher yields of citric acid. Also the threat of contamination is reduced. 5. Temperature and Incubation Period: will depend on the fungal strain used for fermentation process. For A. niger a temperature of 26-28°C is optimum for citric acid production and the incubation period generally varies from 7 to10 days. 6. Fermentation Vessel: Since the production process is highly sensitive to the concentration of iron in the fermentation media and due to the low pH at which the process is carried out, pans and trays used for stationary fermentation should be corrosion resistant. As a result trays and pans used in the process are made of stainless steel or aluminum. Another important parameter in this process is the ratio of surface area to volume of the medium as it decides the rate of

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bioconversion of sugar into citric acid. The lower the ratio, higher is the yield of citric acid. Therefore, shallow pans having a depth of 1-3cm are used. 7. Yield: of the citric acid ranges from 60-80grams of anhydrous citric acid per 100gms of sugar. 8. Recovery: There are three main impurities present in the mother liquor that needs to be removed for the final product. These include unconverted sugars, other acid products of fermentation and trace salts. Figure.7.1 explains the process used for citric acid recovery. b) Submerged Fermentation: Involves culturing the fungus in dispersed form in a fermentation media. Fermentation is carried out in special bioreactors having sterilizable tank capacity of thousands of gallons and equipped with an agitator and a sparger. In this process, fungal spores are produced under aseptic conditions and are used to inoculate media designed to develop cellular mass rather than to produce citric acid (Martin and Waters 1952). A part of this is then transferred to a production medium designed specifically to favor citric acid production. High aeration rate of 0.5-1.5 volumes per minute is maintained and samples are withdrawn at regular intervals to check the progress of fermentation process. This method also utilizes pretreated beet molasses and inorganic salts as explained above. Followed by fermentation process, citric acid is recovered as shown in Figure.7.1.

Figure 7.1. Citric Acid Recovery.

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Loveleen Kaur and Robinka Khajuria Table 7.1. Microbes associated with organic acid production Organic acid Citric acid Lactic acid Itaconic acid Acetic acid Gluconic acid Kojic acid Propionic acid Succinic acid Fumaric acid Pyruvic acid Mallic acid Tartaric acid α- ketoglutaric acid

Microorganism Aspergillus niger/ Candida lipolytica Lactobacillus delbrueckii Aspergillus terreus Acetobacter aceti Aspergillus niger Aspergillus oryzae Propionibacterium shermanii Bacterium succinicum Rhizopus delemar Pseudomonas aeruginosa Lactobacillus brevis Gluconobacter suboxydans Candida hydrocarbofumarica

2. Itaconic Acid Itaconic acid, or methylenesuccinic acid, is an organic compound that exists as a white crystalline powder. It is a naturally occurring compound which is non-toxic and readily biodegradable. Historically, itaconic acid was obtained by the distillation of citric acid. In 1931, itaconic acid was first shown to be a metabolic product of Aspergillus itaconicus and soon after, it was discovered that some strains of Aspergillus terreus also excrete this organic acid. Nowadays, high-yield mutants of A. terrus are extensively used for commercial production processes. Some of the industrially important strains include A. terreus ATCC 10020, A. terreus ATCC 10029 and A. terreus NRRL 265. Itaconic acid is primarily used as a co-monomer in the production of acrylonitrile-butadiene-styrene and acrylate latexes with applications in the paper and architectural coating industry. Itaconic acid is produced by way of the TCA cycle from cis-aconitic acid via decarboxylation (Figure 7.2). First step involves production of citric acid which then undergoes dehydration by aconitate hydratase forming cis-aconitic acid. This step is followed by decarboxylation of cis-aconitic acid by aconitate decarboxylase leading to formation of itaconic acid.

Figure 7.2. Biochemistry of Itaconic acid formation.

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The different process parameters to be maintained during the fermentation process are discussed below (Nelson et al. 1952; Steiger et al. 2013; Huang et al. 2013): 1. Carbon source:Among sugars, glucose and sucrose give satisfactory yield. However due to low cost, cane-molasses and beet molasses are preferred carbon sources for commercial production. 2. Inorganic Salts: Ammonium salts such as NH4NO3 or (NH4)2SO4 are used as source of nitrogen. Production of itaconic acid is sensitive to the concentrations of iron, copper, magnesium and zinc. 3. pH: Initial pH of production media is usually adjusted in the range of 1.8 to 2 by using hydrochloric acid, nitric acid or sulfuric acid. It is necessary to neutralize the accumulated itaconic acid which is done by adding ammonia in a stepwise manner. 4. Temperature and Incubation Period: Temperature within a range of 30-35°C is optimum for itaconic acid production and the fermentation process takes 3-7 days for completion. 5. Aeration and Agitation: It is essential to supply sterile air with constant agitation of the broth in submerged fermentations. 6. Yield: of the citric acid ranges from 5-80grams of itaconic per 100gms of sugar. 7. Recovery: Involves filtration for separating mycelia from the fermentation broth. Acidification, clarification and other purification treatments are followed by crystallization of the product. Solvent extraction is also employed for the recovery of itaconic acid

3. Gluconic Acid Gluconic acid (2, 3, 4, 5, 6-Pentahydroxycaproic acid) is used in food and beverage, pharmaceutical, detergent and construction industries. As a food additive, it is an acidity regulator. It is also used in cleaning products where it dissolves mineral deposits especially in alkaline solution. The gluconate anion chelates Ca2+, Fe2+, Al3+, and other metals. Calcium gluconate, in the form of a gel, is used to treat burns from hydrofluoric acid; calcium gluconate injections may be used for more severe cases to avoid necrosis of deep tissues. Quinine gluconate is a salt between gluconic acid and quinine, which is used for intramuscular injection in the treatment of malaria. Iron gluconate injections have been proposed in the past to treat anaemia. Gluconic acid is produced by A. niger using submerged fermentation processes only (Ramachandran et al. 2006; Porges et al. 1941). The different process parameters to be maintained during the fermentation process are discussed below: 1. Inoculum: consists of either sporulated culture or spores germinated in seed tanks. Direct sporulation avoids the cost of installation and operation of seed tanks while use of germinated spores reduces the operation time for the main reactor. 2. Carbon source: D-glucose is the most preferred carbon source for the production of gluconic acid. It can either be supplied as a solution of crystalline glucose or added as syrup obtained from starch by the action of α-amylase and amyloglucosidase. The concentration of sugar used will depend upon the type of gluconate salt to be

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Loveleen Kaur and Robinka Khajuria produced. For example for the production of sodium gluconate, initial sugar concentration of 28-30% can be used. Table 7.2 gives the basic composition of production media used for gluconic acid. 3. pH: The pH is maintained at about 5.5-6.0 by CaCO3 during the production of the calcium salt and about 6.8-7.2 by NaOH in the production of sodium salt. 4. Aeration and Agitation: It is critical to maintain maximum concentration of oxygen dissolved in the solution. This is achieved by vigorous agitation with a turbomixer or by the action of cavitator with aeration rate of 1-1.5 volumes air/volume solution/minute. 5. Recovery: The first step in the gluconic acid recovery involves separation of the mycelium from the solution by filtration. This filtrate is then used for the recovery of sodium or calcium gluconate and gluconic acid. Calcium gluconate is recovered by heating the filtrate with slight excess of calcium hydroxide, followed by decolorization with carbon and filtration. The filtrate is cooled to a temperature below 20°C and seeded with calcium gluconate crystals leading to rapid crystallization of the compound. Sodium gluconate is recovered by concentrating the filtrate to 42-45 per cent solids and adjusting the pH to 7.5 with NaOH. The salt is drum-dried.

4. Lactic Acid Lactic acid (C2H4OHCOOH) was first isolated in 1780 by the Swedish chemist Carl Wilhelm Scheele. It is a carboxylic acid which has a hydroxyl group adjacent to the carboxyl group, thus making it an alpha hydroxy acid. Lactic acid is chiral and has two optical isomers L-(+)-lactic acid and D-(−)-lactic acid. In animals, L-lactate is constantly produced from pyruvate via the enzyme lactate dehydrogenase (LDH) in a process of fermentation during normal metabolism and exercise. It does not increase in concentration until the rate of lactate production exceeds the rate of lactate removal, which is governed by a number of factors, including monocarboxylate transporters, concentration and isoform of LDH, and oxidative capacity of tissues. In industry, lactic acid fermentation is performed by lactic acid bacteria, which convert glucose and sucrose to lactic acid. The different process parameters to be maintained during the fermentation process are discussed below: 1. Microorganims: Lactic acid fermentation is carried out by some fungi and bacteria. The most common lactic acid producing bacteria include Lactobacillus bulgaricus, L. delbrueckii, L. casei, L. pentosus. Other bacteria which produce lactic acid include: Leuconostoc mesenteroides, Pediococcus cerevisiae, Streptococcus lactis, Bifidobacterium bifidus. The selection of microorganism eventually depends on the kind of carbon source used in the fermentation process. 2. Carbon sources: Glucose, maltose, sucrose or lactose are the most commonly used carbon sources. Crude substrates such as corn starch, rice starch, potato starch are also used as carbon sources but require a pretreatment with enzymes or acids to bring about hydrolysis to maltose and glucose.

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Table 7.2. Composition of media used for Gluconic acid production Ingredient Cornsteep Liquor MgSO4.7H2O KH2PO4 Urea (NH4)2HPO4 H2SO4 to adjust pH to 4.5 Antifoam agent as needed

Quantity( g/l) 3.7 0.17 0.20 0.10 0.40

3. Nitrogen source and Growth factors: Ammonium salts such as ammonium hydrogen phosphate give satisfactory results. Lactobacilli require complex nutrients such as vitamin B-complexes which are supplied by enriching the media with crude vegetable sources. 4. pH and Temperature: pH of the media should be maintained within a range of 5.55.6. pH of the media has a direct effect on the yield and rate of lactic acid fermentation. The accumulation of acid is carried out by constantly adding a slurry of calcium hydroxide as increase in acidity will affect the bacteria. The temperature for the fermentation depends upon the microorganism employed in the process. For example, L. bulgaricus and L. delbrueckii require an optimum temperature of 45 to 50°C while L. casei and L. pentosus require a temperature of 30°C. 5. Aeration and Agitation: The supply of sterile air is not required as the fermentation process using Lactobacilli is anaerobic. Complete removal of air is not necessary as these bacteria are facultative aerobes. 6. Time and Yield: The duration of fermentation process in 5-10 days with yield in the range of 93-95 per cent of the weight of glucose supplied. 7. Recovery: Recovery of lactic acid depends upon the type of grades needed for different purposes. Food-Grade Lactic Acid: recovery involves filtration followed by acidification of the filtrate with sulfuric acid for regenerating lactic acid while calcium is precipitated as calcium sulfate and washed. This wash is combined with filtrate and treated with activated vegetable carbon to remove organic impurities. This is followed by evaporation to achieve a crude lactic acid concentration of 25 per cent. The refining and evaporation steps are repeated to achieve a concentration of 50-65 per cent. Technical-Grade Lactic Acid: Calcium present is removed as calcium sulfate dihydrate by precipitation. This is followed by filtration to remove precipitates of calcium sulfate dihydrate. Filtrate is concentrated to 35-40 per cent by evaporation. Plastic-Grade Lactic Acid: can be obtained by esterification with methanol.

AMINO ACIDS Amino acids due to their functionality and the special features arising from chirality, have gained tremendous interest in the chemical industry sector. Of the 20 standard protein amino

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acids, the 9 essential amino acids-L-valine, L-leucine, L-isoleucine, L-lysine, L-threonine, Lmethionine, L-histidine, L-phenylalanine, and L-tryptophan cannot be synthesized in animals and humans and must be ingested with feed or food. In terms of market volume, development over the last 20 years has increased tremendously in feed amino acids such as L-lysine, Lmethionine, L-threonine, and L-tryptophan, which constitute the largest share (56%) of the total amino acid market. This is followed by the food sector, which is primarily dominated by three amino acids: L-glutamic acid in the form of monosodium glutamate (MSG) as a flavor enhancer and L-aspartic acid and L-phenylalanine: used as starting materials for the peptide sweetener L-aspartyl L-phenylalanyl methyl ester (Aspartame). The remaining proteinogenic amino acids are used in the pharmaceutical and cosmetics industries (Table 7.3). The rapid development of the amino acid market since the 1980s is due to the development of cost effective production and isolation methods for amino acid products. Among the different methods used for amino acid production, microbial fermentation is the most used production method due its economic and ecological advantages. The following section will discuss the processes used for production of industrially important amino acids using microbial fermentation.

1. Monosodium Glutamate (MSG) MSG was first isolated as glutamic acid in 1866 and has since become the basis of a trillion dollar worldwide industry. It was first isolated as a pure substance by a German chemist Ritthausen through the acidic hydrolysis of gliadin, a component of wheat gluten. However, in 1908 Japanese chemist Kikunae Ikeda found that glutamic acid was responsible for the flavor-enhancing properties of the kelp like seaweed, ―konbu‖, or Laminaria japonica, that had been used for many centuries in Japan in the preparation of soup stocks. By extracting 40 kilograms of the seaweed with hot water, Ikeda obtained 30 grams of (S)glutamic acid, which he then identified as the taste-enhancing component of konbu. Ikeda immediately patented a process for isolating monosodium glutamate from wheat flour, and in 1909 the first monosodium glutamate was produced commercially under the trade name Ajinomoto. In the early 1950s it was discovered that E. coli excreted small quantities of amino acids, and that the quantity could be increased by addition of ammonium salts to the culture medium. Soon thereafter, bacteria were discovered that could produce large quantities of (S)-glutamic acid (Figure 7.3) Later it was found that a particular bacterium, named Corynebacterium glutamicum, could give (S)-glutamic acid in a yield of about 30% from carbohydrate according to the stoichiometry of the reaction given in Figure 7.4. The accumulation of (S)-glutamic acid in the culture medium is determined not only by its rate of biosynthesis but also by its escape through the cell membrane. When biotin, a vitamin essential for cell growth is present in sufficient concentration for an optimal rate of proliferation, the cell membrane is impermeable to glutamate, giving an inferior accumulation of glutamate. Since both beet and cane molasses are rich in biotin, these materials could not be used as sources of glucose in microbial fermentations until biotin inhibiting additives were discovered. The less than optimal rate of proliferation of the bacteria in the biotin-limited fermentation was then partially overcome by the addition of sugar to increase the carbohydrate content of the culture medium. In this way the ultimate concentration of (S)glutamic acid that could be achieved was raised to about 80 g/L. The necessary nitrogen could

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be supplied by ammonium salts, urea, or, by gaseous ammonia, which not only provides the nitrogen but also maintain the pH of the culture medium between 7 and 8 without diluting the culture medium. Table 7.3.

Figure 7.3. Structure of Glutamic acid and Monosodium glutamate.

Figure 7.4. Formation of glutamic acid from carbohydrates by Corynebacterium glutamicum.

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Since the fermentation is aerobic, oxygen is supplied by aeration, and the fermenter is stirred. The medium and all other components are sterilized, and all operations and variables, including temperature, pH, and dissolved oxygen concentration, are automatically controlled during the 35–45 hour time for fermentation. The bacterial culture is grown up in stages from lyophilized seed through reinvigoration in a test tube, shake flask culture, 15 hours in a 10,000 litre seed fermenter, and then the 200,000 litre main fermenter. At the end of the fermentation the fermented broth is sterilized and then centrifuged to remove the microorganisms and any other solids. The clear liquid is then concentrated under reduced pressure, the pH is adjusted to 3.2, the isoelectric point of glutamic acid, and the resulting crystals of (S)-glutamic acid are converted to MSG. The advantage of the fermentation method is that it reliably produces only the desired S enantiomer of glutamic acid. In this well-developed process the accumulation of (S)- glutamic acid can be as high as 100 g/L (10 metric tons per 200,000 liter fermenter), and the yield as high as 60%.The disadvantage is that it is fundamentally a batch process.

2. L-Lysine Soon after C. glutamicum was commercialized for MSG production, auxotrophs were developed that excreted commercial quantities of lysine. By 1958, Kinoshita and colleagues had shown that lysine was excreted by homoserine auxotrophs. For example, a homoserine auxotroph (strain ATCC13287) was patented in 1961 that yielded 44 g lysine per litre with a conversion efficiency of 26% from sugar (g lysine/g sugar). Further increases in yield were made incrementally with the introduction of additional amino acid and vitamin auxotrophies plus development of strains resistant to antimetabolites. Typical strains today provide conversion efficiencies of over 50%. Overproduction of lysine is directly related to the regulatory circuits that govern lysine biosynthesis. The approaches used for creating amino acid overproducers are paradigms of rational strain improvement as contrasted with approaches that rely on random mutagenesis and screening. The pathway (Figure 7.5) leading to lysine (also threonine, isoleucine, methione) biosynthesis is initiated with the conversion of aspartate to aspartyl-P via the enzyme aspartokinase (AK). The phosphorylated aspartate is then converted to aspartyl-semialdehyde (ASA) that can be converted to homoserine by homoserine dehydrogenase (HSD) or to diaminopimelic acid (DAP) by a series of five enzymatic conversions, and thence to lysine. Homoserine is the starting point for making threonine and isoleucine as well as methionine. The major control points for the metabolic flux to individual amino acids occurs at the level of aspartokinase and homoserine dehydrogenase. AK is feedback inhibited by the accumulation of threonine plus lysine. Homoserine dehydrogenase is feedback inhibited by threonine and its synthesis is repressed by methionine. Mutations that lead to the overproduction of lysine include HSD-, HSDleaky, and lysine analogue resistant mutants. HSD- mutants are homoserine auxotrophs that effectively lack the ability to make threonine, methionine and isoleucine, thus eliminating the feedback inhibition of AK by threonine. HSDleaky mutants make an HSD that is less effective at making homoserine so that threonine does not accumulate to levels sufficient to shut down AK. The benefit of the latter mutants is that additional amino acids do not need to be added to growth media to make up for their complete lack of synthesis in HSD null mutants. Analogue

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resistant mutants resist the feedback inhibition effects of allosteric effectors. In the case of AK, mutants were derived that resisted analogues of lysine and/or threonine. To derive such mutants, mutagenized cultures are incubated in the presence of the analogue; those that grow ―resist‖ the analogue and are screened for their ability to make lysine. The rationale behind the approach is that wild-type AK recognizes the lysine analog 2-aminoethyl-L-cysteine (AEC) as lysine. The enzyme is inhibited as if lysine had accumulated in the cell and the cell fails to grow due to lysine starvation. Analogue resistant mutants have AKs that do not recognize AEC as lysine; these enzymes remain uninhibited, continue to make aspartyl-P and then lysine and the cells continue to grow. Such mutants, since they no longer respond to intracellular lysine levels, continue to synthesize lysine at high levels.

Lysine Fermentation The industrial production of L-lysine is carried out in a manner similar to that for Lglutamate production. Sugar cane or beet molasses is used as the substrate for production. If the biotin level is low in the starting material, it must be added to a level of about 30 µg/l. If an auxotroph for any amino acid is used, that amino acid must be present so that the cells may grow but should not be present at high enough concentration to exert inhibitory physiological effects such as feedback inhibition of the pathways used for production. L-Threonine L-Threonine production has been accomplished by genetically manipulating strains of E. coli. The initial strains constructed for his purpose were derived in 1969 as mutants resistant to the L-threonine analogue, α-amino- β-hydroxyvaleric acid (AHV) by Shiio and Nakamori in Japan. Since that time several strains have emerged as production strains based on a combinations of auxotrophic and analogue resistant mutations that have the collective effect of funneling carbon to L-threonine production. Detailed understanding of threonine metabolism in E. coli was essential for rationally designing threonine overproducers. The regulation of threonine biosynthesis in E. coli is more complex than that in C. glutamicum. Unlike Corynebacterium, E. coli has three aspartate kinases, AKI, AKII and AKIII. Two (AKI and AKII) are multidomain proteins that also have homoserine dehydrogenase activity responsible for the third step of the pathway. AKI is feedback inhibited by threonine and its synthesis is repressed by a combination of threonine and isoleucine. The synthesis of AKII is repressed by methionine. AKIII is feedback inhibited and repressed by lysine (Figure 7.6). The second step of the pathway is catalyzed by aspartate semialdehyde dehydrogenase (ASD). Its expression is repressed by an accumulation of high levels of lysine, threonine and methionine, with lysine being the most effective. The last two enzymes, homoserine kinase (HK; thrB) and threonine synthase (TS; thrC) are co-expressed along with AKI (thrA) as part of the thr ABC operon. This operon is controlled by transcriptional attenuation. A 21 amino acid long leader peptide between the promoter and first gene has seven threonines and four isoleucines. The rate of transcription dictates whether the genes are transcribed. If the rate of transcription is fast (lots of charged trp-tRNA and iletRNA), then transcription is terminated; if it is slow, leading to pausing of the ribosome during translation, transcription continues and the enzymes are made. Aside from these regulatory mechanisms, other systems also affect threonine production. These include the breakdown of threonine by threonine deaminase (ilvA) or threonine dehydrogenase (tdh), and transport systems that control the uptake or efflux of threonine from the medium.

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Figure 7.5. Aspartate family of amino acids showing the branched pathways leading to lysine methionine, threonine and isoleucine. Lysine plus threonine exert concerted feedback inhibition (dashed lines) on aspartyl kinase (AK) and threonine feedback inhibits homoserine dehydrogenase (HSD). Methionine represses the synthesis of HSD (dots & dashes).

Figure 7.6. Aspartate family of amino acids in E. coli. *HSD is part of the multidomain proteins AKI and AKII are is under the same regulatory controls as AKI and AKII.

With such a complex regulatory scheme, several approaches have been taken to ―open up‖ the pathway to threonine production. These generally involve deriving auxotrophs for amino acids that might compete for carbon flow through the pathway, or analogue resistant mutants that have enzymes that have lost the ability to be feedback inhibited by the product of the pathway. Analogue resistant mutants of E. coli were derived by 1969 using the threonine analogue α-amino- β- hyroxyvaleric acid (AHV). This compound has been used for virtually all subsequent work. In addition to analogue resistance that removed the feedback inhibition on AKI, auxotrophs were made that required isoleucine. By placing the threonine operon onto multicopy plasmids and introducing it into such a strain, strains were obtained that produced about 65 g/l of threonine with yields of 48% (g thr/g sugar).

L-Aspartate and L-Alanine L-Aspartate is used in foods and pharmaceuticals. The production of aspartate from immobilized E. coli cells has been done since 1973. Cells immobilized in various gels including polyacrylamide or κ-carrageenan, polyurethane has been the method of choice. Aspartic acid is made by the enzyme aspartate ammonia lyase (aspartase) in the presence of ammonium fumarate. Once immobilized, the cells are quite stable retaining aspartase activity for well over 600 days even at 37°C. The process is carried out at pH 8.5 with ammonium

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fumarate as the substrate. Immobilized Pseudomonas dacunhae cells can convert aspartate to alanine using the pyridoxalphosphate dependent aspartate β-carboxylase. This process was industrialized in Japan in 1982 and is used today for making L-alanine.

ANTIBIOTICS 1. Penicillin Penicillins are a group of β-lactam having antibacterial properties. The basic structure of penicillin comprises of a lactam ring and a thizolidine ring fused to form 6-aminopenicillanic acid (Figure 7.7). Natural penicillins such as Penicillin V and G are effective against several Gram positive bacteria. They cause cell death by inhibiting the bacterial cell wall synthesis.

Production Process The lyophilized culture of spores is cultivated for inoculum development which is transferred to prefermenter, and then to fermenter. Penicillin production is an aerobic process and therefore, a continuous supply of oxygen to the growing culture is very essential. The process requires an aeration rate of 0.5-1vvm, pH 6.5, optimal temperature of 25 to 270C and is carried out by submerged fermentation. The medium used for fermentation comprises of corn steep liquor (4-5% dry weight) and carbon source usually lactose. An addition of yeast extract, soy meal or whey is done for a good nitrogen supply. Phenylacetic acid which serves as a precursor for penicillin biosynthesis is continuously fed along with sugar for good yield of penicillin. For efficient synthesis of penicillin,the growth of the organism from spores must be in loose form and not as pellets. The growth phase is around 40 hours with a doubling time of 6-8hours. After the growth phase is stabilized, the penicillin production exponentially increases with appropriate culture conditions. The penicillin production phase can be extended to 150-180 hours. As the fermentation is completed, the broth containing about 1% penicillin is processed for extraction. The mycelium is removed by filtration. Penicillin is recovered by solvent (n-butylacetate or methylketone) extraction at low temperature (0.75mm).

TERTIARY TREATMENT Adsorption: is the accumulation of molecules from a substance dissolved in a solvent onto the surface of an adsorbant particle. Adsorption techniques are used to remove soluble organics from drinking waters and wastewaters. Tiny concentrations of both natural and synthetic organic compounds produce serious taste and odor problems in water. Long term exposure to these organics may lead to potential health hazards. Thus, it has to be removed. Biological treatment processes are efficient in removing organics but they are inefficient in removing small concentration of organics, as they form the nutrient source for the organisms. Adsorption is a surface phenomenon and effective adsorbents have a highly porous structure so that their surface area to volume ratio is very high. The solute molecule is held in contact with the adsorbent by a combination of physical, ionic and chemical forces. When the adsorbent is left in contact with a solution, the amount of adsorbed solute increases on the surface of the adsorbent and decreases in the solvent. When the number of molecules of solute is equal on the adsorbent and in the solvent it represents the adsorption equilibrium. The rate of adsorption is governed by the rate of diffusion of solute into the capillary pores of the adsorbent particle. The rate decreases with increasing particle size and increase with increasing solute concentration and temperature. Low molecular weight solutes are more easily adsorbed than the high molecular weight solutes. Activated Carbon: is the most popular adsorbent and is produced from coal, wood, or vegetable fibre sources. Dehydration and carbonization are achieved by slow heating in the absence of air. Activation is done by the application of steam, air or carbon dioxide at a temperature of about 950C. It is used in two forms: Powder Form of Activated Carbon (PAC): The particles are less than 100mm. This technique is used for intermittent removal of occasional organics by adsorption. PAC is added to sedimentation tanks or to the surfaces of sand filters. When the capacity of PAC for adsorption is lost it cannot be regenerated and must be discarded. Granular Activated Carbon (GAC): It is effectively used for the regular removal of organics. The size of the particle is about 0.5-2mm. GAC is used either as a down flow bed, similar to sand filters, instead of sand filters or if the water is turbid, as a separate stage after sand filtration. In the latter case, adsorption of carbon surface by turbidity particles reduces its

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adsorptive capacity. GAC form is expensive and can be regenerated by a process similar to its manufacture. Regeneration reduces the adsorptive capacity. Reverse Osmosis: When a semi permeable membrane separates fresh and saline water, the fresh water passes through the membrane and joins the saline water. In the case of reverse osmosis, when a pressure above the osmotic pressure is applied on the saline water, only the fresh water passes through the membrane. A wide range of membranes are available for the reverse osmosis. They include cellulose acetate, tricellulose acetate, polyamide, polyimide. Upto 60% of treated waste water can be recycled. The remaining 40% along with other streams which cannot be recycled can be collected into pond. The effluent so collected may be toxic and can be reduced by chemical treatment.

Disinfection Tertiary treatment also includes disinfection. It is the final process to which water is subjected prior to distribution. Two types available are chemical and physical disinfection.

Chemical Disinfection Various chemical agents are used which includes chlorine, chlorinated lime, oxidants like ozone and potassium permanganate and halogens.

Figure 11.7. Schematic diagram of Upflow Anaerobic Sludge Blanket Reactor.

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Loveleen Kaur and Robinka Khajuria

Chlorine: and its compounds are common chemical disinfectants. They are less harmful and more effective comparative to other agents. Chlorination process is of two types namely pre-chlorination and post-chlorination. In the former method chlorine is applied prior to any other treatment, usually for controlling algae, taste and odor. In the later method, chlorine is applied after other treatment processes, especially after filtration. The chlorine dosage must be sufficient to leave a residual of 0.2 – 2.0mg/l free chlorine in water is diethyl paraphenylenediamine method and o-toluidine method. Chlorinated Lime: it is commonly known as bleaching powder. Before the discovery of liquid chlorine, chlorinated lime was widely used for chlorination. It is loose combination of slaked lime and chlorine gas. When added to water it decomposes to give hypochlorous acid. Chlorinated lime is unstable and on exposure to sir, light and moisture reduces the chlorine content rapidly. Ozone: It is powerful oxidizing agent and highly unstable. It must be manufactured on site by passing dry air through a high voltage, high frequency electrical discharge. It has more rapid effect than chlorine in destroying viruses and bacteria including spores. It is also effective in eliminating compounds that give objectionable taste and colour to water. The treatment with ozone should leave 1-2mg/l residual ozone. But it usually leaves very low level of residuals and thus there is no protection against new contamination of the water after disinfection. The high installation and operation costs further reduce its use as disinfectant. Potassium Permanganate: It is also powerful oxidizing agent. It has been found to be effective against cholera pathogens but not others. It is not satisfactory disinfectant as it leaves stain in vessels.

Physical Disinfection UV Radiation: Electromagnetic radiation of UV range can be used to destroy microorganisms. This process is effective in certain small water supplies where the water is highly polished i.e., filtered and dematerialized. Actually irradiation must strike the organism to kill it. In this process some of the radiation energy is absorbed by the organism and other constituents in the medium surrounding the organisms. So if sufficient dosages of UV reach the organism, water can be disinfected. The germicidal effect of UV radiations is thought to be associated with its absorption by certain organic components essential for the functioning of cells. Dissipation of energy by excitation causes disruption of unsaturated bonds, particularly of the purines and pyrimidines, thus leading to lethal biochemical changes. UV treatment does not alter the water chemically. Only energy is added, which produces heat, resulting in a temperature rise in the treated water. UV rays can penetrate the cell wall of microorganisms. The germicidal efficiency of UV rays is maximum at the wavelength of 250260nm. There is an abrupt decrease in the efficiency at 290-300 nm which continues upto visible range.

REFERENCES Bailey, J. E. & Ollis, D. F. (1986). Biochemical Engineering 2nded,McGraw-Hill Book Co, New York. Doran, P. M. Bioprocess, (1995). Engineering Principles. Academic Press, New York

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Metcalf, Eddy.(1979). Wastewater engineering, treatment disposal reuse.2nd ed. McGrawHill Book Co, New York. Shuler, M. L. & Kargı, F. (1992). Bioprocess engineering, basic concepts. Prentice Hall, New Jersey Stanbury, P. F., Whitaker, S. J. & Hall, A. (1995). Principles of Fermentation Technology, 2nd ed. Pergamon Press, Oxford.

AUTHORS’ CONTACT INFORMATION Dr. Loveleen Kaur, Assistant Professor, School of Biotechnology and Biosciences, Lovely Professional University, Phagwara-140002 Punjab, India Email: [email protected] Er. Robinka Khajuria, Assistant Professor, School of Biotechnology and Biosciences, Lovely Professional University, Phagwara-140002 Punjab, India Email: [email protected]

INDEX # 21st century, 103

A access, 71, 120, 121 acetaldehyde, 67, 129 acetic acid, 5, 9, 12, 16, 66, 137, 156, 157, 158, 159, 160, 169, 170, 199, 203, 208 acetone, 9, 14, 62, 79, 115, 131, 132, 135, 151, 158, 165 Acetone-Butanol, 131 acidic, 20, 29, 65, 119, 137, 144, 149, 151, 161, 180, 201, 202, 203, 204, 206, 212 acidity, 95, 131, 141, 143, 157, 159, 160, 198, 201, 203, 209 acrylate, 140 acrylonitrile, 140 activated carbon, 118, 149, 150 Activated sludge process, 219 activation energy, 74 AD, 11, 77 adaptation(s), 49, 101 additives, 5, 119, 144, 210, 211, 217 adhesives, 5 adjustment, 88, 118 adsorption, 72, 105, 118, 124, 218, 219, 226 adsorption isotherms, 124 advancements, ix, 83 adverse effects, 187 Aeration, 82, 84, 134, 141, 142, 143, 219 aerobic bacteria, 157 Africa, 188, 189 agar, 9, 19, 22, 28, 29, 30, 31, 35, 56, 57, 58 aggregation, 113 aging process, 132, 159 Agitator, 83

agriculture, 4, 177 AIDS, 164, 189 alanine, 64, 149 alcohol production, 13 alcohols, 14, 61, 68, 88, 135, 157, 159 aldehydes, 62 algae, x, 5, 56, 171, 179, 180, 185, 186, 189, 219, 220, 222, 223, 228 Algal Biomass, 185 alimentary canal, 205 alkalinity, 217 alkane, 181 allergic reaction, 197 allergy, 214 alters, 114 aluminium, 161, 188, 217 amine(s), 29, 62, 214 amino, x, 4, 5, 15, 30, 40, 43, 45, 47, 48, 54, 55, 62, 64, 66, 67, 68, 71, 115, 121, 129, 143, 144, 146, 147, 148, 149, 179, 180, 183, 185, 187, 189, 198, 211 amino acid(s), x, 4, 5, 15, 30, 40, 43, 45, 47, 48, 54, 55, 62, 64, 66, 68, 71, 115, 121, 129, 143, 144, 146, 147, 148, 180, 183, 185, 187, 189, 198, 211 amino groups, 62 aminoglycosides, 150 aminogram, 187 ammonia, 63, 141, 145, 148, 150, 173, 174, 184, 185 ammonium, 30, 41, 49, 63, 78, 114, 118, 131, 143, 144, 148, 151, 173, 186 ammonium salts, 63, 78, 144, 173, 186 amylase, 62, 66, 67, 130, 135, 141 anaerobic bacteria, 202 anaerobic digestion, 219, 224 anaerobic sludge, 224 ancestors, 155 androgen, 165 anemia, 152, 161 anger, 10

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Index

animal disease(s), 4 annealing, 123 antibiotic(s), x, 5, 8, 9, 10, 14, 19, 30, 31, 38, 41, 42, 43, 46, 50, 57, 63, 64, 65, 76, 77, 151 anti-cancer, 11 anticoagulant, 137 antioxidant, 212 antitumor, 197 APA, 10, 149 aquaculture, 188 Arabidopsis thaliana, 11 arginine, 39, 64 Aristotle, 12 aromatic compounds, 164 asbestos, 84 ascorbic acid, 152, 211 aseptic, 32, 69, 72, 79, 80, 82, 84, 86, 87, 93, 139, 174 Asia, 11 Aspartame, 144 aspartate, 146, 147, 148 aspartic acid, 144 Aspergillus terreus, 140, 153, 154 assessment, 214 assimilation, 49, 63, 78, 220 asthma, 197 atmosphere, 82, 207, 222, 223 atmospheric pressure, 115 atoms, 165, 167 attachment, 100, 121 attitudes, 194 autolysis, 64, 68, 87, 180 automate, 177 automation, 172, 176, 191

B Bacillus licheniformis, 8, 45 Bacillus subtilis, 51, 67, 135, 178 bacterial cells, 22, 25, 27, 41, 54 bacterial colonies, 22 bacterial fermentation, 86, 153 bacterial pathogens, 57 bacterial strains, 193, 202, 203 bacteriocins, 25 bacteriophage, 10 bacterium, 11, 17, 25, 26, 27, 43, 53, 54, 56, 144, 170, 201, 208, 220 Baffles, 83, 84 banks, 35, 36 barriers, 113 base, 30, 45, 50, 80, 85, 100, 159, 179, 191 beef, 57, 64, 182

beer, ix, 7, 9, 12, 14, 65, 70, 100, 130, 132, 133, 134, 156, 157, 193 beet molasses, 136, 138, 139, 141, 147, 153, 173 Belgium, 36 benefits, 156, 183, 194, 196 benzene, 114, 130 beverages, ix, 7 bicarbonate, 94 bile, 19 bioavailability, 205 biocatalysts, 5, 100, 169 biochemical processes, 218 biochemistry, 14, 171 bioconversion, 139, 155 biodegradation, 4 biodiesel, 5 biofuel(s), 129 biogas, 9 bioinformatics, 3 biological processes, 5, 152 biological systems, 3, 100 biomass, 18, 56, 58, 59, 61, 80, 100, 101, 103, 106, 156, 163, 164, 167, 171, 173, 176, 177, 180, 182, 183, 184, 185, 186, 187, 190, 220, 222 biomolecules, 105, 121 biopolymer, 4 bioremediation, 4 biosafety, 36 biosynthesis, 37, 39, 44, 46, 50, 58, 61, 77, 144, 146, 147, 149, 150, 152, 160 biosynthetic pathways, 155 biotechnology, ix, 3, 4, 7, 10, 11, 14, 15, 16, 77, 104, 125, 157 biotin, 41, 144, 147, 173 Biotransformation, vii, 155, 156, 160, 163, 165, 166, 169, 170 bleaching, 228 blends, 130 blood, 27, 56, 57, 58, 64, 137, 189, 197, 198, 214 blood pressure, 197, 198, 214 body weight, 189 boils, 9, 122 bonds, 53, 228 bounds, 4, 14 bowel, 193 breakdown, 147, 172, 173 breeding, 46, 49, 223 brevis, 140, 202, 203, 209 Britain, 182 budding, 12, 172 building blocks, 151 businesses, 15 butadiene, 14, 16, 140

Index butadiene-styrene, 140 by-products, 13, 31, 37, 43, 60, 61, 64, 171, 182, 199

C Ca2+, 141 cabbage, 202, 203, 204, 205, 206, 209 calcium, 29, 65, 68, 113, 131, 135, 141, 142, 143, 160, 173, 186, 197, 198, 205 calcium carbonate, 29, 68, 131 cancer, 9, 11, 164, 189, 197, 205, 214 cancer cells, 205 canker sores, 205 capillary, 24, 226 carbohydrate(s), 55, 62, 63, 64, 65, 132, 144, 145, 151, 181, 185, 212 carbon atoms, 165 carbon dioxide, 7, 12, 13, 129, 133, 172, 174, 177, 185, 200, 203, 204, 208, 209, 223, 226 carbonization, 226 carbonyl groups, 62 carboxyl, 137, 142 carboxylic acid, 137, 142 carcinogenesis, 191, 197 carotene, 117, 189, 191 carotenoids, 45 casein, 64, 135 castor oil, 68, 88 catabolism, 41, 65, 211 catalysis, 14, 160 category a, 73 cation, 54 Caucasus, 198 cell culture, 15, 36, 84, 102 cell death, 149 cell line(s), 36 ccell membranes, 114 cell metabolism, 4 cell surface, 113 cellulose, 19, 62, 63, 66, 72, 82, 130, 170, 179, 227 cellulose derivatives, 72 Central Europe, 79 Centrifugation, 110 cephalosporin, 43 ceramic, 72, 90 CH3COOH, 158 challenges, 47 cheese, 7, 9, 11, 14, 161, 177, 181, 193, 198, 199, 200, 201, 203, 213, 214 chelates, 141 chemical(s), 3, 4, 5, 12, 13, 15, 18, 19, 25, 31, 32, 53, 54, 56, 57, 60, 63, 66, 68, 71, 76, 79, 105, 119, 121, 124, 134, 137, 143, 149, 151, 152, 156,

235

157, 158, 159, 160, 164, 165, 166, 167, 171, 195, 197, 199, 210, 211, 217, 226, 227, 228 chemical characteristics, 160, 210 chemical industry, 143 chemical interaction, 124 chemical properties, 105, 119, 157, 158, 199 chemical reactions, 13, 152, 159, 167 children, 189 China, 9, 11 chiral center, 155 chirality, 143 chitin, 183, 185 chitinase, 114 chlorination, 151, 228 chlorine, 151, 227, 228 chlorobenzene, 114 chloroform, 165 chlorophyll, 209, 223 cholera, 228 cholesterol, 43, 183, 189, 197, 212, 214 choline, 183 chopping, 210, 211 chromatographic technique, 31, 119, 121, 165 chromatography, 105, 115, 119, 120, 121, 165 chromium, 80, 81 circulation, 88, 100, 122, 134, 159, 216 Citric acid, 10, 67, 77, 137, 140, 153 cladding, 81 classes, 13, 38, 46, 118 cleaning, 5, 14, 65, 75, 80, 112, 137, 141, 160, 161, 174, 216 cleavage, 167 climate, 188, 222 clone, 22, 25 cloning, 10, 11, 15, 25, 44 clothing, 15 CO2, 54, 59, 131, 157, 177, 185, 200, 223 coagulation process, 217 coal, 56, 129, 226 cobalamin, 152 cobalt, 65, 152, 153 coding, 38, 46, 49 coenzyme, 50, 152 coffee, 193 collagen, 152, 210, 211 collisions, 217 colon, 54, 197 colon carcinogenesis, 197 colonization, 196 color, 29, 38, 185, 206, 207, 208, 209, 210, 211, 212, 217 combustion, 3, 212

236

Index

commercial, 13, 14, 15, 29, 36, 43, 46, 79, 80, 129, 137, 138, 140, 141, 146, 152, 155, 171, 172, 175, 179, 196, 208, 211 commodity, 188 community(s), 4, 21, 25, 26, 27, 50, 171, 182 compaction, 100 competition, 25, 27, 75 complexity, 50, 105 composition, 19, 56, 58, 59, 60, 62, 64, 68, 70, 142, 179, 180, 185, 198 compounds, 5, 6, 18, 20, 29, 32, 38, 47, 53, 54, 55, 67, 68, 88, 113, 115, 116, 118, 132, 151, 152, 155, 156, 157, 159, 164, 168, 173, 178, 197, 203, 228 compression, 115 computer, 175 computer systems, 175 computing, 204 conception, 13, 83 condensation, 62 conditioning, 28, 133 conduction, 122, 123 conductors, 90 configuration, 165, 166 conservation, 15, 35, 50, 51 constant rate, 135 constituents, 72, 102, 189, 224, 228 construction, 5, 11, 43, 80, 141, 160 consumers, 15, 172, 194, 196 consumption, 61, 80, 98, 101, 129, 178, 182, 184, 196, 197, 205, 207, 209, 215 containers, 33, 35, 72, 159, 176, 207 contaminant, 21, 69, 76, 84, 217 contamination, 32, 69, 70, 71, 72, 75, 86, 88, 97, 102, 138, 176, 187, 188, 207, 212, 228 contour, 113 control measures, 176 convention, 35 Convention on Biological Diversity, 3 convergence, 165 cooking, 130, 210 cooling, 18, 33, 65, 71, 74, 84, 86, 90, 121, 134, 135, 175 cooling process, 33 copper, 65, 141, 160, 173, 205 Copyright, 190 correlations, 12 corrosion, 81, 138 cortisol, 166, 167 cosmetic(s), 5, 144 cost, 5, 14, 15, 18, 31, 58, 60, 62, 63, 66, 75, 77, 97, 138, 141, 144, 159, 171, 173, 177, 179 cotton, 9, 64, 68, 84, 187, 188

covering, 206 crop(s), 4, 9, 130 cryopreservation, 32 crystalline, 121, 140, 141 crystallization, 105, 141, 142, 165 crystals, 33, 121, 123, 142, 146 cultivation, 14, 19, 25, 27, 47, 50, 53, 57, 58, 62, 64, 90, 134, 161, 182, 185, 186, 187, 188 culture conditions, 27, 149 culture media, x, 13, 17, 18, 20, 27, 28, 33, 56, 58, 62, 157 culture medium, 18, 19, 20, 21, 24, 56, 58, 63, 66, 144, 151, 207 cycles, 115, 160, 171, 203 cycling, 123 cysteine, 54, 55, 64, 147 cystine, 64 cytochrome(s), 54, 168

D dairy industry, 62, 161 data collection, 90 Decanter Centrifuge, 114 decomposition, 223 defects, 211, 212 deficiency, 69, 160, 185 degradation, 3, 19, 71, 74, 149, 167, 172, 173 dehydration, 32, 33, 140 denaturation, 61, 72 Denmark, 13, 36 deposition, 68, 88 deposits, 14, 36, 141, 161 depth, 106, 134, 139, 187, 216, 218, 224 derivatives, 3, 45, 63, 68, 72, 88, 161, 163, 164, 168, 169 desiccation, 105 desorption, 121, 124, 217 destruction, 71, 72, 73, 74, 123 detection, 29, 30, 31, 47 detergents, ix, 4, 114 developing countries, 189 deviation, 90 dialysis, 103, 168 diaphragm, 89, 93 diarrhea, 197, 201 diatoms, 3, 55 diet, 151, 156, 182, 189, 190, 194 dietary fiber, 185, 205 Differential media, 57 diffusion, 27, 82, 217, 226 diffusion rates, 82 digestibility, 180, 197

Index digestion, 114, 185, 193, 197, 199, 205, 213, 224, 226 digestive enzymes, 194 diluent, 138 discomfort, 197 discs, 83, 85, 89, 112, 222 diseases, 4, 179, 193, 197, 205 disinfection, 70, 172, 227, 228 dispersion, 83 displacement, 88, 115 dissolved oxygen, 94, 146, 172, 175, 219, 220, 223, 224 distillation, 11, 116, 130, 132, 140 distilled water, 33 distribution, 22, 71, 75, 90, 93, 105, 116, 119, 175, 206, 216, 227 diuretic, 164 diversity, 25, 26, 48, 50, 51, 53, 194 DNA, ix, 10, 11, 14, 21, 25, 26, 42, 43, 44, 45, 46, 50, 51, 177, 191, 214 DNA polymerase, 10 DNA sequencing, 10 DOI, 16, 212 donors, 54 dosage, 149, 228 double blind study, 214 dough, 171, 172, 177 draft, 11, 98, 100, 104 drainage, 215 draught, 98 drinking water, 215, 226 drug discovery, 48 drug metabolism, 164, 169 drugs, 15, 46, 155 dry ice, 123 dry matter, 198 drying, 32, 33, 34, 105, 122, 123, 124, 136, 186, 187, 188, 210, 211, 212 DSM, 163, 169 dyes, 18, 19, 57

E Earth Summit, 35 ecology, 25, 48 economic status, 31 economics, ix, x, 53, 75, 76 effluent(s), ix, x, 85, 102, 188, 215, 216, 219, 220, 222, 224, 227 egg, 58, 187 Egypt, 9, 155 elastomers, 5 election, x

237

electric charge, 93, 218 electrical resistance, 91, 93 electricity, 3, 226 electrodes, 68, 88, 91, 93, 94, 96 electrolyte, 33, 93, 94 electromagnetic, 90, 93 electron(s), 10, 54, 72, 129, 157, 193 electrophoresis, 15, 26, 49 elucidation, 46 empirical methods, 210 EMS, 37 encoding, 38, 44, 45, 163 encouragement, x, 216 energy, 3, 5, 6, 15, 27, 41, 53, 54, 56, 59, 61, 63, 74, 82, 86, 89, 98, 111, 129, 153, 173, 181, 182, 185, 209, 226, 228 energy consumption, 98 energy supply, 3 energy transfer, 182 engineering, ix, 10, 11, 15, 44, 45, 47, 48, 49, 50, 51, 79, 81, 104, 136, 154, 229 England, 9 environment(s), ix, 5, 15, 17, 18, 19, 26, 27, 29, 50, 53, 55, 56, 57, 79, 80, 83, 87, 129, 188, 201, 202, 220, 225 environmental conditions, 27, 53, 165, 177 environmental degradation, 3 epidermis, 210 equilibrium, 94, 118, 226 equipment, 18, 24, 74, 106, 116, 174 erythropoietin, 15 ester, 144, 159 estrogen, 164, 165, 170 ethanol, 5, 6, 12, 14, 46, 49, 62, 63, 67, 115, 129, 130, 131, 132, 134, 137, 155, 156, 157, 158, 159, 165, 171, 172, 173, 174, 175, 176, 177 ethers, 167 ethyl acetate, 114, 165 ethyl alcohol, 16, 203 ethylene, 72 ethylene oxide, 72 eukaryotic, 172 Europe, 10, 79, 189 European Union, 157 evaporation, 121, 122, 143, 159 evidence, 12, 194 evolution, 4, 9, 46, 47, 50, 131, 213 excitation, 228 exclusion, 38, 105, 214 excretion, 38, 41, 101 exercise, 142 expertise, 70 exploitation, 15, 77

238

Index

explosives, 10 exporters, 49 exposure, 22, 72, 73, 74, 123, 187, 206, 208, 226, 228 external environment, 87 extraction, x, 25, 59, 65, 105, 113, 115, 116, 117, 118, 132, 136, 141, 149, 160, 165, 170 extracts, 14, 28, 56, 62, 64, 66

F factories, 12, 88 Facultative Pond, 223 FAD, 153, 187 families, x farms, 188 fat, 65, 185, 198, 201, 210, 211, 212 fatty acids, 66, 68, 88, 193, 199, 207, 210 FDA, 11 FDA approval, 11 feedback inhibition, 38, 40, 41, 45, 77, 146, 147, 148 feedstock(s), 5, 63, 77, 137, 171, 173 fermentation technology, 78 fiber(s), 4, 182, 183, 185, 189, 205 fidelity, 51 films, 68, 81 filters, 68, 72, 87, 107, 108, 217, 222, 226 filtration, 29, 33, 71, 72, 87, 105, 106, 107, 108, 109, 110, 111, 113, 132, 133, 135, 141, 142, 143, 149, 151, 163, 176, 177, 184, 217, 219, 226, 228 Finland, 181 fish, 68, 133, 183, 188 fish oil, 68 flatulence, 197, 205 flavor, 43, 132, 133, 134, 144, 171, 177, 185, 198, 199, 201, 203, 206, 208, 209, 210, 211, 212 flexibility, 155 flocculation, 105, 186, 217, 220 flooding, 83 flora, 134, 194, 201, 205, 207, 209 flotation, 216, 217 flour, 144, 150, 151, 172 flowers, 172 fluctuations, 75, 91 fluid, 83, 89, 91, 98, 106, 109, 113, 117, 118, 176, 216 fluid extract, 118 fluidized bed, 100, 163 fluorescence, 94 foams, 68, 88 folate, 55, 205 folic acid, 198

food, ix, 3, 4, 5, 6, 9, 11, 123, 135, 137, 141, 144, 152, 156, 160, 171, 177, 178, 179, 182, 185, 188, 189, 190, 193, 194, 197, 201, 203, 204, 222 food additive(s), 5, 137, 141 food industry, 135, 137, 190, 222 food production, 177 food products, 182 food security, 3 food spoilage, 4 force, 45, 93, 109, 110 formaldehyde, 72 formamide, 26 formation, 4, 13, 14, 33, 37, 38, 41, 43, 59, 61, 62, 67, 78, 79, 84, 86, 88, 93, 97, 101, 106, 109, 113, 121, 124, 140, 156, 159, 161, 163, 169, 175, 176, 185, 199, 208, 211, 217 formula, 59, 161, 204 fouling, 110 fragments, 46, 160 France, 10, 11, 12, 13, 36, 178, 188, 189 free radicals, 55, 152 freezing, 32, 49, 123 freshwater, 50 friction, 93 frost, 160 fructose, 62, 155, 172, 173 fruits, 7, 134, 172 full capacity, 205 funding, 35 fungi, x, 3, 4, 5, 14, 17, 19, 32, 33, 34, 35, 36, 42, 47, 62, 66, 72, 78, 100, 135, 136, 142, 163, 171, 179, 180, 182, 220, 222, 224 fungus, 45, 46, 135, 137, 139, 149, 168, 171, 172, 180, 181, 182, 183, 191 fusion, 43, 44, 47, 49, 50

G gamma radiation, 38 gel, 15, 26, 33, 35, 49, 56, 118, 120, 141 gel permeation chromatography, 120 gene therapy, 11 genes, 10, 11, 27, 38, 44, 46, 49, 147 genetic code, 10 genetic engineering, ix, 10, 11, 15, 44, 47 genetic information, 10, 25, 26 genetic programming, 163 genetic traits, 9 genetics, 9, 10, 37 genome, 11, 25, 44, 45, 46, 48, 49 genomics, 3, 44, 46 genotype, 37 genus, 157, 158, 164

239

Index geometry, 80 Germany, 12, 13, 36, 170, 178, 186, 193 germination, 132 ginger, 193 gland, 84 global warming, 129 glucoamylase, 130 Gluconic acid, 141, 143, 169, 170 glucose, 44, 45, 62, 63, 64, 102, 129, 130, 131, 137, 141, 142, 143, 144, 150, 151, 152, 153, 155, 157, 160, 161, 162, 163, 168, 169, 170, 172, 173, 181, 183, 203, 211 glucose oxidase, 160, 163, 169, 170 glutamate, 42, 45, 48, 144, 145, 147 glutamic acid, 5, 41, 48, 63, 64, 67, 68, 144, 145, 146 glutathione, 54 glycerol, 10, 14, 33, 48, 67, 77 glycine, 153 glycogen, 64 glycol, 33, 43, 88 glycolysis, 129 goods and services, 3 gout, 184 grades, 76, 143 granules, 64, 135, 176, 225 graphite, 118 GRAS, 196 grasses, 223 gravitational force, 110 gravity, 102, 110, 122, 216, 217, 225 green alga, 189 Green Revolution, 10 greenhouse, 129 greenhouse gas, 129 growth factor, 30, 55, 57, 66 growth hormone, 10, 11, 15 growth rate, 19, 101, 102, 103, 171, 179, 180 guidelines, 35, 182

H habitat(s), 21, 25, 27, 50, 172, 204 hair, 189 halogen(s), 81, 227 hardness, 65 harvesting, 80, 176, 180, 186, 187, 206 hazards, 226 HE, 57 healing, 81, 86 health, x, 4, 11, 51, 61, 72, 182, 183, 189, 193, 194, 196, 205, 226 heat removal, 86

heat transfer, 68, 73, 83, 91, 97 heavy metals, 19 height, 84, 98, 219 helium, 94 heme, 27, 54 hemoglobin, 64 hemp, 14 hepatitis, 11, 15 heredity, 9 high blood pressure, 198 high fat, 198 histidine, 144 history, x, 7, 11, 16, 38, 129 HIV, 164 homogeneity, 48 hormone(s), 11, 15, 119, 155, 163, 170 host, 20, 36, 45, 209 housing, 84 human, 4, 10, 11, 15, 36, 62, 129, 152, 155, 156, 170, 176, 178, 182, 184, 188, 189, 193, 196, 197, 212 human body, 11, 152, 189, 193 human estrogen receptor, 170 human genome, 11 human health, 11 human subjects, 212 humidity, 19, 210, 212 humus, 222 Hungary, 48 Hunter, 48 hybrid, 10, 43, 46 hydrocarbons, 63, 121, 165, 212 hydrocortisone, 165, 167 hydrofluoric acid, 141 hydrogen, 54, 55, 63, 67, 95, 138, 143, 166, 167 hydrogen peroxide, 55 hydrogen sulfide, 54 hydrogenation, 165, 167 hydrolysis, 13, 62, 64, 130, 142, 144, 149, 190 hydrophobicity, 105, 113, 121 hydroxide, 68, 114, 142, 143, 186, 206, 217, 219 hydroxyl, 142, 164, 166, 167 hydroxyl groups, 167 hygiene, 110, 207 hypersensitivity, 213 hypertension, 214

I ideal, 18, 19, 59, 68, 92, 98, 152, 176, 188, 204 identification, 4, 46, 47, 56, 57 illumination, 185 imitation, 151

240

Index

immobilization, 100, 121, 160, 163 immobilized enzymes, 100 immune response, 197 immune system, 21, 197 immunity, 193, 213 immunostimulatory, 197, 214 improvements, ix, 15 impulses, 93 impurities, 58, 61, 65, 139, 143, 149, 150, 151, 217, 218, 219 in vitro, 10, 25, 50 incubation period, 138 incubation time, 27 Independence, 179 independent variable, 69 India, 36, 78, 186, 189, 190 individuality, 21 individuals, 21, 36, 197 inducer, 66 inducible enzyme, 19 induction, 93 industrial sectors, 3, 14 industrial wastes, 177, 222 industrialization, 129 industry(s), ix, 4, 5, 13, 14, 16, 37, 42, 59, 60, 62, 64, 67, 68, 71, 76, 88, 97, 101, 124, 129, 135, 137, 140, 141, 142, 143, 144, 152, 155, 157, 160, 161, 163, 177, 181, 190, 194, 215 infants, 197 infection, 68, 197 ingest, 198 ingredients, 56, 61, 65, 73, 177, 194, 200 inhibition, 30, 31, 38, 40, 41, 45, 57, 77, 147, 148, 170 inhibitor, 41, 67 initiation, 197, 206 injections, 141 injury, 49 inoculation, 13, 22, 79, 101, 138, 169, 174, 201 inoculum, 21, 22, 24, 58, 75, 87, 132, 149, 174, 219 inositol, 153, 173 insecticide, 9 insects, 35 insertion, 42 insulin, 10, 11, 15 integration, 46, 159 integrity, 114, 115 interface, 68, 159 interference, 88 interferon, 11, 15 intestinal tract, 201 intramuscular injection, 141 inventions, 3

investment, 14 ion exchangers, 119 ionic polymers, 119 ionizing radiation, 72 ions, 55, 62, 63, 65, 95, 119 iron, 54, 65, 90, 138, 141, 160, 161, 173, 205, 217, 219 irradiation, 228 isolation, ix, x, 17, 18, 19, 20, 21, 23, 24, 25, 27, 28, 29, 30, 31, 41, 50, 51, 56, 105, 121, 144, 163, 165 isoleucine, 44, 64, 66, 67, 144, 146, 147, 148 isomers, 142 isoprene, 16 isotherms, 124 issues, 180 Itaconic acid, 140

J Japan, 36, 144, 147, 149, 186, 187, 189, 213 joints, 79, 80 jumping, 10

K keratin, 64 ketones, 62 kidney, 185 kill, 37, 228 kinetics, 41 KOH, 94 Korea, 193 koumiss, 11 Krebs cycle, 137

L lactase, 197 lactate dehydrogenase, 142 lactic acid, 5, 6, 11, 12, 13, 44, 46, 51, 63, 64, 76, 137, 140, 142, 143, 154, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 213, 214 Lactobacillus, 46, 49, 140, 142, 178, 179, 196, 200, 201, 202, 203, 206, 207, 209, 210, 224 lactose, 62, 142, 149, 181, 197, 198, 199, 200, 211, 213 lakes, 188 laws, 9 leaching, 105 lead, 4, 37, 49, 63, 64, 68, 71, 75, 77, 84, 86, 88, 93, 146, 173, 181, 184, 203, 206, 208, 217, 224, 226

Index leakage, 84, 107 lecithin, 65 legislation, 14 lens, 14 lesions, 41 leucine, 144 liberation, 185 life sciences, ix lifetime, 94 ligand, 121, 170 light, 37, 53, 93, 116, 185, 187, 222, 224, 228 lipids, 55, 114 lipoproteins, 114, 214 liquid chlorine, 228 liquid interfaces, 159 liquid phase, 112, 117, 123, 222, 226 liquids, 12, 87, 88, 89, 112, 116, 217 liver, 68 livestock, 9, 11 loci, 46 logging, 91 love, v, x low temperatures, 20, 207, 209 lubricants, 4, 5 lutein, 205 Lyophilization, 33, 122, 123 lysine, 38, 45, 49, 51, 60, 62, 64, 144, 146, 147, 148, 180, 189 lysis, 25 lysozyme, 41, 114

M magnesium, 65, 138, 141, 173, 198, 205 magnet, 84 magnetic field, 91, 94, 217 magnetic particles, 217 magnetization, 94 magnets, 84 Maillard reaction, 62 majority, 5, 21, 25, 27, 204 malaria, 141 malnutrition, 189 malt extract, 62, 68 maltose, 62, 142, 153, 172, 178 mammals, 194 man, 3, 47, 111, 153 management, 58 manganese, 65, 173, 205, 219 manipulation, 18, 21, 24 mannitol, 203 manufacturing, 3, 4, 14, 161, 171, 194 marine diatom, 3

241

marketing, 75, 76, 209 Maryland, 36 mass, 6, 10, 68, 88, 90, 91, 93, 97, 98, 100, 104, 105, 115, 121, 139, 169, 181, 182, 186, 187, 188 materials, 3, 4, 5, 15, 36, 57, 58, 60, 61, 62, 70, 75, 76, 79, 118, 122, 123, 144, 155, 157, 171, 173, 176, 177, 179, 181, 210, 211, 213, 216, 222, 223 materials science, 4 mathematical methods, 46 matrix, 25, 26, 118, 119, 121, 220, 225 matter, 12, 61, 73, 87, 105, 110, 130, 172, 198, 215, 217, 220, 222, 223 measurement(s), 46, 71, 90, 94, 95, 102 meat, x, 12, 14, 58, 64, 68, 161, 182, 183, 203, 209, 210, 211, 212, 214 media, ix, x, 13, 17, 18, 20, 21, 22, 24, 27, 28, 29, 31, 33, 38, 41, 47, 49, 50, 53, 55, 56, 57, 58, 59, 60, 62, 65, 66, 68, 70, 71, 73, 77, 82, 83, 86, 96, 108, 109, 115, 118, 134, 135, 138, 139, 141, 142, 143, 146, 157, 171, 174, 204 medical, 150 medicine, 15 medium composition, 68, 70 melting, 123 melts, 56 membranes, 68, 72, 109, 114, 119, 227 Mercury, 90 Mesopotamia, 155 Metabolic, 15, 45, 47, 48, 49, 51, 86 metabolic intermediates, 14 metabolism, 4, 30, 51, 55, 57, 69, 142, 147, 159, 164, 169, 173, 203, 220 metabolites, x, 5, 6, 7, 14, 37, 38, 41, 44, 45, 46, 48, 50, 58, 64, 129, 156, 159, 160, 177 metabolized, 56, 62, 63, 64, 67, 88, 203 metal ion(s), 55, 62, 121 metal salts, 138 metals, 19, 90, 107, 138, 141 meter, 90, 93 methanol, 63, 98, 123, 143, 165, 181 methodology, 42, 49, 77, 105, 160 methyl group, 167 methylene chloride, 165 Mexico, 155, 186, 187, 188 mice, 21, 213 microbial cells, 33, 47, 59, 135, 220 microbiota, 197 micrometer, 24 microscope, 9, 10, 11, 24, 157 microscopic investigations, 12 Middle East, 11 migration, 25 milk sugar, 199

242

Index

mixing, 61, 74, 76, 79, 80, 82, 83, 94, 97, 98, 101, 113, 186, 210, 211, 217, 224 MMS, 37 models, 89, 169 modifications, 80, 88, 97, 98, 167 moisture, 87, 198, 228 molasses, 60, 62, 130, 131, 132, 136, 138, 139, 141, 144, 147, 153, 172, 173, 174, 175, 181 mold(s), 4, 7, 11, 134, 168, 172, 179, 194, 199, 205, 208, 210, 212 molecular biology, 10, 25 molecular mass, 105 molecular oxygen, 129, 166, 193 molecular weight, 68, 109, 119, 120, 226 molecules, 28, 42, 44, 46, 48, 109, 119, 120, 121, 123, 124, 129, 130, 155, 156, 164, 226 molybdenum, 65, 81, 173 monomers, 5 monosodium glutamate, 144 Moon, 190 morphology, 38, 51, 55 mosquitoes, 223 motivation, x MR, 103 mucin, 27 mucus, 172 multidimensional, 26 multiplication, 24, 61, 174, 179 mutagenesis, 38, 44, 45, 46, 48, 50, 146 mutant, 38, 41, 42, 49, 50, 152, 168 mutation(s), ix, 14, 31, 37, 38, 40, 41, 42, 44, 45, 46, 147 mycelium, 33, 142, 149, 151, 153, 169 mycobacteria, 150 mycology, 190 Mycoproteins, 182 myoglobin, 211

N NaCl, 56, 64, 210 NAD, 27, 55 NADH, 55 naphthalene, 150 National Research Council, 14 native population, 219 natural habitats, 21 natural selection, 9 NCTC, 36 necrosis, 141 Netherlands, 190 neutral, 116, 119, 204, 218 nickel, 81, 90

nicotinic acid, 41 nitrates, 186 nitric oxide, 211 nitrite, 210, 211, 214 nitrogen, 19, 30, 32, 41, 54, 55, 58, 59, 60, 62, 63, 64, 66, 68, 78, 94, 102, 123, 124, 138, 141, 144, 149, 150, 151, 153, 173, 179, 184, 186, 187, 191, 220 nitrogen compounds, 55 nitrosamines, 152, 211 non-polar, 118 Nozzle sparger, 85 nuclei, 217 nucleic acid, 25, 54, 55, 179, 180, 184, 187 nucleotides, 54, 55, 66 nucleus, 9, 149, 165 null, 146 nutrient(s), 13, 19, 25, 27, 29, 30, 32, 47, 53, 56, 57, 58, 59, 70, 71, 73, 74, 79, 80, 87, 101, 102, 103, 132, 134, 137, 143, 151, 173, 174, 175, 183, 185, 187, 188, 189, 193, 194, 198, 204, 205, 222, 226 nutrient media, 27, 47, 73 nutrition, 55, 161, 189, 191, 194

O obesity, 189 obstacles, 75 OH, 217 oil, 4, 11, 35, 65, 68, 88, 117, 129, 153, 176, 178 olive oil, 63, 68 operations, x, 4, 75, 80, 82, 105, 122, 146, 163, 174 operon, 44, 147, 148 opportunities, 4 optimization, 15, 58, 69, 77, 163, 167, 168, 169 organelle, 114 organic compounds, 53, 54, 55, 151, 155, 226 organic matter, 219, 220, 222, 223 organic solvents, 114, 116, 118, 165 organism, 6, 8, 17, 18, 19, 21, 27, 30, 31, 37, 41, 44, 55, 57, 69, 101, 115, 149, 150, 151, 156, 163, 164, 172, 182, 208, 220, 228 Orifice sparger, 85 osmosis, 227 osmotic pressure, 227 osteoporosis, 164 overlap, 224 overproduction, 38, 45, 146 ox, 164 oxidation, 53, 63, 72, 153, 156, 157, 161, 163, 168, 185, 186, 188, 208, 210, 211, 219, 220, 221, 222, 223, 224 Oxidation Pond, 221, 222

Index oxidative stress, 28 oxygen, 13, 37, 53, 55, 59, 68, 69, 82, 83, 84, 88, 94, 98, 103, 129, 138, 142, 146, 149, 157, 166, 172, 173, 175, 193, 204, 205, 212, 222, 223, 224 oxygen sensors, 94 ozone, 227, 228

P pain, 197 Pakistan, 213 pantothenic acid, 173 parallel, 46, 47, 89 parents, 9 pasteurization, 4, 13, 133 patents, 14 pathogens, 35, 57, 197, 228 pathology, 37 pathways, 3, 7, 38, 39, 40, 41, 44, 50, 147, 148, 155, 162, 172, 174 PCR, 11, 25 peanut meal, 68, 151 Pectinases, 67, 136 penicillin, 8, 10, 13, 14, 50, 57, 60, 64, 66, 67, 69, 77, 78, 80, 116, 149, 150, 153, 155 pepsin, 12 peptide(s), 62, 64, 68, 121, 144, 147, 199, 214 perfusion, 82 periodontal, 51 permeability, 38, 41, 68, 114, 207 permeable membrane, 94, 96, 227 permeation, 120 permit, 22, 59, 80, 220 pernicious anemia, 152 peroxide, 55 petroleum, 63, 129, 178, 181 phage, 69, 76 pharmaceutical(s), 3, 4, 14, 15, 63, 72, 77, 141, 144, 148, 149, 152, 155, 156, 160, 163 phenolic compounds, 38 phenylalanine, 38, 64, 144 Philippines, 10 phosphate, 15, 41, 55, 58, 63, 67, 116, 124, 135, 143, 150, 151, 160, 168, 170, 173, 197, 214 phosphates, 15, 54, 68, 116, 170 phospholipids, 54 phosphorous, 55, 102, 179 phosphorus, 58, 65, 138, 198 phosphorylation, 55 photolysis, 187 photosynthesis, 53, 178, 222, 223 phylum, 51 physical properties, 56, 220

243

physiology, 48, 55 phytoplankton, 224 pigs, 182 pitch, 83, 175 placebo, 214 plant growth, 170 plants, 3, 4, 5, 53, 129, 155, 172, 187 plasmid, 15, 163 plasminogen, 15 plasmolysis, 64 plastics, 4, 5 platform, 15 platinum, 90 PM, 48 point mutation, 46 Poland, 10 polar, 118 pollution, 15, 188, 220 polyacrylamide, 25, 120, 148 polyimide, 227 polymer(s), 3, 5, 6, 116, 118, 119, 169, 186 polymer matrix, 118 polymerase, 10, 11, 49 polymerase chain reaction, 11, 49 polymerization, 16 polyphenols, 208 polypropylene, 88 polysaccharides, 5, 65, 119, 120 polystyrene, 94 polyunsaturated fat, 185 polyurethane, 148 ponds, 185, 186, 187, 188, 222, 223, 224 pools, 38 population, 17, 19, 21, 28, 29, 37, 71, 132, 151, 177, 182, 202, 204, 219, 223 population growth, 177 potassium, 56, 57, 65, 68, 138, 149, 151, 173, 205, 211, 227 potato, 142 poultry, 182 precipitation, 105, 118, 138, 143, 149, 151, 184, 217 prednisone, 165, 166, 167 preparation, 24, 53, 59, 79, 144, 164, 171, 195, 196 preservation, x, 11, 32, 33, 209 preservative, 156, 204 pressure gauge, 93, 95 prevention, 4, 15, 161, 197 primary function, 211 Primary Screening, 29, 30 principles, 3, 47, 79, 94, 105 private sector, 15 probability, 18, 24 probe, 90, 93, 94

244

Index

probiotic(s), 193, 196, 197, 201, 205, 212, 213 process control, 89, 172, 175, 176 processing stages, 61 producers, 19, 29, 30, 38, 41, 46 progesterone, 164, 166, 167, 169 programming, 163, 168 project, 11, 35 proliferation, 144, 197 proline, 62, 64 promoter, 38, 44, 147 propagation, 58, 174 propane, 137 Proteases, 135, 136 protection, 228 protein components, 68 protein engineering, 45 protein hydrolysates, 64 protein sequence, 10 protein structure, 10 protein synthesis, 150 proteins, x, 5, 6, 54, 55, 56, 62, 64, 68, 71, 72, 87, 114, 117, 118, 119, 121, 147, 148, 172, 177, 178, 179, 183, 187, 189, 191, 199, 201, 204 proteomics, 3, 44, 46 Pseudomonas aeruginosa, 140 public service, 37 pulp, 3 pumps, 94, 188, 220 pure water, 204 purification, x, 4, 18, 20, 59, 75, 79, 105, 117, 119, 121, 135, 141, 171, 219 purines, 55, 62, 228 purity, 22, 32, 76, 105, 116, 135, 156 pyridoxine, 41

Q quality control, 32 quality of life, 4, 7, 129 quality standards, 35, 157 quartz, 93, 219

R race, 138 radiation, 38, 71, 72, 103, 123, 228 radicals, 55, 152 radius, 118 rancid, 212 raw materials, 15, 61, 62, 75, 76, 157, 173, 176, 179, 181, 211

reactions, 13, 16, 30, 38, 39, 54, 55, 71, 152, 153, 155, 159, 164, 165, 167, 172, 197, 211, 224 reading, 204 reagents, 217 recombinant DNA, ix, 11, 14, 42, 43, 44, 46 recombination, 37, 42, 44, 46, 48, 49, 51 reconstruction, 45 recovery, 4, 14, 18, 27, 31, 37, 58, 60, 61, 75, 76, 105, 106, 112, 115, 116, 117, 121, 130, 139, 141, 142, 143, 151, 159, 160 recovery plan, 116 rectification, 130 recycling, 171, 177 red wine, 134 reducing sugars, 62, 211 regulations, 80 regulatory controls, 148 relevance, 14 renewable energy, 129 reparation, 59, 171 repression, 38, 41, 49, 58, 77 reproduction, 6, 10, 43, 153, 172 requirements, x, 17, 25, 53, 55, 57, 59, 61, 65, 69, 72, 80, 83, 86, 90, 103, 111, 123, 164, 187 researchers, 4, 178, 205 residuals, 228 residues, 171, 217 resins, 70, 150 resistance, 38, 41, 44, 72, 73, 81, 90, 91, 93, 106, 107, 118, 148, 160 resolution, 120 resources, 15, 35, 50, 171 respiration, 51, 54, 55, 193 response, 77, 197, 213 restriction enzyme, 10 reverse osmosis, 227 RH, 212 rheology, 177 Rhizopus, 140, 164, 166, 167, 178, 179 riboflavin, 44, 46, 49, 152, 153 ribonucleic acid, 182 ribosomal RNA, 170, 185 ribosome, 147, 150 rings, 84, 85, 167 risk(s), 32, 70, 71, 82, 88, 159, 176 RNA, 27, 170, 184 rods, 118 room temperature, 32, 34, 194, 201 roots, 7, 16 Rotameter, 91 Rotating biological contractor, 222 rotifers, 224 routes, 156

Index rowing, 129 rubber(s), 14, 82, 86, 89 rules, 156 ruthenium, 94

S safety, 3, 35, 61, 89, 93, 106, 155, 188, 206, 209, 210 saline water, 227 Salmonella, 19, 57, 211, 214 salt concentration, 115, 204, 206, 209, 211 salts, 19, 51, 54, 56, 57, 63, 64, 65, 78, 81, 118, 134, 135, 138, 139, 141, 143, 145, 149, 160, 173, 179, 186, 217 saturated fat, 182, 183, 185, 198 saturation, 204, 216 savings, 15 scarcity, 177 science, ix, 4, 7, 36 scope, 5 SCP, 85, 98, 171, 177, 179, 180, 181, 182, 186, 187, 188 seasonal factors, 179 Second World, 178 security, 3 sedative, 164 sediment(s), 50, 216, 220 sedimentation, 110, 113, 216, 217, 219, 220, 222, 224, 226 seed, 68, 141, 146, 174, 175, 184 selectivity, 57, 155 senses, 89 sensing, 88, 90, 93, 94, 96 sensitivity, 19, 41 sensors, 90, 94 septum, 106 sequencing, 10, 25, 27, 46, 47 serine, 63, 135 serum, 58, 197, 214 servers, 84 services, 3, 35 sewage, 9, 20, 61, 185, 186, 188, 215, 216, 222, 224 shape, 93, 106, 120, 123, 172, 223 shear, 82, 97, 113, 115, 222 shear rates, 97 sheep, 11, 28, 195 shelf life, 177, 199, 207, 208, 210 shock, 115 shock waves, 115 shortage, 14, 182 showing, 18, 20, 24, 148, 202 side chain, 167 signalling, 28

245

silica, 33, 35, 118 silicon, 55, 68 silicones, 68, 88 silver, 94 skin, 68, 134, 189, 198, 208 slag, 87 sludge, 100, 173, 216, 217, 219, 220, 222, 223, 224, 225 smallpox, 9 smoking, 210, 212 SO42-, 55 social attitudes, 194 society, 3, 15, 129 sodium, 19, 56, 57, 67, 68, 82, 114, 142, 149, 160, 185, 206, 210, 211 sodium hydroxide, 68, 206 solid phase, 118, 121, 123 solid state, 182 solubility, 5, 31, 82, 88, 115, 118, 165, 217 solution, 35, 72, 90, 93, 95, 103, 109, 116, 121, 123, 141, 142, 159, 161, 206, 207, 208, 226 solvents, 114, 116, 118, 123, 132, 149, 165 South America, 189 soybeans, 65 Sparger, 83, 84 species, 11, 17, 19, 20, 24, 25, 27, 28, 31, 33, 35, 38, 42, 43, 45, 48, 49, 57, 72, 97, 129, 135, 136, 157, 158, 163, 164, 166, 167, 172, 179, 182, 187, 202, 203, 204, 205, 206, 209, 210, 222 specific gravity, 216 spindle, 89 spore, 38, 43, 72, 73, 74, 138 sputum, 20 stability, 45, 66, 68, 77, 114, 118, 123, 160, 163, 211 stabilization, 222 starch, 13, 14, 62, 63, 64, 66, 129, 130, 131, 132, 135, 141, 142, 151, 153, 172, 181, 182, 193 starvation, 147 state(s), 22, 32, 33, 77, 97, 101, 103, 110, 123, 168, 182, 209 Steam traps, 88, 89 steel, 79, 80, 81, 93, 112, 138, 159, 173, 176, 188, 218 sterile, 22, 24, 32, 34, 35, 71, 72, 76, 87, 88, 103, 141, 143, 174, 176 Sterilization, 61, 69, 72, 73, 74, 75, 86, 87 Steroid, 14, 156, 163, 164, 165, 167, 168, 169, 170 sterols, 66, 164, 165 stigma, 165 stimulation, 43 stock, 175 stoichiometry, 59, 144 stomach, 201, 205

246

Index

stomach ulcer, 205 storage, 5, 6, 32, 33, 58, 61, 63, 109, 175, 194, 204, 206, 213 storage media, 58 strain improvement, x, 14, 37, 38, 41, 43, 46, 47, 50, 51, 146 Streptomycin, 8, 60, 78, 150 stress, 6, 28, 49, 97, 115, 177, 189 structural gene, 38, 40 structural protein, 72 structure, 5, 10, 53, 105, 118, 124, 149, 150, 152, 165, 216, 226 style, 132 styrene, 140 Submerged culture, 134, 159 substrate(s), 4, 5, 13, 33, 34, 37, 38, 43, 45, 59, 60, 61, 62, 63, 64, 65, 66, 70, 75, 76, 77, 79, 94, 98, 100, 103, 130, 131, 134, 137, 142, 147, 149, 155, 156, 157, 159, 165, 166, 167, 170, 172, 173, 177, 178, 179, 181, 182, 220 succession, 134, 158 sucrose, 14, 35, 62, 65, 130, 131, 138, 141, 142, 153, 165, 172, 173, 211 Sudan, 155 sugar beet, 130 sugarcane, 130, 181 sulfate, 30, 43, 63, 113, 114, 118, 131, 143, 150, 186, 217 sulfur, 54, 55, 64, 65, 134, 138, 151, 211 sulfur dioxide, 134 sulfuric acid, 30, 68, 130, 138, 141, 143 Sun, 188 supplementation, 153 suppression, 78, 197, 198 Supreme Court, 11 surface area, 84, 86, 118, 123, 138, 156, 225, 226 surface energy, 82 surface tension, 68, 88 surfactants, 5, 68 survival, 32, 35, 55, 197, 213 survivors, 37 susceptibility, 94 suspensions, 27, 35, 217 sustainability, 4 sustainable development, 3 Sweden, 36 swelling, 114 symptoms, 197 synthesis, 10, 39, 41, 42, 55, 146, 147, 148, 149, 150, 152, 155, 156, 166, 167 synthetic polymers, 217

T takeover, 208 tanks, 79, 97, 141, 153, 164, 173, 174, 175, 176, 187, 216, 217, 219, 224, 226 tannins, 134 target, 46, 58, 61, 156 taxonomy, 157 techniques, ix, x, 3, 25, 26, 31, 37, 41, 44, 45, 46, 50, 72, 91, 102, 105, 108, 119, 121, 160, 165, 172, 194, 216, 226 technology(s), ix, x, 3, 4, 10, 11, 14, 43, 44, 45, 47, 50, 78, 136, 170, 171, 172, 177, 178, 182, 221, 225 teeth, 12 tempo, 14 tension, 68, 88, 94 testing, 30, 31, 182 testosterone, 165 textiles, 3 texture, 123, 182, 194, 198, 199, 203, 206, 209, 210, 211 thallium, 57 therapy, 11, 205 thermal resistance, 72 thermal stability, 118 thermolysis, 115 thermostability, 45 threonine, 38, 44, 49, 64, 66, 67, 144, 146, 147, 148, 180, 189 tissue, 15, 105, 152, 209, 211 tissue plasminogen activator, 15 tofu, 161 tones, 155 total product, 172 toxic substances, 56 toxicity, 82, 165 toxin, 45 trace elements, 55, 62, 65, 138, 160, 173, 197 trade, 144, 175, 182 training, 11 traits, 9 transcription, 48, 147 transcripts, 27 transducer, 91, 93 transformation(s), 4, 11, 43, 155, 156, 157, 159, 164, 165, 168, 169 transformation processes, 155 translation, 147 transport, 61, 118, 147, 160, 176 transport costs, 176 transportation, 33, 58, 63, 160, 181

247

Index treatment, ix, x, 4, 11, 13, 14, 28, 59, 65, 71, 76, 85, 102, 130, 141, 151, 153, 164, 180, 185, 190, 194, 198, 204, 206, 215, 216, 217, 219, 220, 221, 224, 225, 226, 227, 228, 229 trial, 58 tricarboxylic acid, 137, 157 tricarboxylic acid cycle, 157 triglycerides, 88 tryptophan, 43, 64, 144 tuberculosis, 10, 150 tumor(s), 197, 213 tumor growth, 213 turbulence, 98, 220 tyrosine, 38, 180

U UK, 12, 36, 213 ultrasound, 115 UN, 3, 189 uniform, 83, 106, 138 United, 172, 187 United States, 172, 187 urban, 215 urban areas, 215 urea, 25, 63, 64, 145 uric acid, 184 USA, 36, 51, 170, 189 USDA, 10, 14 USSR, 50, 198 UV, 37, 228 UV radiation, 228

V vaccine, 9, 11, 15 vacuum, 33, 35, 107, 108, 109, 121, 122, 123, 124, 135, 176, 185, 211 validation, 71 valine, 64, 144 valueless, 29 valve, 89, 90, 91, 115, 218 vapor, 116, 117, 122, 123, 124 variables, 70, 97, 109, 146 variations, 23 varieties, 9, 15, 198, 210 vector, 163 vegetable oil, 117 vegetables, 7, 206, 209 velocity, 73, 75, 91, 109, 110, 115, 210, 216, 217 ventilation, 222 vessels, 56, 79, 81, 84, 85, 112, 116, 133, 174, 228

Vinegar, 11, 156, 158 viruses, 14, 20, 36, 72, 106, 119, 224, 228 viscosity, 61, 69, 83, 84, 106, 110, 135 vitamin A, 189, 198 vitamin B1, 63, 152 vitamin B12, 63, 152 vitamin C, 5, 10, 152, 205 vitamins, 5, 30, 43, 46, 55, 62, 64, 66, 71, 129, 151, 154, 173, 182, 187, 189, 193, 197, 198, 205 Vorticella, 220

W Washington, 48, 103 waste, x, 4, 5, 9, 32, 59, 61, 62, 65, 76, 79, 156, 177, 179, 181, 185, 186, 215, 216, 217, 220, 222, 226, 227 waste disposal, 9, 76, 79 waste treatment, 59, 76 waste water, x, 4, 65, 185, 186, 215, 216, 217, 220, 222, 226, 227 wastewater, 102, 190, 220, 225 water quality, 188 water supplies, 65, 228 water vapor, 124 wear, 159, 215 weight gain, 179 West Africa, 189 wetting, 68 wholesale, 172 wild type, 38, 43 wind power, 188 wires, 86, 93 withdrawal, 80, 103 wood, 80, 130, 159, 160, 181, 212, 226 wool, 87 workers, 79, 80, 98, 179 World Health Organization, 185 World War I, 14, 131 worldwide, 144, 157, 186, 197

X xanthan gum, 63

Y yarn, 84 yeast(s), x, 4, 5, 7, 9, 10, 12, 13, 14, 36, 43, 47, 57, 61, 64, 66, 68, 70, 79, 85, 100, 109, 129, 130, 132, 134, 149, 153, 163, 167, 168, 169, 170, 171,

248

Index

172, 173, 174, 175, 176, 177, 178, 179, 181, 182, 190, 191, 195, 198, 214 yield, 10, 13, 14, 18, 24, 27, 29, 38, 41, 44, 45, 47, 58, 59, 61, 62, 66, 70, 71, 74, 76, 130, 131, 137, 138, 139, 140, 141, 143, 144, 146, 149, 164, 167, 176, 177, 182, 185, 186, 188 young mammals, 194

Z zeolites, 118 zinc, 65, 141, 173 zymase, 172