Basic Biotechniques for Bioprocess and Bioentrepreneurship 0128161094, 9780128161098

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Basic Biotechniques for Bioprocess and Bioentrepreneurship

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Basic Biotechniques for Bioprocess and Bioentrepreneurship

Edited By Arvind Kumar Bhatt Department of Biotechnology, Himachal Pradesh University, India

Ravi Kant Bhatia Department of Biotechnology, Himachal Pradesh University, India

Tek Chand Bhalla Department of Biotechnology, Himachal Pradesh University, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-816109-8 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Linda Versteeg-Buschman Editorial Project Manager: Timothy J. Bennett Production Project Manager: Niranjan Bhaskaran Cover designer: Miles Hitchen Typeset by STRAIVE, India

Contents Contributors Foreword Preface

xv xix xxi

Part I Isolation, screening and culture maintenance 1. Isolation of microorganisms

16 17 17 17

2. Screening strategies 1

Mahinder Kumar Gupta 1. Introduction 2. Clinically important microorganisms 2.1 Site for isolation 2.2 Sampling 3. Agriculturally important microorganisms 3.1 Preliminary assessment 3.2 Identification of sites and sample collection 3.3 Storage and pretreatment of soil samples 4. Dairy related microorganisms 4.1 Sample collection 4.2 Preservation of samples 5. Extremophiles 5.1 Thermophiles 5.2 Psychrophiles 5.3 Alkaliphiles/acidophiles 5.4 Piezophiles 5.5 Radiophiles 5.6 Xerophiles 5.7 Metallophiles 5.8 Halophiles 5.9 Microaerophiles 6. Transport of samples 7. Industrially important microorganisms 7.1 Sources of microorganisms 7.2 Enrichment and isolation 7.3 Cultivation of microorganisms 7.4 Cultivation of extremophiles

7.5 Cultivation of anaerobic microorganisms 7.6 Intracellular bacterial culture 8. Conclusion References

3 3 3 4 4 9 9 9 10 10 10 11 11 11 11 12 12 12 13 13 13 13 14 14 14 14 16

Chayanika Putatunda, Preeti Solanki, Shruti Pathania, Anil Kumar, and Abhishek Walia 1. Introduction 2. Conventional strain screening techniques 2.1 Culture-dependent methods 2.2 Conventional screening of antimicrobials 3. Alternative cultivation methods 4. Molecular method of microbial strain screening strategies 4.1 Randomly amplified polymorphic DNA 4.2 Restriction fragment length polymorphism 4.3 Pulse field gel electrophoresis 4.4 Amplified fragment length polymorphism 4.5 RioPrinter 4.6 Multilocus sequence typing 4.7 MALDI-TOF 5. High-throughput screening techniques 5.1 Advantages of HTS technology 5.2 Analytical measurement 6. “Omics”-based screening techniques 7. Virtual screening strategies 8. Some potential application of novel screening strategies 8.1 In medicine and drug discovery 8.2 Environmental applications 9. Conclusions and future perspectives References

23 24 24 24 25 26 27 29 29 29 30 30 31 31 32 32 33 36 40 40 40 41 41

v

vi

Contents

3. Identification, morphological, biochemical, and genetic characterization of microorganisms

4. Microbial activity and productivity enhancement strategies Shashi Kant Bhatia, Vijay Kumar, Virender Kumar, Ravi Kant Bhatia, and Yung-Hun Yang

Nivedita Sharma, Nisha Sharma, Shakshi Sharma, Pushpinder Sharma, and Bindu Devi 1. Introduction 2. Isolation of microorganisms 2.1 Methods of isolation 3. Identification of microbes 3.1 Principles of taxonomy 3.2 Strategies used to identify microbes 3.3 Morphology of bacteria 3.4 Morphology of fungi 4. Biochemical characterization of microbes 4.1 Indole test 4.2 Methyl red test 4.3 Voges Proskauer test 4.4 Citrate test 4.5 Triple sugar-iron (TSI) agar test 4.6 Carbohydrate fermentation test 4.7 Oxidative fermentative (O-F) test 4.8 Amino acid decarboxylase test 4.9 Litmus milk test 4.10 Hydrogen sulfide test 4.11 O-nitrophenyl-β-D-galactopyranoside (ONPG) test 4.12 Phenylalanine deaminase test 4.13 Catalase test 4.14 Oxidase test 4.15 Gelatin hydrolysis test 4.16 Starch hydrolysis test 4.17 Lipid hydrolysis test 4.18 DNA hydrolysis test or deoxyribonuclease (DNase) test 4.19 Coagulase test 5. Genetic characterization of microorganisms 5.1 Microorganisms whose study is encompassed by microbial genetics 5.2 Determination of DNA sequences 5.3 Microbial fingerprinting methods 6. Conclusion References

47 47 47 52 52 53 59 61 63 63 63 63 65 65 67 68 69 69 70 70 72 72 73 73 74 74 74 75 75

75 76 79 82 82

1. Introduction 2. Isolation of microbes 3. Statistical design for culture and reaction condition optimization 4. Induction strategy 5. Immobilization 5.1 Adsorption 5.2 Covalent binding 5.3 Entrapment 5.4 Cross-linking 5.5 Other methods of immobilization 6. Mutagenesis for enhancement of enzyme activity and productivity 6.1 Physical and chemical mutagenesis 6.2 Directed evolution 6.3 Site directed mutagenesis 7. Metabolic engineering 7.1 Improvement of microbes for utilization of carbon source 7.2 Construction of new metabolic pathway 7.3 Increased cofactor production and regeneration 7.4 Improvement of robustness to stress 8. Co-culture strategy 9. Conclusion Acknowledgment References

85 85 86 87 88 90 91 91 91 92 92 93 95 95 95 96 96 96 98 98 99 99 99

5. Culture maintenance, preservation, and strain improvement Aman Kumar, Srijana Mukhia, Anil Kumar, Kiran Dindhoria, Neha Baliyan, and Rakshak Kumar 1. Introduction 2. Culture media for different aspects 2.1 Classification by physical nature 2.2 Classification by chemical composition 2.3 Classification by purpose/ functional use 3. Sterilization techniques 3.1 Heat sterilization 3.2 Gas sterilization

105 106 106 106 107 108 108 110

Contents vii

3.3 Sterilization by radiation 3.4 Filter sterilization 4. Maintenance and preservation of pure cultures 4.1 Metabolically active methods 4.2 Metabolically inactive methods 4.3 Microbial culture collections 5. Strain improvement 5.1 Characteristics of an improved strain 5.2 Methods for microbial strain improvement 6. Conclusion Acknowledgment References

110 110 111 112 112 114 114 116 117 120 120 120

Part II Laboratory techniques & instrumentation

Arshad Jawed 125 126 127 127 128 130 130

7. Centrifugation: Basic principle, types

138 138 139 142 142 142 142

8. Spectroscopy—Principle, types, and applications

1. Introduction 2. General types of spectra 2.1 Continuous spectra 2.2 Discrete spectra 3. Principle of spectroscopy 3.1 Optical instruments in spectroscopy 3.2 How spectroscopy different from spectrometry 4. Types of spectroscopy 4.1 Ultraviolet and visible spectroscopy 4.2 Infrared spectroscopy 4.3 Mass spectrometry 4.4 Nuclear magnetic resonance (NMR) spectroscopy 5. Conclusion References

145 145 145 145 146 147 147 147 147 150 155 158 162 162

9. Protein purification: Basic principles and techniques

Gaurav Sood, Minakshi Sharma, and Rajesh Kaushal 1. Introduction 2. Basic principles of centrifugation, centrifugal force, and sedimentation coefficient 2.1 Calculation of centrifugal force 2.2 Calculation of angular velocity 2.3 Calculation of relative centrifugal field (RCF) 2.4 Sedimentation coefficient 3. Instrumentation of a centrifuge 3.1 Types of rotors

138

Sweta Sinha, Christine Jeyaseelan, Gunjan Singh, Tanya Munjal, and Debarati Paul

6. Biomolecules: Types, homogenization, bead beater, sonication 1. Introduction 2. Classification of cell disruption processes 2.1 Physical disruption methods 2.2 Chemical disruption 2.3 Large-scale cell disruption: The bead mill 3. Conclusion References

3.2 Material used in rotor construction 3.3 Various types of centrifugation technique 3.4 Types of centrifuges 3.5 Separation methods in different types of centrifugation 3.6 Applications of centrifugation techniques 3.7 Care of centrifugation equipment 3.8 Safety aspects while operating a centrifuge References

133

133 134 134 135 135 136 137

Alla Singh, Krishan Kumar, Dharam Paul Chaudhary, Neeraj Kumar, and Deepak Pandey 1. Introduction 2. Need of protein purification and determination of protein identity 3. Basic principles of protein purification 4. Other considerations 5. Alternative systems 6. Importance of recombinant protein market 7. Conclusion References

165 165 166 168 168 169 169 170

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Contents

10. Chromatography: Basic principle, types, and applications Mahesh Kumar Gupta and Pradip Kumar Biswas 1. Basic principle 2. General terms used in chromatography 3. Types of chromatography 3.1 Liquid chromatography 3.2 Affinity chromatography 3.3 Ion-exchange chromatography 3.4 Size exclusion chromatography (gel filtration chromatography) 3.5 Hydrophobic interaction chromatography and reverse phase chromatography (hydrophobic surface area) 3.6 Multimodal or mixed-mode chromatography (multiple properties) References

173 174 174 174 176 177 180

181

181 182

11. Electrophoresis: Basic principle, types, and applications Babita Rana and Gopal Krishna Joshi 1. Introduction 2. Types of electrophoresis 2.1 Gel electrophoresis 2.2 Zone electrophoresis 2.3 Free flow electrophoresis 2.4 Capillary electrophoresis 3. Conclusion References

183 183 183 189 189 191 192 192

Part III Genomic and proteomic analysis 12. DNA, RNA isolation, primer designing, sequence submission, and phylogenetic analysis Rupali Sharma, Shashwat Sharad, Gillipsie Minhas, Deep Raj Sharma, Kulsajan Bhatia, and Neel Kamal Sharma 1. Introduction 2. Isolation of DNA 2.1 Phenol-chloroform extraction of DNA 2.2 Detergent-based isolation of DNA 2.3 Density-gradient centrifugation

197 197 197 198 198

3. Isolation of RNA 3.1 Guanidium thiocyanate-based isolation of RNA 3.2 TRIzol reagent isolation 4. PCR and primer designing 4.1 Primer design for PCR 4.2 Polymerase chain reaction 4.3 Parameters for primer pair design 4.4 Primer design tips 4.5 Probes design tips 4.6 Product amplicons 5. Submission of sequence to GenBank 6. Phylogenetic analysis 6.1 Methods of phylogenetic analysis 6.2 Character-based methods 6.3 Calculation of the degree of divergence 6.4 Molecular clock hypothesis 6.5 Advantages of phylogenetic analysis 6.6 Modern methods in phylogenetic analysis 7. Conclusion References

198 199 199 199 199 200 200 201 201 202 202 203 203 204 205 205 205 205 206 206

13. RDT and genetic engineering: Basic of RDT method, PCR, and application Komaljeet Gill, Shivanti Negi, Neerja Rana, and Pankaj Kumar 1. 2. 3.

Introduction Recombinant DNA technology Genetic engineering and RDT differences 4. Polymerase chain reaction 4.1 Denaturation 4.2 Primer annealing 4.3 Elongation 4.4 Applications of PCR 5. RDT/genetic engineering linkage to bioentrepreneurship 6. Three pillars of bioentrepreneurship 7. Recombinant DNA technology market-growth and trends 8. RDT linked bioentrepreneurship in the health sector 9. RDT linked bioentrepreneurship in food and agriculture sector: Helping to feed the world 10. Industrial and environmental bioentrepreneurship 11. Conclusion References

207 208 209 209 209 209 210 210 212 213 214 214

215 215 215 216

Contents

14. Protein sequence analysis Deepak Sharma and Abhishek Chaudhary 1. Introduction 2. Proteomics 3. Bioinformatics tools for protein sequence analysis 3.1 Dynamic Bayesian networks 3.2 Support vector machines 3.3 Neural network 4. Sequence aligning programs 4.1 Basic local alignment search tool 4.2 FASTA 4.3 Clustal 4.4 Other programs 5. Alignment-free sequence analysis 6. Conclusion References

217 217 218 219 219 219 220 220 220 221 221 221 221 222

15. Computational strategies and tools for protein tertiary structure prediction Raj Kumar and Ajay Sharma 1. 2. 3. 4. 5.

Introduction Homology modeling Fold recognition/threading Ab initio protein structure prediction Hybrid methods and current trend in protein structure prediction 6. Conclusion References

225 225 227 236 237 238 238

16. Docking strategies Arushi Sharma and Ragothaman M. Yennamalli 1. 2. 3. 4. 5. 6.

Introduction Scoring functions Protein-ligand docking strategies Protein-protein docking strategies Protein-nucleic acid docking strategies Different software and tools used for docking 6.1 AutoDock 6.2 AutoDock Vina 6.3 DOCK 6.4 GOLD 6.5 GLIDE 6.6 GlamDock 6.7 FlexAID 6.8 iGEMDOCK 6.9 FlexX

243 245 246 247 248 249 249 249 249 249 250 250 250 250 250

6.10 Fleksy 6.11 ParaDockS 6.12 FLIPDock 6.13 PharmDock 6.14 FRED 6.15 RosettaLigand 6.16 Flexible CDOCKER 6.17 LigandFit 6.18 rDock 6.19 Lead Finder 6.20 GalaxyDock 2 6.21 MS-DOCK 6.22 BetaDock 6.23 EADock 6.24 FLOG 6.25 Hammerhead 6.26 SwissDock 6.27 Docking Server 6.28 1-Click Docking 6.29 DOCK Blaster 6.30 BLIND Docking Server 6.31 ParDOCK 6.32 FlexPepDock 6.33 ClusPro 6.34 PatchDock 6.35 MEDOCK 6.36 BSP-SLIM 6.37 BioDrugScreen 6.38 KinDOCK 6.39 idTarget 6.40 Pose & Rank 7. Future prospects 8. Conclusion References

ix

250 251 251 251 251 251 251 251 252 252 252 252 252 252 253 253 253 253 253 253 254 254 254 254 254 254 254 255 255 255 255 255 256 256

17. A beginner’s guide to measuring binding affinity during biomolecular interactions Hannah I. Martin, Vidushi Agnihotri, Ragothaman M. Yennamalli, and Aurijit Sarkar 1. Introduction 2. An introduction to major methods of detecting protein-ligand complexes 2.1 Fluorescence assays 2.2 Differential scanning fluorimetry (DSF) 2.3 Isothermal titration calorimetry 2.4 Surface plasmon resonance 2.5 Nuclear magnetic resonance spectroscopy 2.6 Frontal affinity chromatography 3. Conclusion References

259 261 261 263 264 265 266 268 269 269

x Contents

Part IV Industrially important enzymes

20. Industrial enzymes: Basic information, assay, and applications

18. Enzymes and their significance in the industrial bioprocesses

Jagdish Singh and Parmjit S. Panesar

Neerja Thakur, Sheetal, and Tek Chand Bhalla 1. Introduction 2. Enzymes in industries: The sources 3. Production of industrial enzymes 3.1 Selection of strain 3.2 Strain improvement 3.3 Processes for enzyme production in industry 3.4 Downstream processing 3.5 Enzyme formulation 4. Applications of industrial enzymes 4.1 Medicine 4.2 Food and brewing industry 4.3 Dairy products 4.4 Animal feed 4.5 Pulp and paper industry 4.6 Polymer and textile industry 4.7 Enzymes in detergent industry 4.8 Leather industry 4.9 Waste treatment 4.10 Bioethanol production 5. Industrial enzymes global market 6. Conclusion References

273 273 275 276 276 277 277 278 278 278 280 280 281 281 281 282 282 283 283 283 283 283

19. Enzyme kinetics: Industrially important enzymes Archana Singh, Pranjali Singh, Ankit Singh, Deepak Pandey, Durgavati Yadav, and Kumar Sandeep 1. Introduction 2. Industrially important biocatalysts 2.1 Whole cell-based biocatalyst 2.2 Enzymes as a cell-free biocatalyst 3. Production of industrially important enzymes 3.1 Acyltransferase 3.2 Sorbitol dehydrogenase 4. Enhancement in enzymes properties, stability, and kinetics 4.1 Protein engineering 4.2 Immobilization of enzymes 5. Conclusion References

285 286 286 286 286 287 287 288 288 288 292 292

1. Introduction 2. Different important industrial enzymes 3. Cellulase 3.1 Sources 3.2 Application 3.3 Assay of cellulase activity 4. α-Amylase 4.1 Sources 4.2 Application 4.3 Assay of amylase activity 5. Glucose oxidase (GOx) 5.1 Sources 5.2 Applications 5.3 Assay of GOx activity: GOx activity using a coupled reaction with dianisidine and horseradish peroxide 6. Invertase 6.1 Sources 6.2 Applications 6.3 Assay of invertase activity 7. Lipase 7.1 Sources 7.2 Applications 7.3 Assay of lipase activity 8. Protease 8.1 Sources 8.2 Applications 8.3 Assay of protease activity 9. Conclusions and future prospects References

295 296 296 296 297 298 299 299 299 300 301 301 302

302 303 303 303 303 304 304 304 305 306 306 306 307 307 307

Part V Techniques in process development 21. Fundamentals of fermentation technology Manya Behl, Saurabh Thakar, Hemant Ghai, Deepak Sakhuja, and Arvind Kumar Bhatt 1. Introduction 2. History and key scientific achievements in the field of fermentation 3. Modes of fermentation operation 3.1 Batch fermentation 3.2 Fed-batch fermentation 3.3 Continuous fermentation

313 313 314 314 314 315

Contents

3.4 Comparison of batch, fed-batch, and continuous mode of fermentation 4. Factors affecting fermentation 4.1 Moisture 4.2 Oxidation reduction potential 4.3 Temperature 4.4 Biological agents responsible for fermentation 4.5 Nutritional requirements 4.6 Hydrogen ion concentration (pH) 4.7 Inhibitors 4.8 Types of reactors 5. Economic aspects of industrial fermentation from a market perspective 5.1 Plant design 5.2 Process design 6. Some examples of commercial fermentation plants 7. Drivers and future of industrial fermentation 8. Conclusion References

22. Sterilization in bioprocesses

316 316 316 316 316 317 318 318 318 318

323 323 324 324

Introduction Principles of sterilization Overview of fermentation process Sterilization in industrial fermentation Different methods of sterilization Which article should be sterilized? Sterilization of culture media 7.1 Heat sterilization 7.2 Batch sterilization 7.3 Continuous sterilization 7.4 Other physical methods 8. Conclusion References

329 329 329 329 330 330 331 331 331 333 334 336 338 338

23. Scale-up: Lab to commercial scale Krunal Dholiya, Ashesh Parmar, and Hafiza Sidra Bashir 1. Introduction 2. What drives the scaling-up process? 2.1 Market 2.2 Product and process 2.3 Production facilities 2.4 Operation 2.5 Getting familiar with numbers 2.6 Doing the research

345 345 348 348 349 350 350 350 351 351 351 352

24. Bioprocess: Control, management, and biosafety issues Subhasish Dutta and Debanko Das

325 325 325

Sampan Attri and Gunjan Goel 1. 2. 3. 4. 5. 6. 7.

2.7 Starting up and scaling-up; two sides of the same coin 3. Role of laboratory scale in optimization and scale-up 4. Challenges of scaling-up 4.1 Solutions of the limitations 4.2 Optimizing the organism 4.3 Optimization of reaction engineering 4.4 Substrate control 4.5 Temperature management 4.6 pH control 4.7 Oxygen mass transfer coefficient and stirring 5. Conclusion References

xi

341 342 343 343 343 343 344 344

1. Introduction 1.1 Primary online sensors 1.2 Primary at-line sensors 1.3 Process analytical technology 2. Bioprocess control and management 2.1 Estimating the health of a developing batch 2.2 KB control system 2.3 Bioprocess monitoring using electrochemical techniques 2.4 In situ online measurement 2.5 Factors that influence bioprocess monitoring 2.6 Future aspects of bioprocess control 3. Biosafety issue 3.1 National and international scenario 3.2 Biosafety levels 3.3 Biosafety perspective of India 3.4 Outcome result and risk approach 4. Conclusion References

355 355 355 355 356 356 357 358 359 359 359 359 360 360 361 362 363 363

25. Demonstration and industrial scale-up Govindarajan Ramadoss, Saravanan Ramiah Shanmugam, and Thirupathi Kumara Raja Selvaraj 1. Introduction 2. A common skeleton for bioprocess development at scale 2.1 Fermentation

365 366 366

xii Contents

2.2 Product formation and recovery 2.3 Product purification and concentration 2.4 Handling of bioprocessing wastes 2.5 Pitfalls and alternative plans 2.6 Account for operating variances 2.7 Operating costs 3. Laboratory-scale strain development—Parameters to be accounted 3.1 Physical stresses encountered 3.2 Mechanical stresses encountered 3.3 Chemical stresses encountered 4. Systems biology approaches to address the effects of scale-related factors 4.1 Gas mixing 4.2 Substrate and nutrient heterogeneity 4.3 Phenotypic and genetic heterogeneity 5. Stable and reliable production in large-scale bioreactors using engineered strains 5.1 Growth-associated product formation and tolerance engineering 5.2 Nongrowth-associated product formation 5.3 Biosensors and/or biocontrollers 6. Conclusions References

367 367 367 367 368 369

369 370 370 370 370 370 371 371

372 372 372 373 373 373

26. Downstream processing of biotechnology products Dattatray Bedade and Shweta Pawar 1. Introduction 2. Precipitation 2.1 Solubility and stability of proteins 2.2 Methods of precipitation 3. Ultrafiltration 3.1 Principle 3.2 Applications of ultrafiltration in bioprocessing 3.3 Fermentation broth 3.4 Separations/recovery 3.5 Product concentration 3.6 Membrane solutions for herbals and natural products 4. Three phase partitioning 5. Aqueous two-phase separation 5.1 Purification/separation of enzyme by ATPS 6. Chromatographic methods 6.1 Ion exchange chromatography 6.2 Gel filtration chromatography

377 377 377 378 379 379 379 380 380 380

6.3 Affinity chromatography 6.4 Expanded bed chromatography 7. Gel electrophoresis 7.1 Polyacrylamide gel electrophoresis 7.2 Formation of polyacrylamide gels 7.3 Use of stacking gels 7.4 Applications 8. Chromatofocusing 8.1 Principle 8.2 Applications 8.3 Limitations 9. Conclusion References

383 384 385 385 385 385 386 386 387 388 388 388 388

27. Waste management and environment Aakarsha Handa and Paulraj Rajamani 1. Introduction 2. What is the bioprocessing industry? 3. Different types of bio-based industry and its waste composition 3.1 Biopharmaceutical industry 3.2 Agriculture and food industry 3.3 Biochemical industry 3.4 Biofuel and bioenergy 4. Impact of bioprocessing industry waste on environment 4.1 Aquatic life 4.2 Gaseous environment 4.3 Terrestrial life 5. Waste management approaches 5.1 Solid waste management 5.2 Liquid effluent management 5.3 Gaseous waste management 6. Future prospects 7. Conclusion References

391 391 392 392 393 394 394 395 395 397 397 397 397 404 406 408 408 409

Part VI Feasibility, marketing and biobusiness 28. Biobusiness opportunities

380 380 381 381 382 382 383

Ashutosh Gupta and Gitika Nagrath 1. Introduction 2. Emerging biobusiness opportunities 2.1 Agriclinics and centers 2.2 Vegetable processing plant 2.3 Biodiesel production 2.4 Biopesticide manufacturing

417 417 417 417 417 418

Contents

2.5 Vermicompost production 2.6 Specialty medicine manufacturing 2.7 Seed coating services 2.8 Wastewater treatment plants 2.9 Biodegradable plastic production 2.10 Food supplements 2.11 Biorefineries 3. Institutional role in promoting biobusiness opportunities 4. Assessment of biobusiness opportunities 5. Biobusiness opportunities—A managerial perspective 5.1 Compatible business environment 5.2 Appropriate marketing mechanism 5.3 Adequate financial planning 5.4 Human resource management 5.5 Legal framework 6. Commercialization of biobusiness opportunities 7. Challenges and suggestions for developing biobusinesses opportunities 7.1 Common challenges 7.2 Challenges of small and medium entrepreneurs 8. Conclusion References

418 418 419 419 419 419 419 419 420 421 421 421 422 422 423 423 423 423 424 424 424

29. Feasibility analysis and business plan Ashok Kumar Bansal 1. Introduction 2. Process and dimensions of feasibility analysis 2.1 Types of feasibility analysis 2.2 Importance of feasibility study 2.3 Sources of information for feasibility studies 3. Cost/benefit analysis 3.1 Categories of costs and benefits 4. Business plan 4.1 Significance of writing the business plan 4.2 Purposes of making a business plan 4.3 Features of an ideal business plan 4.4 Inputs required for a business plan 4.5 Preparing the business plan 4.6 Outline of a business description 4.7 How to assess the progress of a business plan? 4.8 Common mistakes to avoid while writing a business plan

427 427 428 429 429 430 430 431

5. Business plan: Sample 6. Conclusion References

435 436

436 439 439

30. Patenting, IPR, and regulatory issues Sarita Devi 1. Introduction 2. Types of intellectual property rights (IPRs) 2.1 Patents 2.2 Copyright 2.3 Industrial design 2.4 Trademark 2.5 Trade secret 2.6 Geographical indicators (GI) 2.7 Layout design for integrated circuits 2.8 Protection of new plant variety 3. History of patent system 3.1 History of patents in India 3.2 The regulation of intellectual property 3.3 The TRIPS agreement as an international regulatory device 4. Conclusion Acknowledgments Conflict of interest Funding References

441 441 441 443 443 443 443 444 444 444 444 445 448 450 452 452 452 452 452

31. Commercialization and technology transfers of bioprocess Amit Seth, Aditya Banyal, and Pradeep Kumar 1. 2. 3. 4. 5. 6.

431 432 432 432 434 434

xiii

7.

Introduction Scale-up Low-cost bioprocess development Quality control in bioprocess development Role of R&D in technology development Bioreactor design development and challenges Good manufacturing practices (GMPs) in biotech industry 7.1 Good manufacturing practices (GMPs) compliance for bioprocess 7.2 Components of GMP 7.3 Development of bioprocess purification process 7.4 Process development in upstream processing

455 455 456 457 457 458 459 459 460 460 460

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Contents

7.5 Process development in downstream processing 8. Product novelty and risk assessment 9. Ethical and environmental implications of process development 10. Market strategies for the commercialization of the product 11. Academia-industry partnership/synergy 12. Conclusion References

4. Marketing strategies 5. Marketing mix 5.1 Product 5.2 Price 5.3 Place or distribution 5.4 Promotion 6. Green marketing 6.1 Evolution of green marketing 6.2 Green marketing process 6.3 Green marketing orientation 6.4 Dimensions of green marketing 6.5 Green marketing tools 6.6 Marketing mix in green marketing 6.7 Green marketing practices in India 6.8 Golden rules of green marketing 6.9 Challenges of green marketing 7. Conclusion References

461 461 462 464 465 466 466

32. Bioproduct marketing strategies Shivani Chauhan, Prakriti Jhilta, and Ravi Kant Bhatia 1. Introduction 2. Development of marketing of bioproducts 3. Classification of markets

471 472 472

Index

473 476 476 477 478 478 479 480 480 480 480 481 482 482 483 483 484 484 485

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Vidushi Agnihotri (259), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India Sampan Attri (329), Department of Biotechnology, Jaypee University of Information Technology, Waknaghat, Solan, India Neha Baliyan (105), Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Ashok Kumar Bansal (427), ICDEOL, Himachal Pradesh University, Shimla, India Aditya Banyal (455), Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India Hafiza Sidra Bashir (341), Centre for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan Dattatray Bedade (377), Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States Manya Behl (313), Department of Biotechnology, Himachal Pradesh University, Shimla, India Tek Chand Bhalla (273), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India Kulsajan Bhatia (197), Government Medical College and Hospital, Chandigarh, India Ravi Kant Bhatia (85,471), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India Shashi Kant Bhatia (85), Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, South Korea Arvind Kumar Bhatt (313), Department of Biotechnology, Himachal Pradesh University, Shimla, India

Pradip Kumar Biswas (173), Revelations Biotech Pvt. Ltd and Karyotica Biosciences, Hyderabad, Telangana, India Abhishek Chaudhary (217), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, India Dharam Paul Chaudhary (165), ICAR-Indian Institute of Maize Research, Ludhiana, India Shivani Chauhan (471), Biotechnology Incubation Centre (BIC-HPU), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India Debanko Das (355), Department of Biotechnology, Haldia Institute of Technology, ICARE Complex, Haldia, West Bengal, India Bindu Devi (47), Microbiology Research Laboratory, Department of Basic Sciences, College of Forestry, Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Sarita Devi (441), Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Krunal Dholiya (341), R&D-Product Development, Framtix Holdings AB, Malm€o, Sweden Kiran Dindhoria (105), Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Subhasish Dutta (355), Center of Innovative and Applied Bioprocessing (CIAB), Mohali, Punjab, India Hemant Ghai (313), Department of Biotechnology, Himachal Pradesh University, Shimla, India Komaljeet Gill (207), Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Gunjan Goel (329), Department of Microbiology, School of Interdisciplinary and Applied Sciences, Central University of Haryana, Mahendergarh, India

xv

xvi

Contributors

Ashutosh Gupta (417), Department of Commerce and Business Management, DAV University, Jalandhar, India

Vijay Kumar (85), Biotechnology Division, CSIRInstitute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Mahesh Kumar Gupta (173), A Metahelix Life Sciences Ltd, A TATA Enterprise, Bangalore, India

Virender Kumar (85), Biotechnology Division, CSIRInstitute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Mahinder Kumar Gupta (3), Department of Microbiology, CSK Himachal Pradesh Agriculture University, Palampur, Himachal Pradesh, India Aakarsha Handa (391), School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India Arshad Jawed (125), College of Nursing & Allied Health Sciences, Jazan University, Jazan, Riyadh, Saudi Arabia Christine Jeyaseelan (145), Amity Institute of Applied Sciences, Amity University, Noida, Uttar Pradesh, India

Hannah I. Martin (259), Department of Basic Pharmaceutical Sciences, Fred Wilson School of Pharmacy, High Point University, High Point, NC, United States Gillipsie Minhas (197), Neuroscience Research Lab, Department of Neurology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India Srijana Mukhia (105), Department of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Prakriti Jhilta (471), Biotechnology Incubation Centre (BIC-HPU), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India

Tanya Munjal (145), Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

Gopal Krishna Joshi (183), Department of Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand, India

Gitika Nagrath (417), Department of Commerce and Business Management, DAV University, Jalandhar, India

Rajesh Kaushal (133), Dr Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Aman Kumar (105), Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Anil Kumar (23,105), School of Chemical and Metallurgical Engineering, University of Witwatersrand, Johannesburg, South Africa; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Krishan Kumar (165), ICAR-Indian Institute of Maize Research, Ludhiana, India Neeraj Kumar (165), Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi, India Pankaj Kumar (207), Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Pradeep Kumar (455), Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India Raj Kumar (225), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India Rakshak Kumar (105), Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

Shivanti Negi (207), Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Deepak Pandey (165,285), Department of Reproductive Biology, All India Institute of Medical Sciences, New Delhi, India Parmjit S. Panesar (295), Food Biotechnology Research Laboratory, Department of Food Engineering & Technology, Sant Longowal Institute of Engineering & Technology, Longowal, Punjab, India Ashesh Parmar (341), Smt GN Pandya Commerce & Science College, Surat, India Shruti Pathania (23), Dr. YSP University of Horticulture and Forestry, Solan, Himachal Pradesh, India Debarati Paul (145), Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India Shweta Pawar (377), Department of Chemical Engineering, Institute of Chemical Technology, Mumbai, India Chayanika Putatunda (23), Department of Microbiology, Om Sterling Global University, Hisar, Haryana, India Paulraj Rajamani (391), School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India

Contributors

Govindarajan Ramadoss (365), School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, India Babita Rana (183), Department of Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand, India Neerja Rana (207), Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Deepak Sakhuja (313), Department of Biotechnology, Himachal Pradesh University, Shimla, India Kumar Sandeep (285), Dr. B. R. Ambedkar Institute Rotary Cancer Hospital—All India Institute of Medical Sciences, New Delhi, India Aurijit Sarkar (259), Department of Basic Pharmaceutical Sciences, Fred Wilson School of Pharmacy, High Point University, High Point, NC, United States

xvii

Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Nivedita Sharma (47), Microbiology Research Laboratory, Department of Basic Sciences, College of Forestry, Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Pushpinder Sharma (47), Microbiology Research Laboratory, Department of Basic Sciences, College of Forestry, Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Rupali Sharma (197), Center for Neuroscience and Regenerative Medicine, Uniformed Services University of the Health Sciences (USUHS), Bethesda, MD, United States

Thirupathi Kumara Raja Selvaraj (365), School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, India

Shakshi Sharma (47), Microbiology Research Laboratory, Department of Basic Sciences, College of Forestry, Dr. Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India

Amit Seth (455), Department of Life Sciences (Botany), Manipur University, Imphal, India

Sheetal (273), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India

Saravanan Ramiah Shanmugam (365), School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, India

Alla Singh (165), ICAR-Indian Institute of Maize Research, Ludhiana, India

Shashwat Sharad (197), Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences (USUHS), Bethesda, MD, United States Ajay Sharma (225), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India

Ankit Singh (285), Babu Banarasi Das National Institute of Technology & Management, Lucknow, India Archana Singh (285), Department of Bioinformatics, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, India Gunjan Singh (145), Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India

Arushi Sharma (243), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India

Jagdish Singh (295), Bioprocess Technology Laboratory, Department of Biotechnology, Mata Gujri College, Fatehgarh Sahib, Punjab, India

Deep Raj Sharma (197), Department of Pediatrics and Neuroscience, Albert Einstein College of Medicine, New York, NY, United States

Pranjali Singh (285), National Institute of Technology, Warangal, India

Deepak Sharma (217), Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, India

Sweta Sinha (145), Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India Preeti Solanki (23), School of Health and Applied Sciences, Apex Professional University, Pasighat, Arunachal Pradesh, India

Minakshi Sharma (133), Chaudhary Sarwan Kumar Himachal Pradesh Agriculture University, Palampur, Himachal Pradesh, India

Gaurav Sood (133), Dr Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India

Neel Kamal Sharma (197), AFRRI, Uniformed Services University of the Health Sciences (USUHS), Bethesda, MD, United States

Saurabh Thakar (313), Department of Biotechnology, Himachal Pradesh University, Shimla, India

Nisha Sharma (47), Microbiology Research Laboratory, Department of Basic Sciences, College of Forestry,

Neerja Thakur (273), Department of Biotechnology, Himachal Pradesh University, Shimla, Himachal Pradesh, India

xviii

Contributors

Abhishek Walia (23), Department of Microbiology, College of Basic Sciences, CSKHPKV, Palampur, Himachal Pradesh, India

Yung-Hun Yang (85), Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, South Korea

Durgavati Yadav (285), Department of Urology, All India Institute of Medical Sciences, New Delhi, India

Ragothaman M. Yennamalli (243,259), Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, India

Foreword Bioentrepreneurship essentially is described as an innovation-led business model based on advanced scientific and technological developments. It has three basic elements: an efficient quality management team, a sustainable source of finance and availability, and the access and use of new or novel tools and technologies to develop the bio-based processes and products that should be good enough for doing the business of biotechnology. The book titled Basic Biotechniques for Bioprocess and Bioentrepreneurship is an excellent referral material for researchers and students alike and also for entrepreneurs interested in biology and biotechnology. The key points include the well-spelled out chapters with the principle, methodology, and expected applicability from a bio-entrepreneur point of view. Each chapter emphasizes techniques so that anybody who wants to do something can use the text and start working. They provide details about various processes likely to emerge from this topic and ways to go ahead for these. The chapters also focus on the entrepreneurial opportunities available currently or likely to be possible in the future. In addition, the legal, ethical, and commercial implications are also addressed. Technological advancements in the fields of science and technology, including biology, biological engineering, and especially in biotechnology, have led to the use of plant, animals, and microbes with materials science and technology to transform laboratory findings into valuable commercial bioprocesses to bring various bioproducts to market through entrepreneurs. I hope that the knowledge disseminated through this book will be of immense use to next-generation biotechnology entrepreneurs, increasing their capabilities to do the business of biotechnology for the benefit of mankind. The authors who have contributed the chapters in this book are the qualified domain experts and the editors of the book are highly acclaimed experts globally. I congratulate the editors to have thought of such an effort and also for successful completion of the same. I am confident that their joint efforts have made this book a unique source of scientific and technological knowledge on this very important topic, and that readers will find the book very useful. Ashok Pandey Centre for Innovation and Translational Research, CSIR-Indian Institute of Toxicology Research, Lucknow, India

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Preface Technological advancements in the fields of science and technology and especially biotechnology, which have existed since time immemorial and gained pace during the late 1980s, have revolutionized the entire universe. The global COVID-19 pandemic provides an excellent opportunity to reflect on the role of biotechnology in saving lives worldwide. The immense utility of biotechnology in the production of various valuable bioproducts has been a result of the tireless efforts of each stakeholder in our long journey to reach the present stage and gives us confidence to move ahead with the assurance of a sustainable environment and livelihood. Such a comprehensive reference book has been a long-sought dream, but it also took a very long time to realize this dream. When we started this book, the thought was to summarize the practical experiences of all professionals currently working since the early years of the biotechnology industry, to transform the laboratory-scale efforts for biotechnological product/process development, and to bring these bioproducts to market through several successful bioentrepreneurs. India has around 65% of the population of those under 35 years, and all these young minds are interested in creating their own start-ups. Biotechnology-based start-ups have played pivotal roles in India’s start-up ecosystem under the “Make In India” initiatives. Hence, it is important to "build" more experienced entrepreneurial leaders that are skilled, well-equipped, and have the foresight and commitment required to establish successful businesses. The insights, guidance, and learnings from various biotech-based entrepreneurs, academicians, and professionals who have worked and contributed in this field for decades have been compiled in this book. This book could definitely help determined youth and other professionals in their entrepreneurial journey through biotechnological initiatives. The thematic areas of Basic Biotechnology Techniques and Bioentrepreneurship are extensively evolving, with the latest scientific technology and breakthroughs appearing practically. Gathering information for such diverse thematic areas and presenting these in a sequence as per our thought required significant efforts from all contributors. This book contains a plethora of knowledge and motivation for next-generation biotechnology entrepreneurs, boosting their chances of success in this fascinating and rewarding yet challenging industry. Detailed discussion on topics such as basic biotechnology techniques, laboratory techniques for sample analysis, scale-up techniques, feasibility analysis, and how to make sector-specific business and marketing plans are thoroughly discussed in the book. All these chapters on various aspects will definitely help budding bio-entrepreneurs in their areas of interest. This book has been in the planning and execution stage for more than 4 years. We would like to thank the authors collectively for putting pen to paper or mouse to pad and sharing their valuable knowledge with readers. The book would not have existed without the efforts of our research group; all PhD scholars, postdoctorates, and masters students in our lab; and the department that is always teaching us to improve and do better. The difficulties faced by the students in the lab and the entrepreneurs in the field have always acted as a strong tonic for us to initiate such a large effort to compile this book. We are also thankful to everyone for encouragement and enthusiasm, and special thanks to Deepak Sakhuja and Hemant Ghai, Start-Up Incubatees at the Biotechnology Incubation Centre, HP University, for their valuable support during editing of this book. In addition, special thanks go to Elsevier Editorial Project Manager Timothy Bennett for his tireless efforts, timely reminders, and prompt responses on a regular basis. He kept the project moving as the book entered the final days of production. We sincerely hope that the book will be quite useful not only to students and entrepreneurs but also to society.

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Part I

Isolation, screening and culture maintenance

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Chapter 1

Isolation of microorganisms Mahinder Kumar Gupta Department of Microbiology, CSK Himachal Pradesh Agriculture University, Palampur, Himachal Pradesh, India

1 Introduction Microorganisms are generally regarded as living forms that are microscopic in size and relatively simple, usually unicellular in structure. Microorganisms are a heterogeneous group of several distinct classes of living beings. Whittaker’s system recognizes five-kingdoms of living things—Monera (bacteria), Protista, Fungi, Plantae, and Animalia. Five kingdoms have been modified further by the development of three domains, or super kingdoms system—the Bacteria, the Archaea (meaning ancient), and the Eukarya. Microorganisms are essentially the basic and preferred modals for genetic studies as well as manipulations. Their enormously vast diversity can accentuate the progress in genetic engineering as well as industrial developments [1]. But all the aforesaid research is based on the purity and identity of the microorganisms. Thus, their isolation is essential for taxonomic and experimental work. It is the process of separating a single species of microorganism from its natural habitat and growing it by itself, without interference from other organisms, on a sterile substratum, i.e., in pure culture. The microorganism can then be distinguished from other species by its individual characters (even if artifacts of laboratory conditions) and propagated to provide experimental material. Many fungi and bacteria can live for years in culture if competitors are excluded and if the nutrient medium is renewed periodically. A named collection of such cultures can be invaluable for comparison with freshly isolated and unidentified microorganisms, although some diagnostic characters may be lost and sporulation may cease after repeated subculture [2].

2 Clinically important microorganisms Humans and animals have abundant normal microflora which usually do not produce disease but maintains a balance that ensures the survival, growth, and propagation of both the bacteria and the host. Disease appears when this balance is disturbed either by lowering of host immunity or invasion of defense barriers of the host by pathogens. Some examples of pathogens are Streptococcus pneumoniae, Salmonella typhi serotype typhi, Treponema pallidum, etc. Some members of normal flora become pathogenic to the host if host defense is breached. Common examples are Staphylococcus aureus and Escherichia coli. E. coli is part of the gastrointestinal microbiota of normal humans but is also a common cause of urinary tract infections, traveler’s diarrhea, and other diseases. Some microorganisms are present in the environment but cause disease only when the host is debilitated or becomes immune-suppressed or immune-compromised. These are called opportunistic pathogens. Examples are Pseudomonas species, Stenotrophomonas maltophilia and many yeasts and molds. In such cases, the pathogens grow with the normal microbes or sometimes overgrown by the latter so it becomes difficult to prove their role in the cause of a particular disease [3]. Since Robert Koch proposed a series of postulates in 1884 to link many specific bacterial species with particular diseases, these have remained a mainstay of microbiology. However, many microorganisms that do not meet the criteria of the postulates have been shown to cause disease. For example, Treponema pallidum (syphilis) and Mycobacterium leprae (leprosy) cannot be grown in vitro, but there are animal models of infection for these agents. Similarly, for Neisseria gonorrhoeae (gonorrhea), there is no animal model of infection even though the bacteria can be cultured in vitro. Experimental infection in humans has been produced that substituted for an animal model [4].

2.1 Site for isolation For isolation of clinically important microorganisms, one must look for the diseased person/animal and his surrounding environment. In the body, most bacteria that cause disease do so first by attaching or adhering to host cells, usually Basic Biotechniques for Bioprocess and Bioentrepreneurship. https://doi.org/10.1016/B978-0-12-816109-8.00001-5 Copyright © 2023 Elsevier Inc. All rights reserved.

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4 PART I Isolation, screening and culture maintenance

epithelial cells. After the bacteria have established a primary site of infection, they multiply and spread directly through tissues or via the lymphatic system to the bloodstream. This infection (bacteremia) can be transient or persistent. Bacteremia allows bacteria to spread widely in the body and permits them to reach tissues particularly suitable for their multiplication. Swabs from the oral cavity and sputum are collected in suspicion of Streptococcus spp., Neisseria spp., Eubacterium spp. and Corynebacterium spp., etc. Blood is the most suitable sample where septicemia and bacteremia is suspected such as that caused by Staphylococcus spp., Streptococcus spp., Enterobacteria spp., etc. [5–7] (Table 1).

2.2 Sampling A properly collected specimen is the single most important step in the diagnosis of an infection, because the results of diagnostic tests for infectious diseases depend on the selection, timing and method of collection of specimens. Bacteria and fungi grow and die, are susceptible to many chemicals and can be found at different anatomic sites and in different body fluids and tissues during the course of infectious diseases. Because isolation of the agent is so important in the formulation of a diagnosis, the specimen must be obtained from the site most likely to yield the agent at that particular stage of illness and must be handled in such a way as to favor the agent’s survival and growth. Acute symptomatic phase is most productive in providing appropriate samples [8]. Recovery of bacteria and fungi is most significant if the agent is isolated from a site normally devoid of microorganisms (a normally sterile area). Any type of microorganism cultured from blood, cerebrospinal fluid, joint fluid, the pleural cavity, or peritoneal cavity is a significant diagnostic finding. Conversely, many parts of the body have normal microbiota that may be influenced by endogenous or exogenous agents. Recovery of potential pathogens from the respiratory, gastrointestinal, or genitourinary tracts; from wounds; or from the skin must be considered in the context of the normal microbiota of each particular site. Microbiologic data must be correlated with clinical information in order to arrive at a meaningful interpretation of the results [9]. A few general rules apply to all specimens: 1. The quantity of sample must be adequate so as to recover the appropriate causative agent. 2. The sample should be representative of the infectious process (e.g., sputum, not saliva; pus from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface). 3. Only sterile equipment and aseptic precautions should be used so as to avoid contamination of the specimen. 4. The specimen must be taken to the laboratory and examined immediately. Special transport media may be helpful in maintenance of the viability of microorganisms. 5. Specimens to diagnose bacterial and fungal infections must be taken before starting antimicrobial therapy. If antimicrobial drugs are given before specimens are taken for microbiologic study, drug therapy may have to be stopped and repeat specimen collection. The type of specimen to be examined is determined by the clinical status of the patient. If symptoms or signs point to involvement of one organ system, specimens are obtained from that source. In the absence of localizing signs or symptoms, repeated blood samples for culturing are taken first and specimens from other sites are then considered in sequence, depending in part on the likelihood of involvement of a given organ system in a given patient and in part on the ease of obtaining specimens.

3

Agriculturally important microorganisms

Soil sustains an immense diversity of microbes, which, to a large extent, remains unexplored. These microbes significantly contribute toward various biogeochemical cycles and hence influence the nutritional composition of soil. Microbial ecology is the branch which examines the diversity of microorganisms and how micro-organisms interact with each other and with their environment to generate and to maintain such diversities. The microbial population of soils is estimated to comprise less than 5% of the total space and consists of seven major groups including bacteria, actinomycetes, fungi, algae, protozoa, nematodes, and bacteriophages. Among these groups bacteria are the most abundant group [10]. Microbial communities, particularly bacteria and fungi constitute an essential component of the biological characteristics in soil ecosystems [11]. However, major microbial activity is confined to “hot spots,” i.e., aggregates with accumulated organic matter, rhizosphere [12]. Bacteria do not occur freely in the soil solution but are closely attached to soil particles or embedded in organic matter; even after adding the dispersing agent bacteria are not completely dislodged from the soil particles and distributed in

TABLE 1 Table of diseases.

S. no.

Microorganism

Diseases caused

1.

Staphylococcus spp.

Folliculitis, osteomyelitis, arthritis, bronchopneumonia, meningitis, intracranial thrombophlebitis, bacteremia, septicemia, pyemia, endocarditis Urinary tract infections

Affected organs/ tissue

Sample to be taken

Skin and soft tissue, musculoskeletal, respiratory, CNS

Pus Nasal swab Sputum Cerebrospinal fluid (CSF)

Endovascular, blood vascular system

Mode of transmission

References (select latest)

Direct contact, through fomites, by dust, by airborne droplets

[1]

Direct contact, though contaminated fingers, dust, fomites, nonbiting insects

[2]

Contaminated fingers, inhalation of contaminated droplets

[3]

[4]

Blood Urinary tract

Urine 2.

3.

4.

5.

6.

Streptococcus spp.

Pneumococcus spp. (now known as Streptococcus spp.) Neisseria spp.

Corynebacterium spp.

Bacillus spp.

Upper respiratory tract infections, pharyngitis, rheumatic fever, pyoderma, glomerulonephritis, neonatal meningitis, endocarditis, dental carries

Throat

Throat swab

Cardiac muscles Skin Kidney Brain Blood vascular system Mouth

Blood Pus Blood CSF Blood

Pneumonia Acute tracheobronchitis Empyema Meningitis

Lungs

Sputum, blood

Brain

CSF

Cerebrospinal meningitis Meningococcal septicemia

Brain Blood

Airborne droplets

Gonorrhea

Genital tract

CSF Blood, nasopharyngeal swab Urethral discharge

Diphtheria Pseudotuberculosis (sheep) Suppurative lymphadenitis (horse) Pyelonephritis (cattle) Pneumonia (foals)

Nasopharynx

Throat swab Sputum Pus

Airborne droplets

[5]

Anthrax (zoonosis) Fatal septicemia Meningitis Foodborne gastroenteritis (food poisoning)

Skin, lungs, intestine Blood, brain Stomach

Contaminated soil Inhalation of dust from infected wool Ingestion of contaminated food

[6]

Oral swab

Sexual contact

Urine Sputum Tissue from carcass Swabs from lesions CSF Fecal samples

Continued

TABLE 1 Table of diseases—cont’d

S. no.

Microorganism

Diseases caused

7.

Clostridium spp. (anaerobe)

8.

Enterobacteria (E. coli, Klebsiella spp., Enterobacter spp., Serratia spp., Proteus spp.)

Affected organs/ tissue

Sample to be taken

Gas gangrene Tetanus Food poisoning (botulism) Acute colitis Braxy (sheep) Malignant edema (cattle and sheep)

Muscle tissue Muscular system, CNS Digestive system Colon

Necrotic tissue, exudates

Urinary tract infections Diarrhea Pneumonia Pyogenic infections Neonatal meningitis Septicemia Bacillary dysentery Enteric fever Food poisoning

Genital tract Stomach Lungs Perianal area Brain Blood Intestine Intestine intestine

Urine Feces Sputum

Food, feces Feces

CSF Blood Feces

Mode of transmission

References (select latest)

Wound contamination Septic absorption Ingestion of preformed toxin

[7]

Hospital instrumentation Ingestion Air-borne droplets Intra-abdominal infections

[8]

Ingestion

9.

Vibrio spp.

Cholera

Gastrointestinal tract

Stool

Contaminated food/water

[9]

10.

Pseudomonas spp.

Nosocomial infections

Gastrointestinal tract

Pus (repeated samples)

Hospital environment

[10,11]

11.

Yersinia spp.

Plague Yersiniosis

Lymph nodes Lungs

Smears from bubo Tissue from lesions

Flea bite Droplet infection

[12]

12.

Pasteurella spp.

Wound infection Meningitis Osteomyelitis

Skin Brain Skeletal system

Pus CSF Tissue

Animal bites Droplet infection

[13,14]

13.

Haemophilus spp.

Meningitis Laryngoepiglottitis Pneumonia Conjunctivitis Chancroid

Brain Epiglottis Lungs Eyes Genitalia

CSF Blood Sputum Ocular discharge Lesions

Droplet infection

[15,16]

Direct contact

14.

Bordetella spp.

Pertussis

Respiratory system

Postnasall swab Pernasal swab

Droplet infection Fomites

[17]

15.

Brucella spp.

Brucellosis

Reticuloendothelial system

Blood, CSF, urine, pus

Ingestion, contact, inhalation, inoculation

[18]

16.

Mycobacterium spp.

Tuberculosis

Lungs, brain, liver, bone marrow, kidney

Sputum, laryngeal swab, gastric levage

Droplet infection

[19]

17.

Nontuberculosis mycobacteria

Chronic pulmonary disease Avian tuberculosis Lymphadenopathy Leprosy

Lungs Lymph nodes Skin

Sputum Smear from ulcer Scrappings from nasal mucosa, skin lesions and ear lobules

Droplet infection

[20]

18.

Spirochetes

Syphilis Yaws Pinta

Genitalia

Serum from lesions CSF

Sexual contact Flyborne disease

[21]

19.

Mycoplasma spp.

Pneumonia Genital infections

Lungs Genital tract

Throat swab, sputum Tissue

Droplet infection Direct contact

[22]

20.

Actinomyces spp.

Actinomycosis

Cheek, submaxillary regions, lungs

Pus from lesions Sputum

Endogenous infection

[23]

21.

Nocardia spp.

Cutaneous, subcutaneous, and systemic lesions

Skin, subcutaneous tissue, internal organs

Pus from lesions Tissue

Soil-borne infection

[24]

22.

Listeria spp.

Neonatal meningitis Abortion, stillbirth Listeriosis

Brain Genital tract Internal organs

Cervical and vaginal secretions, blood, CSF

Contact with infected animals Ingestion

[25]

23.

Campylobacter spp.

Diarrhea

Gastrointestinal tract

Feces

Zoonotic disease

[26]

24.

Helicobacter spp.

Mucosa associated lymphoid tissue lymphoma

Gastric mucosa

Feces

Direct contact

[27]

25.

Rickettsia spp.

Typhus fever Spotted fever

Vascular endothelial cells

Saliva, feces Smears from peritoneum

Arthropod borne disease

[28]

26.

Chlamydia spp.

Trachoma Genital chlamidiasis Psittacosis (parrots)

Eyes Genital tract Brain

Ocular secretions Genital secretions CSF

Fomites Direct contact

[29]

Fungal infections 27.

Cryptococcus spp.

Cryptococcosis

Lungs, brain

Secretions from lesions, sputum, CSF

Inhalation, direct contact

[30]

28.

Candida spp.

Candidosis

Skin, mucosa, and internal organs

Scrapings from lesions

Opportunistic endogenous infection

[31]

29.

Dermatophytes

Dermatomycosis

Skin, nails, and hair

Scrapings from ringworm lesions

Contaminated products

[32]

30.

Aspergillosis spp.

Asthma Bronchopulmonary aspergillosis

Respiratory system

Sputum, lung tissue

Opportunistic infection

[33]

32.

Penicillium spp.

Penicilliosis

Respiratory system

Sputum

Opportunistic infection

[34]

8 PART I Isolation, screening and culture maintenance

suspension as individual cells. Moreover, they play a major role in organic matter decomposition, biotransformation, biogas production, nitrogen fixation, etc. The bacteria which contribute significantly toward the fertility of agricultural lands have been referred to as plant growth promoting rhizobacteria (PGPR), capable of promoting plant growth by colonizing the plant root [13]. These are also termed as plant health promoting rhizobacteria (PHPR) or nodule promoting rhizobacteria (NPR) and are associated with the rhizosphere which is an important soil ecological environment for plant-microbe interactions. Most PGPRs are members of fluorescent Pseudomonas. The best-known PGPRs are Rhizobia, which produce nodules in leguminous plants. A variety of bacteria have been used as soil inoculants intended to improve the supply of nutrients to crop plants. Species of Rhizobium (Rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium, and Sinorhizobium) have been successfully used worldwide to permit an effective establishment of the nitrogen-fixing symbiosis with leguminous crop plants. On the other hand, nonsymbiotic nitrogen fixing bacteria such as Azotobacter, Azospirillum, Bacillus, and Klebsiella sp. are also used to inoculate a large area of arable land in the world with the aim of enhancing plant productivity. In addition, phosphate solubilizing bacteria such as species of Bacillus and Paenibacillus (formerly Bacillus) have been applied to soils to specifically enhance the phosphorus status of plants [14]. In addition to advantageous partners, the pathogens are also important inhabitants of soil. These include plant, human as well as animal pathogens. Bacteria as plant pathogens can cause severe economically damaging diseases, ranging from spots, mosaic patterns or pustules on leaves and fruits, or smelly tuber rots to plant death. Some cause hormone-based distortion of leaves and shoots called fasciation or crown gall, a proliferation of plant cells producing a swelling at the intersection of stem and soil and on roots. Commonly isolated are Pseudomonas syringae, Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas sps., Erwinia amylovora, Xylella fastidiosa, etc. Besides, there are some human pathogens like Clostridium, Bacillus, Listeria, Campylobacter, Burkholderia, Legionella and Mycobacterium, etc. that are frequently observed in soil [14]. Actinomycetes share the characters of both bacteria and fungi, and they are commonly known as “ray-fungi” because of their close affinity with fungi. They are Gram-positive and release antibiotic substances into the soil. The earthy odor of newly wetted soils has been found to be a volatile growth product of actinomycetes. Their presence in the soil is governed by pH of the soil and the favorable pH is neutral or alkaline pH (6.0–8.0). Actinomycetes are one of the predominant members of soil microbial communities and they have beneficial roles in soil nutrients cycling and agricultural productivity [15]. In the order of abundance in soils, the common genera of actinomycetes are Streptomyces (nearly 70%), Nocardia and Micromonospora, Actinomycetes, Actinoplanes, Micromonospora, and Streptosporangium are also generally encountered. Soil algae (both prokaryotes and eukaryotes) luxuriantly grow where adequate amounts of moisture and light are present. They play a significant role for the improvement of agricultural soils through biological nitrogen fixation, reclamation of alkaline soil, sewage treatment, bio control of agricultural pests, formation of microbiological crust, etc. Commonly observed algae genera in soils are Nostoc, Anabaena, Phormidium, Oscillatoria, Chlorella, etc. [16]. Fungi share a major part of the total microbial biomass in the aerated and cultivated soils because of their large diameter and extensive network of mycelium. However, the population of soil fungi ranges from 2
104 to 1
106 propagules per gram dry soil and its number differs according to isolation procedure and composition of media. Fungi dominate in low pH or slightly acidic soils which are undisturbed for long. These are classified into root inhabiting and soil inhabiting fungi by Garrett [17]. These are associated with nutrient supply toward plants as mycorrhizae thus enhancing the plant productivity [18] and providing protection against soil-borne diseases through antibiotic production [19]. The common genera encountered in soil are Zygomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes. Fungi may play a pathogenic role causing a variety of plant diseases. Common examples of pathogenic fungi are Phytophthora, Rhizoctonia, Pythium, Vertcullium, etc. In moist soil most of the protozoans remain in encysted form. The population of each group is 103 per gram of wet soil. The role of soil protozoa is predatory, as these eat upon bacteria and thereby regulate their population. These are among the nutrient suppliers to the plants and have been found to significantly affect the plant growth. Predation by protozoa has a significant effect in controlling bacterial populations in soil, and the degradation of bacteria undoubtedly contributes to the maintenance of soil fertility [20]. Likewise, protozoa play an integral part in the cycling of nutrients in aquatic food chains [21,22]. The common protozoans found in agriculture soils are Acantbamoeba, Naegleria, Tetrabymena, Hartmannella, etc. With the advancements in soil ecology the role of nematodes has gained importance. Nematodes derive nutrients for their growth and reproduction from the cell contents and cytoplasm of protozoa, bacteria, fungi, etc., thus maintaining their populations. Nematode communities play a significant role in regulating decomposition and nutrient cycling [23] and are centrally placed in the soil food web [24]. Common nematodes in soils are Caenorhabditis, Aphelenchoides, Tylenchus, Tylencholaimus, and Ditylenchus, etc. Besides, they may be pathogenic to plants, e.g., Meloidogyne.

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Recently, bacteriophages and plant viruses have also been proved to be among the important soil inhabitants. Bacteriophages are the viruses that infect bacteria and thus provide protection to the plant against bacterial diseases. These may be used as biocontrol agents in crop production [25,26].

3.1 Preliminary assessment Site assessment prior to sample collection should include exploratory soil sampling. On a potential field site, it can be done with hand probes or augers, or by exposing the upper part of the soil with a spade. Profiles can reveal evidence of deposition, erosion, and previous land use, and can sometimes serve as a rough gauge of time since previous major soil disturbances. Indeed, determining past human impacts on the soil may be critical to understanding current soil conditions, how a soil will change in response to disturbance and how a soil’s physical and biological features will change with time.

3.2 Identification of sites and sample collection Sample number calculation: If data are distributed normally, then the number of samples that are necessary for a given level of accuracy can be found relatively simply by using the relationship, i.e., n ¼ t2C2/E2 where, n ¼ the number of samples to be collected, t ¼ Student’s t statistic that is appropriate for the level of confidence and number of samples being collected, C ¼ the coefficient of variation (standard deviation divided by the mean), E ¼ the acceptable error as a proportion of the mean [27]. Composite sampling: Compositing or combining sampling units into a single sample for analysis is an effective method for obtaining an accurate estimate of the population mean while reducing cost and analytical time. The requirements for compositing samples are (1) the sample volume represents a homogeneous sample, (2) each sample contributes an equal amount to the composite, and (3) there are no interactions between the sample units within the composite that would significantly affect the composite value. When these conditions are met, values from composites agree well with the means obtained from single sampling units [28]. Sample collection and its frequency: The rhizosphere region and rhizoplane provide better sites for the isolation of bacteria than the bulk soil [29]. Several studies indicated that structural and functional diversity of rhizosphere populations is affected by plant species, root exudates and rhizo-depositions [11,30]. Soil types, growth stage of plants, cropping practices, and environmental factors also influence the composition of the microbial community in the rhizosphere [31]. Bacterial diversity of the wheat rhizosphere was observed to be more diverse than chickpea rhizosphere. A representative number of soil samples are taken from the study site and either combined to make a composite sample or treated as individual. Typically, a series of random samples is taken across representative areas that are described by uniform soil type, soil texture and habitat characteristics. Samples of agricultural soils are often taken from specific soil depths (e.g., 0–20 or 0–30 cm) [32]; samples of forest soils are taken from specific soil horizons (e.g., litter horizon, A horizon).

3.3 Storage and pretreatment of soil samples Biological analyses should be performed as soon as possible after soil sampling to minimize the effects of storage on soil microbial communities. Moist soil can be stored for up to 3 weeks at 4°C when samples cannot be processed immediately. If longer storage periods are necessary, the samples taken to measure most soil biochemical properties (soil microbial biomass, enzyme activities, etc.) can be stored at 20°C; the soil is then allowed to thaw at 4°C for about 2 days before analysis. The soil disturbance associated with sampling may itself trigger changes in the soil population during the storage interval. Observations on stored samples may not be representative of the undisturbed field soil. If samples are stored, care should be taken to ensure that samples do not dry out and that anaerobic conditions do not develop. Soil samples are often sieved through a 2-mm mesh screen to remove stones, roots, and debris prior to analysis. Wet soil samples have to be either sieved through a 5-mm mesh or gently pre dried before using the 2-mm mesh sieve. For Streptomycetes, pretreatment is required. Pretreatment of soil samples implemented by physical and chemical treatment to enhance selective isolation, liberate spores from vegetative cells and reduce growth of other microorganisms. Different types of chemical treatment: calcium carbonate treatment, calcium chloride treatment, chitin treatment, sodium dodecyl and yeast extract, phenol and antibiotics (antifungal and antibacterial) treatment and physical method: air dry, heat dry, moist heat, electromagnetic wave, UV treatment are applied as pretreatment for isolation of Streptomyces [33].

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4

Dairy related microorganisms

Milk is a highly nutritious food that can be obtained from a variety of animal sources such as cows, goats, sheep and buffalo, as well as humans, for human consumption. Being rich in proteins, fats, carbohydrates, vitamins, minerals, and essential amino acids, it provides an ideal hub for the growth of many microorganisms [34,35]. Generally, lactic acid bacteria (LAB) are a dominant population in bovine, goat, sheep, and buffalo milk, prior to pasteurization. The most common LAB genera in milk include Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus. Psychrotrophic microorganisms, which survive during cold storage, are also a major component and frequently include Pseudomonas and Acinetobacter spp. Other strains of non-LAB genera are also encountered in milk, as well as various yeasts and molds [34]. Human milk on the other hand is typically dominated by Streptococcus, Staphylococcus, Lactobacillus, and Bifidobacterium spp. [36]. Milk in healthy udder cells is thought to be sterile but thereafter becomes colonized by microorganisms from a variety of sources, including the teat apex, milking equipment, air, water, feed, grass, soil, and other environments [37,38]. There were 25 genera detected in these milk samples, and many of these, including Aerococcus, Streptococcus, Propionibacterium, Acinetobacter, Bacillus, Ochrobactrum, Pseudomonas, Psychrobacter, Staphylococcus, Sphingomonas, Enterobacter, Pantoea, Brachybacterium, Corynebacterium, Kocuria, Microbacterium, and Pseudoclavibacter, were also detected in different areas throughout the farm including test surfaces, milking parlors, hay, air and dust. The microorganisms, which are present in milk, but were not detected in the farm environment, included Lactococcus, Lactobacillus and Enterococcus as well as Leucobacter, Deinococcus, and Paracoccus. Similarly, a large number of other taxa were detected in the farm environment, but not in milk [35]. This may be due to strict hygiene standards maintained at the farms. Besides raw milk, processed milk products are also home to a complex microbial ecosystem. These aerobic microorganisms have a strong impact on the appearance, odor, flavor, and texture development of the respective cheese products or certain proteolytic LAB, may cause flavor defects (e.g., bitterness and putrid flavors) in milks and cheeses. The presence of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus in raw milks and cheeses constitutes a health risk [39–44].

4.1 Sample collection While collecting samples from animals, the first streams of milk from each quarter is discarded for mammary gland stimulation, and subsequently the teats are dipped in iodine tincture. Then teats are cleaned and disinfected using 70% ethanol, the first three streams are discarded and the milk samples are collected into sterile plastic tubes without preservatives. Approximately 50 mL of milk is collected in a single sterile 50-mL centrifuge tube from each animal. Milk samples from cows with mastitis are collected from the mastitis quarter and milk samples from healthy cows are collected at random from one of the cow’s hind quarters. Samples are kept on ice until transported to the laboratory. A small aliquot approx. 2-mL is separated for culture analysis and the remaining 48-mL sample is stored at 20°C for further processing. While sampling from milk supplies, liquid milk in cans and bulk tanks should be thoroughly mixed to disperse the milk fat. Representative samples of packed products must be taken for any investigation on quality. Presterilized plungers and dippers should be used in sampling milk from milk cans. On the spot sterilization may be employed using 70% alcohol swab and flaming or scaling in hot steam or boiling water for 1 min. In case of cheese, curd, and other dairy products, representative samples should be collected from the distribution centers in sterile conditions. Samples should be collected in duplicates. Samples should be sealed and properly labeled indicating the identification of product, the nature of the product, name, and signature of the authorized person responsible for taking the samples. After collection, samples should be immediately transferred to the laboratory, usually within 24 h.

4.2 Preservation of samples Milk and milk products can be transported in refrigerated conditions (e.g., ice packs) to avoid growth of contaminants. Raw milk, sterilized milk from production units, semisolid, or solid milk products and cheese should be transported and stored at 40°C. Butter and butterfat should also be transported and stored at 40°C in the dark. Sterilized milk in unopened containers, dried milk, and dried milk products can be stored at room temperature. Milk-based ice creams should be transported and stored at 180°C.

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5 Extremophiles Biocatalysts, an extremophile (from Latin extremus meaning “extreme” and Greek philia meaning “love”), is an organism that thrives in physically or geochemically extreme conditions that are detrimental to the majority of life on Earth. The term extremophile was first introduced by McElroy in 1974 [45]. Most extremophiles belong to the domains of archaea, bacteria, and eukarya [46]. With the notion that extremophiles are capable of surviving in extreme environments, it is assumed that their enzymes are adapted to function optimally under such conditions. These enzymes show unique features as extreme thermal stability, resistance against chemical denaturants such as detergents, chaotropic agents, organic solvents, and extremes of pH [47]. It has emerged as a prospective area for enzymologists to exploit these microbes for various industries [48].

5.1 Thermophiles Thermophilic microorganisms (optimum growth temperature of 500°C or above) have attracted great attention among extremophiles because they are sources of thermostable enzymes. They can be generally classified into moderate thermophiles (growth optimum; 50–600°C), extreme thermophiles (growth optimum; 60–800°C) and hyperthermophiles (growth optimum; 80–1100°C). Thermophiles have been isolated from the different ecological zones (e.g., hot springs, deep sea) of the earth. The organisms with the highest growth temperatures (103–1100°C) are members of the genera Pyrobaculum, Pyrodictium, Pyrococcus, and Melanopyrus belonging to archaea, among fungi, the Ascomycota and Zygomycota family have high growth temperature while in case of bacteria, Thermotoga maritima and Aquifex pyrophilus exhibit the highest growth temperatures of 90°C and 950°C, respectively [49,50]. These properties imply extremely important implications because enzymes that have been isolated from such microorganisms show unique features are extremely thermostable and usually resistant against some chemical denaturants [51]. For thermophiles, the best sites for sample collection are hot springs, geothermal sites, oil reservoirs and deep sea hydrothermal vents [52,53]. There are two types of samples collected while isolating thermophiles, i.e., soil and water samples. Soil samples may be the desert soil, sediments of hot springs, or oil reservoirs [54]. Soil samples are processed as discussed before while dealing with agronomically important microbes differing in incubation time as well as temperature. Surface water samples are collected in sterile containers. The microorganisms are isolated using standard plate count technique or membrane filtration technique. In standard plate count technique, the water sample is diluted serially, poured in media, and incubated at required high temperature. After incubation the number and types of colonies reveal the microorganisms in the sample. In membrane filtration technique, the sample is filtered through a 0.45 filter and then the filter is carefully placed on the selective media and incubated.

5.2 Psychrophiles Psychrophilic (growth temperature of 150°C or lower) or psychrotolerant (able to grow at temperature close to the freezing point of water but fastest growth rate at above 200°C) microorganisms are found inhabiting the low temperature environments of the Earth, including polar regions, glaciers, ocean deeps, shallow subterranean regions, upper atmosphere, refrigerated appliances and on and in plants and animals inhabiting cold regions [55]. These microorganisms mainly belong to the family of bacteria (e.g., Pseudoalteromonas, Vibrio, Pseudomonas, Arthrobacter, and Bacillus) [56,57], archaea (e.g., Methanogenium and Halorubrum), fungi (such as Penicillium and Cladosporium) [58] and yeast (such as Candida and Cryptococcus) [59]. The soil, ice, and water samples are usually collected from polar regions, glaciers and oceans. The soil samples are processed as previously discussed in agricultural microorganisms keeping the temperature 0–100°C. Water samples are filtered through 0.22 or 0.45 membrane filters and the membrane is incubated in the culture medium at low temperature (0–100°C).

5.3 Alkaliphiles/acidophiles Alkaliphiles are the class of extremophiles that live in alkaline environments at a pH of 8 or more such in soda lakes and carbonate-rich soils. Acidophiles on the other hand, tend toward acidic conditions with a pH optimum for growth at, or below, pH 3. One of the most striking properties of such organisms is their use of proton pumps to maintain a neutral pH internally. Thus, intracellular enzymes from these microorganisms do not need to be adapted to extreme growth conditions, while their extracellular enzymes need to function at low or high pH environments depending upon their source. For pH adaptation, alkaliphiles and acidophiles utilize several strategies. Active mechanisms to achieve this may involve

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secondary proton uptake by membrane-associated antiporters. Passive mechanisms include negatively charged cell-wall polymers in alkaliphiles and unusual bioenergetics, unusual permeability properties, positive surface charges, high internal buffer capacity, overexpression of H + export enzymes, and unique transporters in acidophiles [60]. Alkaliphilic microorganisms coexist with neutrophilic microorganisms, as well as occupying specific extreme environments in nature. To isolate alkaliphiles, alkaline media must be used. Sodium carbonate is generally used to adjust the pH to around 10, because alkaliphiles usually require at least some sodium ions. Alkaliphilic bacteria have been isolated from deep sea sediments [61], hydrothermal areas [62], soda lakes [63,64]. Most of the truly alkalophilic microorganisms have either been isolated from specific, enriched environments such as indigo dye balls, potato processing-plant effluents, or alkaline lakes or have been isolated upon suitable enrichment culturing of soil [65,66]. Soil or water samples for isolation of acidophilic bacteria may be collected from fruit orchards, yogurt, acidic mine waters, and acidic geothermal springs [67–69].

5.4 Piezophiles Microorganisms that prefer high-pressure conditions are termed piezophiles (barophiles). Oceans or deep-sea are home to these microorganisms and they are mainly distributed among the genera Shewanella, Methanococcus, Pyrococcus, and Moritella [70,71]. It is believed that enzymes isolated from piezophiles are stable at high pressure and do not need specific pressure-related adaptations. Piezophilic microorganisms and their enzymes have considerable potential for use in biotechnology, in particular for food industries, where high pressure is applied for processing and the sterilization of food materials. Sampling: Water, sediment, and organisms are important samples from which deep-sea microorganisms can be isolated. Multibottle rosette sampler, sterile bag water sampler and deep sea camera equipped sampler have been used worldwide. Deep-sea sediment samples obtained using the submersible should be mixed with culture medium (e.g., marine broth medium) and then the cultivation could be initiated in the high-pressure vessel under the environmental pressure and temperature conditions. After several days of cultivation, the mixed cultures could be transferred into the fresh media to continue to cultivate under different conditions, if necessary [72]. The microbes can be cultivated by pressure bag method or deep bath system, which are specially designed for isolating and purification of obligate piezophiles.

5.5 Radiophiles Microorganisms that are highly resistant to high levels of ionizing and ultraviolet radiation are called radiophiles. These radiation-resistant microorganisms show high potential in the treatment of radioactive environmental wastes. Numerous radiation-resistant bacteria have been isolated by incubating the culture in the presence of high gamma radiation. Radiophiles are receiving a lot of attention recently, because of their ability to survive under conditions of starvation, oxidative stress and high amounts of DNA damage [73]. Deinococcus radiodurans, the most radiation resistant organism known and the only one for which a system of genetic transformation and manipulation has been developed, is currently being engineered for remediation of radioactive wastes [74]. Samples for isolating radiophiles include soil and water from radioactive sites, hot springs, Antarctic dry valleys [75,76]. But the cultivation of such microorganisms requires continuous exposure to radiation for up to 6000 rad/h and can survive very low temperatures [77–79].

5.6 Xerophiles The organisms, which have the ability to grow in extremely dry conditions or in the presence of very low water activity (aw0.85), are considered to be xerophiles. However, only some specialized genera among bacteria, yeasts, fungi, lichens, algae are able to survive under such an environment. Xerophiles are considered to be responsible for spoiling dry foods and stored grains, spices, nuts and oilseeds. Xerophilic fungi range from marginal (which grow over normal media) like Aspergillus and Penicillium to extreme Xeromyces bisporous, Chrysosporium species, Eremascus species, etc. [80]. There are two kinds of sites from which xerophiles can be isolated. One is preserved and dehydrated food and the other is hypersaline sites [81]. Xerophilic organisms have been isolated from soil samples collected from stored and desiccated food, black tea [82].

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5.7 Metallophiles Microorganisms that can grow in the presence of high metal concentrations (otherwise essential as trace elements) are called metallophiles. Since pollution by heavy metals (Cu, Cr, Zn, Cd, Co, Pb, Ag, Hg) poses a threat to public health, fishery, and wild-life, there has been an increasing interest in metallophiles for removal of the toxic heavy metals from soils, sediments, and wastewaters [83]. Metallophiles also show high potential in bio-mining of expensive metals from effluents of industrial processes [84]. Heavy metals generally exert an inhibitory action on microorganisms by blocking essential functional groups, displacing essential metal ions or modifying the active conformation of biological molecules [85]. However, certain microorganisms are able to undo these effects and show resistance either by accumulating the metals in the form of particular protein-metal association, or heavy metal efflux systems [86]. Metal rich biotopes are the reservoirs for metallophiles. These include metal-rich geographical belts [87], industrial dumping sites [88,89], geothermal environments [90], and volcanic areas [91].

5.8 Halophiles Halophiles are extremophilic microorganisms that can grow optimally in saline environments (media containing 0.5–5.2 M NaCl). In this system, nonhalophiles are those that grow best in media containing 2000 bases. Amplified fragments separated by agarose gel electrophoresis and polymorphisms are detected after ethidium bromide staining. Interpreting of generated banding pattern done by scoring—present (1) or absent (0) polymorphic bands in individual lanes. The scoring can be done based on the banding profiles and the criteria for selecting scoring bands: (1) reproducibility—need to repeat experiment; (2) thickness; (3) size; and (4) expected segregation observed in a mapping population [67]. The main advantage of RAPDs is that they are quick and easy to assay, require low quantities of template DNA to perform the reaction and random primers are commercially available as no sequence data for primer construction are needed. Jarocki et al. [68] evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR, and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level.

TABLE 1 Different techniques for the identification of microbial strains. Microbial identification techniques Culturable

Nonculturable

Traditional media

Biochemical

Molecular

Microscopic (light field, bright, dark fluorescence, phase contrast, SEM, TEM, ATM, CLSM, inverted microscopy)

Traditional

PCR RT PCR RFLP RAPD AFLP PFGE MALDI-TOF Ribotyping

Mass spectroscopy Spectrometry

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TABLE 2 Enlist the varied software and commercial kits required to perform molecular techniques for strain identification and differentiation. Techniques

Software utilized for analysis

RAPD

For primer designing: Metagenomes (RPD-M), Perl script (tool), PRIMEX Result analysis Gel compare Software—comparison of the similarity between isolates banding patterns and construction of a dendagram, GelQuest, PAST software, NTSYS and DARWIN softwares, BioNumerics Software

RFLP

Software for restriction enzyme designing: Webcutter and NEBCutter; WatCut; Restriction site analysis; Sequence extractor; Restriction analyzer; SNP cutter Result analysis PyElph;GelJ: http://gelj.sourceforge.net/; GeneMapper Software; GeneQuest

PFGE

Restriction enzyme software http://insilico.ehu.es/; NEBcutter; WebDSV Result analysis software BioNumerics software; Fingerprinting-II software Matlab software; Phoretix gel analysis software

AFLP

GeneMapper software; GenAlEx; Popgene and TFPGA; NTSYSpc; NTSYS

Rio-Printer

Qualicon (DuPont)

MALDI-TOF

BioTyper (Bruker Daltonics, Billerica, MA); MASCOT; MATLAB; VITEKR _ MS(bioMerieuxInc Cromwell), (Matlab), Process, MALDIquant, or SpecAlign

Multilocus sequence typing (MLST)

BIGSdb; mlstdbNET; agdbNet

TABLE 3 General flow diagram of varied molecular techniques. RAPD

RFLP

AFLP

PFGE

Isolation of Isolation of Isolation of Bacterial bacterial DNA bacterial DNA bacterial DNA culture

RioPrinter Bacterial colony

MALDI TOF

MLST

Intact cells, Culture MALDI matrix bacterial (e.g., ferulic cell Addition of Extraction of acid or Addition of Digestion with Digestion of agarose to form genomic DNA sinapinic acid) selected restriction DNA plug and restriction Extract nucleotide enzymes enzyme digestion DNA Cell lysis and Adaptor extraction Cell extracts, Gel PCR reaction Ligation Southern blotting bacterial cells Sanger electrophoresis are lysed via sequenci Whole genome physical (e.g., ng Electrophoresis Amplification extraction Hybridization sonication or Denaturation with PCR bead-beating Alignme Digestion with Autoradiography nt of Interpretation Southern Electrophoresis RE Supernatant reads of results hybridization added into matrix Interpretation Electrophoresis Interpretation of results Interpretation of results of results Analysed with MALDI

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4.2 Restriction fragment length polymorphism RFLP method in molecular biology was evolved for detecting variation at the DNA sequence level of various biological samples. The principle of this method is based upon the comparison of restriction enzyme cleavage profiles following the existence of a polymorphism in a DNA sequence related to other sequences [69]. The similarity of pattern generated can be used to differentiate species (strains) from one another. The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and digest DNA wherever a specific short sequence occurs, in a process known as restriction digestion. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis or by southern blotting of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences. In bacteriology, if two strains differ in the distance between cleavage sites for a particular RE, the length of the restriction fragments is different between the strains [70]. The similarity of the generated patterns of restriction fragments can be used to differentiate strains and to analyze the genetic relatedness [69]. RFLP patterns are mainly determined through the specific combination of REs and nucleic acid probes. RFLP analysis was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurized milk and fresh cheese, digested with three restriction endonucleases (DdeI, HaeIII, HindI) to study variation in strains. Todd [66] studied identification and differentiation of closely related species of Bifidobacterium genus, DNA was digested with single endonuclease (Hae III) resulted in efficient discrimination of the tested species and subspecies allowing the construction of a dichotomous key.

4.3 Pulse field gel electrophoresis PFGE is an electrophoresis technique used to separate large DNA molecules (10 kb–10 Mb), as they are not separated by normal conventional electrophoresis. Higher size DNA molecules generally move together in a gel forming a single band that is difficult to realize the separation [71]. In, PFGE electric field is applied in varied directions leading to different pattern band formation within the gel. In case of bacterial strain identification, bacterial culture was added into suspension buffer, followed by addition of molten agar to make a plug, than lysis of cell and its digested by restriction cells forming 16S ribosomal DNA (rDNA) and the banding patterns of PFGE in a group of strains reflect DNA polymorphism at the RE recognition sites. Restriction enzymes’ analysis during PFGE steps is very important because the length of DNA fragments derived from the analysis will predict a pattern to distinguish between two bacterial strains [72]. When the DNA fragments are generated by enzymes—that usually are eight base pairs or a little more—if they are much larger than this size, a special form of electrophoresis will be needed to separate them. Important support of this approach is the ability of DNA to travel throughout the gel and create differences based on travel length. Electric current forces DNA which is negatively charged, to move toward the positive pole within the gel. Smaller pieces of DNA in comparison with larger pieces have more free movement [71]. Therefore, direct electric current will move ahead of larger parts. Plugs should be of appropriate size, plugs that are too small can easily be damaged and resulted in band distortion, whereas a larger plug resulted in thick, distinct band that are difficult to analyze, whereas variation in plug size among samples on the same gel may result in different fluorescence intensity that are even difficult to analyze. The BioNumerics software offers an integrated platform for the analysis of PFGE fingerprints. BioNumerics uses industry leading database engines that allow you to store all your epidemiological info and gel images in one database. Convenient wizards help you to define new fingerprint types, choose the optimal settings for normalization, resolution, background subtraction, smoothing, and band finding. The entire gel image preprocessing is contained in a powerful tab-based window, allowing easy access to re-edit the processing at any stage without losing any editing in another step. PFGE has proved to be a useful tool for the identification of a wide LAB strains variety and especially for species belonging to the genus Lactobacillus [73]. Several restriction enzymes have been used for obtaining profiles of Oenococcus oeni strains: NotI [74,75], ApaI and SfiI [76–79]. Similarly, Zou et al. [80] utilizes the bioinformatics approaches for PFGE database to enhance the data mining of PFGE fingerprints resulted in fast and accurate prediction to elucidate Salmonella serotype implicated in food-borne diseases.

4.4 Amplified fragment length polymorphism AFLP is a versatile technique for genome-wide screening of genetic diversity and can be applied to almost any organism. The technique relies on detecting genetic polymorphisms through differential endonuclease restriction digestion of genomic DNA. The rapidity and large amount of data generated by this approach, as well as robustness and repeatability of AFLP analysis, a commonly used tool in population genetic and ecological studies. Typically include five main steps: (a) restriction of genomic DNA and ligation of adaptors (to restricted fragments); (b) preselective PCR amplification of a

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PART I Isolation, screening and culture maintenance

subset of the restricted fragments; (c) selective PCR amplification, reducing further fragment number; (d) electrophoretic separation of amplified DNA fragments; (e) scoring and interpretation of the data [81]. The power of AFLP analysis derives from its ability to quickly generate large numbers of marker fragments for any organism, without prior knowledge of genomic sequence. Rare-cutting enzymes employed in AFLP include EcoRI, AseI, HindIII, ApaI, and PstI. MseI and TaqI are the commonly used frequent-cutters. Three types of restriction fragments are generated following digestion: (i) fragments cut by the rare-cutting enzyme on both ends; (ii) fragments cut with the frequent cutting enzyme on both ends; and (iii) fragments that have been cut by both the rare-cutter and frequent-cutter. Using EcoRI and MseI restriction enzymes as examples, EcoRI, MseI-MseI, and EcoRI-MseI fragments would be produced during restriction enzyme digestion. Genomic DNA of high purity is required for AFLP to ensure complete digestion by the restriction endonucleases. Incomplete restriction of DNA generates partial fragments, predominantly of high molecular weight. Amplification of fragments that are not fully digested generates an altered banding pattern, and may be misinterpreted as false polymorphisms [81]. The strengths of AFLPs lie in their high genomic abundance, considerable reproducibility, the generation of many informative bands per reaction, their wide range of applications, and the fact that no sequence data for primer construction are required. AFLPs may not be totally randomly distributed around the genome as clustering in certain genomic regions, such as centromere, has been reported for some crops. AFLPs can be analyzed on automatic sequencers, but software problems concerning the scoring of AFLPs are encountered on some systems [82]. The taxonomical and phylogenetic relationship among multiple strains of E. chrysanthemi and E. carotovora subspecies studied using AFLP fingerprinting, for various purposes, i.e., to either identify number of unknown isolates, to discriminate between closely related strains and to provide species- and subspecies-specific banding profiles [83].

4.5 RioPrinter This is an automated Southern blot device that uses a labeled ssDNA probe from the 16sRNA codon. The RioPrinter uses a restriction enzyme, and strains can be identified and/or characterized by analyzing the ribosomal DNA banding pattern. Every time a sample is run, the RioPrinter system produces an exact genetic snapshot of the microorganism that is linked to historical data. This genetic snapshot is akin to a “fingerprint.” The DNA fingerprint is generated from regions of the rRNA genes (5S, 16S, 23S, and the spacer region including Glu-tRNA) that is unique to the microorganism at the strain level. Ribotyping is a method for bacterial identification and characterization that, unlike certain previously described molecular typing methods, employs rRNA-based phylogenetic analysis. Given that that rRNA genes (such as 16S rRNA) are highly conserved within a bacterial species, identifying 16S rRNA gene polymorphisms reflects the evolutionary lineage of the bacterial species, and can shed light on bacterial classification, taxonomy, epidemiological investigation, and population biology. Ribotyping typically involves a multistep process starting with restriction enzymes that target the genomic sequence of interest, followed by southern blot transfer and hybridization with probes, and analysis of ribotype RFLP bands. However, with advances in molecular tools and knowledge of genomic sequences, several modifications to this technique have been published. It is important to note that for the purpose of primer and probe design, ribotyping requires some prior knowledge of the genome sequence under study. In one study, PCR-ribotyping was employed to characterize 99 strains of Clostridium difficile isolated from patients with nosocomial diarrhea. Following DNA extraction and PCR amplification of select regions of the 16S rRNA and 23S rRNA genes, amplified products were fractionated by electrophoresis. The banding pattern revealed dierent PCR-ribotypes with high reproducibility and discriminatory power. In a modification of this method, PCR-ribotyping was directly employed on stool samples for detection and typing of C. difficile strains. Primer modifications targeting both, the 16S-23S rRNA intergenic spacer region and 16S and 23S genes itself, resulted in increased specificity for direct typing [84]. With these new primers, PCR-ribotype could be detected directly from stool samples in 86 out of 99 cases, with a high degree of concordance with PCR-ribotyping done from isolated colonies.

4.6 Multilocus sequence typing MLST is a method using DNA sequencing to uncover allelic variants in several conserved genes (usually seven genes), and is currently one of the most popular genotyping methods for characterizing bacterial strains. MLST examines multiple housekeeping genes whose sequences are constrained because of the essential function of the proteins they encode; the variation observed in these sequences is therefore neutral or nearly neutral. Typically, fragments of 450–500 bp of seven genes are sequenced and each different sequence for a given gene is attributed a number. Each strain is, therefore, assigned a seven-number allelic profile designated as sequence type (ST) [84]. MLST is suitable for long-term investigation of bacterial population structures particularly when subtyping bacterial species with a high rate of genetic recombination, such as

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N. meningitidis, S. pneumoniae [85], and Enterococcus faecalis [86]. MLST analysis indicates that recombinational replacements in many species contribute more to clonal diversification than do point mutations [87]. DNA sequences are easily stored in online databases, which allow convenient exchange of strain typing data both within and between laboratories. These databases also facilitate the global epidemiological survey of bacterial infections [84,87]. MLST has been applied to more than 23 bacterial species and is regarded as a reference genotyping method for many bacteria [72]. However, MLST also has drawbacks. First, alleles are assigned to a numbering system that is not representative of the actual gene sequence, which makes the phylogenetic analysis of tested strains poorly credible [72]. Second, the use of highly conserved housekeeping genes in MLST often fails to detect the variability of closely related strains. Finally, sequencing of seven genes is costly and time consuming.

4.7 MALDI-TOF Mass spectrometry is an analytical technique in which chemical compounds are ionized into charged molecules and ratio of their mass to charge (m/z) is measured. MALDI-TOF has been proposed as a promising alternative for the dereplication of recurrent bacterial isolates [88] and has been used as a cost- and time-effective alternative to 16S rRNA gene sequencing [89,90]. MALDI-TOF MS is the latest next generation tool being used for the rapid identification and classification of microorganisms. It is based on the ionization of the microbial cells with short laser pulses and then accelerating the particles in a vacuum system using an electric field. After the ionization, a molecular fingerprint in the form of a spectra profile is obtained, which is specific for each microorganism. This spectrum is then compared to an existing database, resulting in its identification by an automated program. Preparation of samples for MALDI-TOF MS involves crystallization with a large molar excess of matrix (usually a UV-absorbing organic acid) on target plates. For microbiological applications mainly TOF mass analyzers are used. MALDI TOF MS-enabled identification nearly always requires cultivation of the microorganism of interest cultured bacterial cells are prepared for MALDI analysis [91]. Either intact cells or cell extracts are prepared for analysis. With intact cells, the MALDI matrix (e.g., ferulic acid or sinapinic acid) is mixed directly with intact cells. With cell extracts, bacterial cells are lysed via physical (e.g., sonication or bead-beating) or chemical (e.g., via exposure to TFA and formic acid/organic solvents) means to release the contents of the cells into the supernatant. The supernatant is added to the matrix and analyzed with MALDI.

5 High-throughput screening techniques Industrial microbiology and biotechnology use different microorganisms and enzymes to produce numerous important and valuable products such as antibiotics, enzymes, fuels, chemicals, vaccines, food products, dyes, and healthcare products. The microorganisms used in industrial microbiology and biotechnology are commonly called cell factories. Naturally isolated microorganisms from any source can very rarely be used for large scale industrial production because of very less yield and weak tolerance power to harsh industrial conditions like high temperature, high pH and for some other reasons which are used at industrial scale. Various strategies have been developed and established in order to overcome these problems, including reconstructing cell factories through modifying the physiological and biological functions of the microorganism at the gene level, and also by providing optimum environments for strain growth and product accumulation in upstream and downstream processes [92,93]. To increase the accumulation of target products, random mutagenesis (including physical and chemical mutagenesis) [94], engineering approaches, adaptive laboratory evolution (ALE) [94], and genetic and metabolic engineering [95] have been developed. It is difficult to develop methods for rapid screening of microbial strains in a large library of mutants because the probability of a beneficial mutation can be very low (