Green technologies are no longer the “future” of science, but the present. With more and more mature industries, such as the process industries, making large strides seemingly every single day, and more consumers demanding products created from green technologies, it is essential for any business in any industry to be familiar with the latest processes and technologies. It is all part of a global effort to “go greener,” and this is nowhere more apparent than in fermentation technology.
This book describes relevant aspects of industrial-scale fermentation, an expanding area of activity, which already generates commercial values of over one third of a trillion US dollars annually, and which will most likely radically change the way we produce chemicals in the long-term future. From biofuels and bulk amino acids to monoclonal antibodies and stem cells, they all rely on mass suspension cultivation of cells in stirred bioreactors, which is the most widely used and versatile way to produce. Today, a wide array of cells can be cultivated in this way, and for most of them genetic engineering tools are also available. Examples of products, operating procedures, engineering and design aspects, economic drivers and cost, and regulatory issues are addressed. In addition, there will be a discussion of how we got to where we are today, and of the real world in industrial fermentation. This chapter is exclusively dedicated to large-scale production used in industrial settings.
Table of Contents
Foreword xvii
About the Editors xix
List of Contributors xxi
Preface xxv
Acknowledgement xxvii
1 Introduction, Scope and Significance of Fermentation Technology 1
Saurabh Saran, Alok Malaviya and Asha Chaubey
1.1 Introduction 1
1.2 Background of Fermentation Technology 2
1.3 Market of Fermentation Products 3
1.4 Types of Fermentation 4
1.4.1 Solid State Fermentation (SSF) 4
1.4.2 Submerged Fermentation (SmF) 7
1.4.3 Solid State (SSF) vs. Submerged (SmF) Fermentation 9
1.5 Classification of Fermentation 9
1.6 Design and Parts of Fermentors 10
1.7 Types of Fermentor 15
1.7.1 Stirred Tank Fermentor 15
1.7.2 Airlift Fermentor 16
1.7.3 Bubble Column Fermentor 17
1.7.4 Fluidized Bed Fermentor 18
1.7.5 Packed Bed Fermentor 19
1.7.6 Photo Bioreactor 19
1.8 Industrial Applications of Fermentation Technology 21
1.9 Scope and Global Market of Fermentation Technology 22
1.10 Conclusions 23
References 24
2 Extraction of Bioactive Molecules through Fermentation and Enzymatic Assisted Technologies 27
Ramón Larios-Cruz, Liliana Londoño-Hernández, Ricardo Gómez-García, Ivanoe García, Leonardo Sepulveda, Raúl Rodríguez-Herrera and Cristóbal N. Aguilar
2.1 Introduction 27
2.2 Definition of Bioactives Compounds 29
2.2.1 Polyphenols and Polypeptides 29
2.2.2 Importance and Applications of Bioactive Compounds 29
2.2.3 Bioactive Peptides 31
2.3 Traditional Processes for Obtaining Bioactive Compounds 33
2.3.1 Soxhlet Extraction 33
2.3.2 Liquid-Liquid and Solid-Liquid Extraction 34
2.3.3 Maceration Extraction 35
2.4 Fermentation and Enzymatic Technologies for Obtaining Bioactive Compounds 35
2.4.1 Soft Chemistry in Bioactive Compounds 35
2.4.2 Biotransformation of Bioactive Compounds 36
2.4.3 Enzymatic and Fermentation Technologies 39
2.5 Use of Agroindustrial Waste in the Fermentation Process 45
2.5.1 Cereal Wastes 46
2.5.2 Fruit and Plant Waste 46
2.6 General Parameters in the Optimization of Fermentation Processes 49
2.6.1 Response Surface Methodology 49
2.6.2 First-Order Model 49
2.6.3 Second-Order Model 49
2.7 Final Comments 52
Acknowledgements 52
References 52
3 Antibiotics Against Gram Positive Bacteria 61
Rahul Vikram Singh, Hitesh Sharma, Anshela Koul and Vikash Babu
3.1 Introduction 61
3.2 Target of Antibiotics Against Gram Positive Bacteria 64
3.2.1 Cell Wall Synthesis Inhibition 65
3.2.2 Protein Synthesis Inhibition 70
3.2.3 DNA Synthesis Inhibition 72
3.3 Antibiotics Production Processes 72
3.4 Conclusion 75
References 76
4 Antibiotic Against Gram-Negative Bacteria 79
Maryam Faiyaz, Shikha Gupta and Divya Gupta
4.1 Introduction 79
4.2 Gram-Negative Bacteria and Antibiotics 80
4.2.1 β-Lactam Drugs 81
4.2.2 Macrolide 82
4.2.3 Aminoglycosides 84
4.2.4 Fluoroquinolones 84
4.3 Production of Antibiotics 85
4.3.1 Strain Development 85
4.3.2 Media Formulation and Optimization 88
4.3.3 Fermentation 90
4.3.4 Downstream Processing and Purification 92
4.3.5 Quality Control 95
4.4 Conclusion 95
References 96
5 Role of Antifungal Drugs in Combating Invasive Fungal Diseases 103
Kakoli Dutt
5.1 Introduction 103
5.2 Antifungal Agents 105
5.2.1 Azoles 114
5.2.2 Polyenes 115
5.2.3 Allylamine/Thiocarbonates 116
5.2.4 Other Antifungal Agents 117
5.3 Targets of Antifungal Agents 120
5.3.1 Cell Wall Biosynthesis Inhibitors 120
5.3.2 Sphingolipid Synthesis Inhibitors 123
5.3.3 Ergosterol Synthesis Inhibitors 125
5.3.4 Protein Synthesis Inhibitors 126
5.3.5 Novel Targets 128
5.4 Development of Resistance towards Antifungal Agents 130
5.4.1 Minimum Inhibitory Concentration 130
5.4.2 Antifungal-Drug-Resistance Mechanisms 131
5.5 Market and Drug Development 134
5.6 Conclusions 136
Acknowledgement 137
References 137
6 Current Update on Rapamycin Production and Its Potential Clinical Implications 145
Girijesh K. Patel, Ruchika Goyal1 and Syed M. Waheed
6.1 Introduction 145
6.2 Biosynthesis of Rapamycin 146
6.2.1 Microbial Strain 147
6.2.2 Optimization of Carbon, Nitrogen Sources and Salts 147
6.2.3 Strain Manipulation to Improve Rapamycin Production 148
6.3 Organic Synthesis of Rapamycin 152
6.4 Extraction and Quantification of Rapamycin 152
6.5 Physiological Factors Affecting Rapamycin Biosynthesis 153
6.5.1 Effect of Media Components 153
6.5.2 Effect of pH on Rapamycin Production 153
6.5.3 Effect of Physical Gravity 154
6.5.4 Effect of Morphological Changes 154
6.5.5 Effect of Dissolved Oxygen (DO) and Carbon Dioxide (DCO2) 154
6.6 Production of Rapamycin Analogs 154
6.7 Mechanism of Action of Rapamycin 155
6.8 Use of Rapamycin in Medicine 157
6.8.1 Anti-Fungal Agent 157
6.8.2 Immunosuppression 158
6.8.3 Anti-Cancer Agent 158
6.8.4 Anti-Aging Agent 158
6.8.5 Role in HIV Treatment 158
6.8.6 Rheumatoid Arthritis 159
6.9 Side Effects of Long-term Use of Rapamycin 159
6.10 Conclusions 159
Acknowledgements 160
References 160
7 Advances in Production of Therapeutic Monoclonal Antibodies 165
Richi V Mahajan, Subhash Chand, Mahendra Pal Singh, Apurwa Kestwal and Surinder Singh
7.1 Introduction 165
7.2 Discovery and Clinical Development 166
7.3 Structure and Classification 167
7.4 Nomenclature of Monoclonal Antibodies 168
7.5 Production of Monoclonal Antibodies 170
7.5.1 Hybridoma Technology 170
7.5.2 Epstein-Barr Virus Technology 172
7.5.3 Phage Display Technology 172
7.5.4 Cell Line Based Production Techniques 173
7.5.5 Chemical Modifications of Monoclonal Antibodies 183
7.5.6 Advances in Antibody Technology 183
7.6 Conclusions 185
References 186
8 Antimicrobial Peptides from Bacterial Origin: Potential Alternative to Conventional Antibiotics 193
Lipsy Chopra, Gurdeep Singh, Ramita Taggar, Akanksha Dwivedi, Jitender Nandal, Pradeep Kumar and Debendra K. Sahoo
8.1 Introduction 193
8.2 Classification of Bacteriocins 194
8.2.1 Bacteriocins from Gram-Negative Bacteria 194
8.2.2 Bacteriocins from Gram-Positive Bacteria 194
8.3 Mode of Action 196
8.3.1 Pore-Forming Bacteriocins 196
8.3.2 Non-Pore-Forming Bacteriocins: Intracellular Targets 198
8.4 Applications 198
8.4.1 Food Bio Preservative 198
8.4.2 Food Packaging (In Packaging Films) 198
8.4.3 Hurdle Technology to Enhance Food Safety 199
8.4.4 Therapeutic Potential 200
8.4.5 Effect of Bacteriocins on Biofilms 200
8.5 Conclusions 202
Acknowledgments 202
Abbreviations 202
References 202
9 Non-Ribosomal Peptide Synthetases: Nature’s Indispensable Drug Factories 205
Richa Sharma, Ravi S. Manhas and Asha Chaubey
9.1 Introduction 205
9.1.1 Non-Ribosomal Peptides as Natural Products 205
9.1.2 Non-Ribosomal Peptides as Drugs 206
9.2 NRPS Machinery 208
9.3 Catalytic Domains of NRPSs 208
9.3.1 Adenylation (A) Domains 208
9.3.2 Thiolation (T) or PCP Domains 209
9.3.3 Condensation (C) Domains 209
9.3.4 Thioesterase (Te) Domains 209
9.4 Types of NRPS 210
9.4.1 Type A (Linear NRPS) 210
9.4.2 Type B (Iterative NRPS) 210
9.4.3 Type C (Non-linear NRPS) 210
9.5 Working of NRPSs 210
9.5.1 Priming Thiolation Domain of NRPS 211
9.5.2 Substrate Recognition and Activation 211
9.5.3 Peptide Bond Formation between NRP Monomers 211
9.5.4 Chain Termination of NRP Synthesis 212
9.5.5 NRP Tailoring 212
9.6 Sources of NRPs 213
9.7 Production of Non-Ribosomal Peptides 216
9.8 Future Scope 218
Acknowledgements 219
References 219
10 Enzymes as Therapeutic Agents in Human Disease Management 225
Babbal, Adivitiya, Shilpa Mohanty and Yogender Pal Khasa
10.1 Introduction 225
10.2 Pancreatic Enzymes 230
10.2.1 Trypsin (EC 3.4.21.4) 230
10.2.2 Pancreatic Lipase (EC 3.1.1.3) 231
10.2.3 Amylases (EC 3.2.1.1) 231
10.3 Oncolytic Enzymes 232
10.3.1 L-Asparaginase (EC 3.5.1.1) 232
10.3.2 L-Glutaminase (EC 3.5.1.2) 233
10.3.3 Arginine Deiminase (ADI) (EC 3.5.3.6) 233
10.4 Antidiabetic Enzymes 234
10.4.1 Glucokinase (EC2.7.1.1)
10.5 Liver Enzymes 235
10.5.1 Superoxide Dismutase (SOD) (EC 1.15.1.1) 235
10.5.2 Alkaline Phosphatase (ALP) (EC 3.1.3.1) 236
10.6 Kidney Disorder 237
10.6.1 Uricase (EC 1.7.3.3) 237
10.6.2 Urease (EC 3.5.1.5) 238
10.7 DNA- and RNA-Based Enzymes 238
10.7.1 Dornase 239
10.7.2 Adenosine Deaminase 240
10.7.3 Ribonuclease 240
10.8 Enzymes for the Treatment of Cardiovascular Disorders 241
10.8.1 The Hemostatic System 242
10.8.2 Enzymes of the Hemostatic System 244
10.9 Lysosomal Storage Disorders 251
10.9.1 α-Galactosidase A (EC 3.2.1.22) 251
10.9.2 Glucocerebrosidase (EC 3.2.1.45) 252
10.9.3 Acid Alpha-Glucosidase (GAA) (EC 3.2.1.20) 253
10.9.4 α-L-iduronidase (Laronidase) (EC 3.2.1.76) 253
10.10 Miscellaneous Enzymes 254
10.10.1 Phenylalanine Hydroxylase (EC 1.14.16.1) 254
10.10.2 Collagenase (EC 3.4.24.3) 255
10.10.3 Hyaluronidase 256
10.10.4 Bromelain 256
10.11 Conclusions 256
References 257
11 Erythritol: A Sugar Substitute 265
Kanti N. Mihooliya, Jitender Nandal, Himanshu Verma and Debendra K. Sahoo
11.1 Introduction 265
11.1.1 Background of Erythritol 265
11.1.2 History of Erythritol 268
11.1.3 Occurrence of Erythritol 268
11.1.4 General Characteristics 268
11.2 Chemical and Physical Properties of Erythritol 271
11.3 Estimation of Erythritol 271
11.3.1 Thin Layer Chromatography (TLC) 273
11.3.2 Colorimetric Assay for Detection of Polyols 273
11.3.3 High-Performance Liquid Chromatography (HPLC) 273
11.3.4 Capillary Electrophoresis (CE) 273
11.4 Production Methods for Erythritol 274
11.4.1 Chemical Methods for Erythritol Production 274
11.4.2 Fermentative Methods for Erythritol Production 274
11.5 Optimization of Erythritol Production 275
11.5.1 One Factor at a Time 276
11.5.2 Statistical Design Approaches 277
11.6 Toxicology of Erythritol 277
11.7 Applications of Erythritol 277
11.7.1 Confectioneries 278
11.7.2 Bakery 279
11.7.3 Pharmaceuticals 279
11.7.4 Cosmetics 279
11.7.5 Beverages 279
11.8 Precautions for Erythritol Usage 279
11.9 Global Market for Erythritol 280
11.10 Conclusions 280
References 281
12 Sugar and Sugar Alcohols: Xylitol 285
Bhumica Agarwal and Lalit Kumar Singh
12.1 Introduction 285
12.1.1 Lignocellulosic Biomass 286
12.1.2 Properties of Xylitol 287
12.1.3 Occurrence and Production of Xylitol 289
12.2 Biomass Conversion Process 289
12.2.1 Pretreatment Methodologies 289
12.2.2 Enzymatic Hydrolysis 292
12.2.3 Detoxification Techniques 293
12.3 Utilization of Xylose 296
12.3.1 Microorganisms Utilizing Xylose 296
12.3.2 Metabolism of Xylose 297
12.4 Process Variables 299
12.4.1 Temperature and pH 299
12.4.2 Substrate Concentration 300
12.4.3 Aeration 301
References 303
13 Trehalose: An Anonymity Turns Into Necessity 309
Manali Datta and Dignya Desai
13.1 Introduction 309
13.2 Trehalose Metabolism Pathways 310
13.3 Physicochemical Properties and its Biological Significance 311
13.4 Trehalose Production 312
13.4.1 Enzymatic Conversion to Trehalose 312
13.4.2 Microbe Mediated Fermentation 314
13.4.3 Purification and Detection of Trehalose in Fermentation Process 316
13.5 Application of Trehalose 317
13.5.1 Role of Trehalose in Food Industries 317
13.5.2 Role of Trehalose in Cosmetics and Pharmaceutics 318
13.6 Conclusions 319
References 320
14 Production of Yeast Derived Microsomal Human CYP450 Enzymes (Sacchrosomes) in High Yields, and Activities Superior to Commercially Available Microsomal Enzymes 323
Ibidapo Stephen Williams and Bhabatosh Chaudhuri
14.1 Introduction 323
14.1.1 Cytochrome P450 (CYP) Enzymes in Humans 323
14.1.2 Human Cytochrome P450 Enzymes and their Role in Drug Metabolism 324
14.1.3 Requirement of Activating Proteins to Form Functional Human CYP Enzymes 325
14.1.4 Use of Yeast Biased Codons for the Syntheses of Human Cytochrome P450 Genes 325
14.1.5 Expression of Human CYP Genes in Baker’s Yeast from an Episomal Plasmid 325
14.1.6 Expression of Human CYP Genes in Baker’s Yeast from Integrative Plasmids 327
14.1.7 The ADH2 Promoter for Production of Human CYP Enzymes in Baker’s Yeast 327
14.1.8 Growth of Yeast Cells Containing Integrated Copies of CYP Gene Expression Cassettes, Driven by the ADH2 Promoter, for Production of CYP Enzymes 328
14.2 Amounts of Microsomal CYP Enzyme Isolated from Yeast Strains Containing Chromosomally Integrated CYP Gene Expression Cassettes are far Higher than Strains Harbouring an Episomal Expression Plasmid Encoding a CYP Gene 328
14.2.1 Preparation of Microsomal CYP Enzymes 328
14.2.2 Measurement of the Amounts of Functional CYPs in Microsomes Isolated from Baker’s Yeast 329
14.2.3 Production of Functional Human CYP1A2 Microsomal Enzyme from Baker’s Yeast 330
14.2.4 Production of Functional Human CYP3A4 Microsomal Enzyme from Baker’s Yeast 330
14.2.5 Production of Functional Human CYP2D6 Microsomal Enzyme from Baker’s Yeast 331
14.2.6 Production of Functional Human CYP2C19 Microsomal Enzyme from Baker’s Yeast 332
14.2.7 Production of Functional Human CYP2C9 Microsomal Enzyme from Baker’s Yeast 333
14.2.8 Production of Functional Human CYP2E1 Microsomal Enzyme from Baker’s Yeast 333
14.2.9 Comments on the Production of Human CYP Enzymes from Baker’s Yeast 334
14.3 Comparison of CYP Enzyme Activity of Yeast-Derived Microsomes (Sacchrosomes) with Commercially Available Microsomes Isolated from Insect and Bacterial Cells 336
14.3.1 Fluorescence-based Assays for Determining CYP Enzyme Activities in Isolated Microsomes 336
14.3.2 Comparison of Enzyme Activity of CYP1A2 Sacchrosomes with Commercially Available CYP1A2 Microsomes Isolated from Insect and Bacterial Cells 336
14.3.3 Comparison of Enzyme Activity of CYP2C9 Sacchrosomes with Those of Commercially Available CYP2C9 Microsomes from Insect and Bacterial Cells 337
14.3.4 Comparison of Enzyme Activity of CYP2C19 Sacchrosomes with Those of Commercially Available CYP2C19 Microsomes from Insect and Bacterial Cells 337
14.3.5 Comparison of Enzyme Activity of CYP2D6 Sacchrosomes with Those of Commercially Available CYP2D6 Microsomes from Insect and Bacterial Cells 338
14.3.6 Comparison of Enzyme Activity of CYP3A4 Sacchrosomes with Those of Commercially Available CYP3A4 Microsomes from Insect and Bacterial Cells 338
14.3.7 Comparison of Enzyme Activity of CYP2E1 Sacchrosomes with One of the Commercial CYP2E1 Microsomes Available from Insect Cells 339
14.4 IC50 Values of Known CYP Inhibitors Using Sacchrosomes, Commercial Enzymes and HLMs 339
14.5 Stabilisation of Sacchrosomes through Freeze-drying 340
14.6 Conclusions 342
References 345
15 Artemisinin: A Potent Antimalarial Drug 347
Alok Malaviya, Karan Malhotra, Anil Agarwal and Katherine Saikia
15.1 Introduction 347
15.2 Biosynthesis of Artemisinin in Artemisia annua and Pathways Involved 348
15.3 Yield Enhancement Strategies in A. annua 351
15.4 Artemisinin Production Using Heterologous Hosts 352
15.4.1 Microbial Engineering 352
15.4.2 Plant Metabolic Engineering 353
15.5 Spread of Artemisinin Resistance 357
15.6 Challenges in Large-Scale Production 358
15.7 Future Prospects 360
References 360
16 Microbial Production of Flavonoids: Engineering Strategies for Improved Production 365
Aravind Madhavan, Raveendran Sindhu, KB Arun, Ashok Pandey, Parameswaran Binod and Edgard Gnansounou
16.1 Introduction 365
16.2 Flavonoids 366
16.3 Flavonoid Chemistry and Classes 366
16.4 Health Benefits of Flavonoids 367
16.5 Flavonoid Biosynthesis in Microorganism 368
16.6 Engineering of Flavonoid Biosynthesis Pathway 370
16.7 Metabolic Engineering Strategies 370
16.8 Applications of Synthetic Biology in Flavonoid Production 371
16.9 Post-modification of Flavonoids 374
16.10 Purification of Flavonoids 374
16.11 Conclusion 375
Acknowledgements 375
References 376
17 Astaxanthin: Current Advances in Metabolic Engineering of the Carotenoid 381
Manmeet Ahuja, Jayesh Varavadekar, Mansi Vora, Piyush Sethia, Harikrishna Reddy and Vidhya Rangaswamy
17.1 Introduction 381
17.1.1 Structure of Astaxanthin 382
17.1.2 Natural vs. Synthetic Astaxanthin 382
17.1.3 Uses and Market of Astaxanthin 383
17.2 Pathway of Astaxanthin 384
17.2.1 Bacteria 384
17.2.2 Algae 384
17.2.3 Yeast 385
17.2.4 Plants 386
17.3 Challenges/Current State of the Art in Fermentation/Commercial Production 386
17.4 Metabolic Engineering for Astaxanthin 388
17.4.1 Bacteria 388
17.4.2 Plants 390
17.4.3 Synechocystis 391
17.4.4 Algae 391
17.4.5 Yeast 392
17.5 Future Prospects 393
References 395
18 Exploitation of Fungal Endophytes as Bio-factories for Production of Functional Metabolites through Metabolic Engineering; Emphasizing on Taxol Production 401
Sanjog Garyali, Puja Tandon, M. Sudhakara Reddy and Yong Wang
18.1 Introduction 401
18.2 Taxol: History and Clinical Impact 403
18.3 Endophytes 403
18.3.1 Biodiversity of Endophytes 405
18.3.2 Endophyte vs. Host Plant: the Relationship 405
18.4 The Plausibility of Horizontal Gene Transfer (HGT) Hypothesis 407
18.5 Endophytes as Biological Factories of Functional Metabolites 409
18.6 Taxol Producing Endophytic Fungi 410
18.7 Molecular Basis of Taxol Production by Taxus Plants (Taxol Biosynthetic Pathway) 412
18.8 Metabolic Engineering for Synthesis of Taxol: Next Generation Tool 416
18.8.1 Plant Cell Culture 417
18.8.2 Microbial Metabolic Engineering for Synthesis of Taxol and Its Precursors 418
18.8.3 Metabolic Engineering in Heterologous Plant for Synthesis of Taxol and Its Precursors 420
18.9 Future Perspectives 421
Acknowledgements 423
References 423
Index 431
