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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.

High Value Fermentation Products, Volume 1: Human Health

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

Ramon Larios-Cruz, Liliana Londono-Hernandez, Ricardo Gomez-Garcia, Ivanoe Garcia, Leonardo Sepulveda, Raul Rodriguez-Herrera and Cristobal 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 -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 -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 -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