Basic Biotechniques for Bioprocess and Bioentrepreneurship

Description

Basic Biotechniques for Bioprocess and Bioentrepreneurship deals with the entire field of industrial biotechnology, starting from the basic laboratory techniques to scale-up, process development, demonstration, and finally its commercialization. The book compiles currently scattered materials on this topic and updates this information based on practical experience and requirements. The book will be an ideal source for new entrepreneurs who wish to start their own commercial units. Front Cover Basic Biotechniques for Bioprocess and Bioentrepreneurship Copyright Contents Contributors Foreword Preface Part I: Isolation, screening and culture maintenance Chapter 1: Isolation of microorganisms 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 Chapter 2: Screening strategies 1. Introduction 2. Conventional strain screening techniques 2.1. Culture-dependent methods 2.2. Conventional screening of antimicrobials 2.2.1. Bioactivity based-screening 2.2.2. Gene-based screening 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 5.2.1. Spectroscopic equipment and measurements 5.2.2. Mass spectrometry 5.2.3. Single cell analysis by microfluidics technology 5.2.4. Electrochemical sensor-based screening 5.2.5. Biosensor-based screening 5.2.6. Statistical data analysis 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 Chapter 3: Identification, morphological, biochemical, and genetic characterization of microorganisms 1. Introduction 2. Isolation of microorganisms 2.1. Methods of isolation 2.1.1. Isolation in liquid medium by serial dilution technique also referred to as end tube method 2.1.2. Isolation on solid medium or isolation by plating media 2.1.3. Single-cell isolation using micromanipulation 2.1.4. Selective methods 3. Identification of microbes 3.1. Principles of taxonomy 3.2. Strategies used to identify microbes 3.2.1. Using phenotypic characteristics to identify microbes Microscopic morphology Procedure of gram staining 3.2.2. Culture characteristics 3.2.3. Streak method 3.2.4. Serology 3.2.5. Fatty acid analysis (FAME) 3.3. Morphology of bacteria 3.3.1. Colony morphology 3.3.2. Cellular morphology 3.4. Morphology of fungi 3.4.1. Unicellular fungi 3.4.2. Molds or filamentous fungi 3.4.3. Microscopic structures 3.4.4. Macroscopic structures 3.4.5. Dimorphic 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 4.7.1. Nitrate reduction broth 4.8. Amino acid decarboxylase test 4.9. Litmus milk test 4.10. Hydrogen sulfide test 4.10.1. Urease 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 5.2.1. Procedure 5.2.2. Agarose gel electrophoresis 5.2.3. Solution/Regents 5.2.4. Quantification of DNA 5.3. Microbial fingerprinting methods 5.3.1. Pulsed field gel electrophoresis (PFGE) 5.3.2. Restriction fragment length polymorphism (RFLP) 5.3.3. Ribotyping 5.3.4. PCR-RFLP 5.3.5. Random amplified polymorphic DNA (RAPD) 5.3.6. Direct amplification fingerprinting (DAF) 5.3.7. Repetitive sequence-based PCR (rep-PCR) 5.3.8. Multilocus sequence typing (MLST) 5.3.9. Random amplified polymorphism deoxyribonucleic acid (RAPD) 5.3.10. Plasmid profile analysis 5.3.11. Amplified fragment length polymorphism (AFLP) 5.3.12. Single-strand conformational polymorphism (PCR SSCP) 5.3.13. Direct amplification fingerprinting (DAF) 5.3.14. Computer-assisted analysis 6. Conclusion References Chapter 4: Microbial activity and productivity enhancement strategies 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 References Chapter 5: Culture maintenance, preservation, and strain improvement 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 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 5.2.1. Mutation 5.2.2. Genetic recombination 5.2.3. Genetic engineering 5.2.4. Genome editing 6. Conclusion References Part II: Laboratory techniques & instrumentation Chapter 6: Biomolecules: Types, homogenization, bead beater, sonication 1. Introduction 2. Classification of cell disruption processes 2.1. Physical disruption methods 2.1.1. Decompression 2.1.2. Osmotic shock 2.1.3. Thermal lysis 2.2. Chemical disruption 2.2.1. Antibiotics 2.2.2. Chelating agents 2.2.3. Chaotropic agents 2.2.4. Detergents 2.3. Large-scale cell disruption: The bead mill 2.3.1. Important operational parameters of bead-milling Agitator speed Effect of feed rate on microorganism disintegration Size of the beads Bead loading Specific weight of the grinding elements Concentration of cell suspension Temperature 3. Conclusion References Chapter 7: Centrifugation: Basic principle, types 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 3.2. Material used in rotor construction 3.3. Various types of centrifugation technique 3.4. Types of centrifuges 3.4.1. Ultracentrifuges 3.5. Separation methods in different types of centrifugation 3.5.1. Differential centrifugation 3.5.2. Density gradient centrifugation 3.5.3. Properties of a good gradient material 3.6. Applications of centrifugation techniques 3.7. Care of centrifugation equipment 3.8. Safety aspects while operating a centrifuge References Chapter 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 4.1.1. Principle 4.1.2. Applications of UV-vis spectroscopy Spectroscopy in environmental analysis UV-vis spectroscopy for water analysis and environmental applications Spectrophotometric analysis of bacterial water contaminants Spectrophotometers for chlorine and fluoride quantification UV-vis spectroscopy for geological studies linked to water contamination Other applications 4.2. Infrared spectroscopy 4.2.1. Source 4.2.2. Some important definitions 4.2.3. Instrumentation 4.2.4. Sample types and preparation 4.2.5. Various types of detectors used 4.2.6. Fourier transform IR (FTIR) spectrometers Advantages of FTIR 4.2.7. Applications of IR spectroscopy 4.3. Mass spectrometry 4.3.1. The mass spectrometer 4.3.2. The nature of mass spectra 4.3.3. The working principle of a mass spectrometer 4.3.4. Applications of mass spectrometry Analysis of biomolecules Analysis of glycans Analysis of lipids Analysis of proteins and peptides Analysis of oligonucleotides 4.4. Nuclear magnetic resonance (NMR) spectroscopy 4.4.1. NMR spectrum 4.4.2. NMR spectrometers 4.4.3. Spin-spin coupling 4.4.4. Applications of NMR 5. Conclusion References Chapter 9: Protein purification: Basic principles and techniques 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 Chapter 10: Chromatography: Basic principle, types, and applications 1. Basic principle 2. General terms used in chromatography 3. Types of chromatography 3.1. Liquid chromatography 3.1.1. Steps involved in liquid chromatography 3.2. Affinity chromatography 3.2.1. Application 3.2.2. Mechanism of affinity binding 3.3. Ion-exchange chromatography 3.3.1. Ion-exchange chromatography resin selection 3.3.2. Functional groups used on ion exchangers 3.3.3. Steps in involved in 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 Chapter 11: Electrophoresis: Basic principle, types, and applications 1. Introduction 2. Types of electrophoresis 2.1. Gel electrophoresis 2.1.1. Agarose gel electrophoresis 2.1.2. Polyacrylamide gel electrophoresis (PAGE) 2.1.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 2.1.4. Native PAGE 2.1.5. Pulse field gel electrophoresis 2.1.6. Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) 2.1.7. Isoelectric focusing (IEF) 2.1.8. Two-dimensional (2D) gel electrophoresis 2.2. Zone electrophoresis 2.3. Free flow electrophoresis 2.3.1. Micro-electrophoresis 2.3.2. Moving boundary electrophoresis 2.4. Capillary electrophoresis 3. Conclusion References Part III: Genomic and proteomic analysis Chapter 12: DNA, RNA isolation, primer designing, sequence submission, and phylogenetic analysis 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 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 Chapter 13: RDT and genetic engineering: Basic of RDT method, PCR, and application 1. Introduction 2. Recombinant DNA technology 3. Genetic engineering and RDT differences 4. Polymerase chain reaction 4.1. Denaturation 4.2. Primer annealing 4.3. Elongation 4.4. Applications of PCR 4.4.1. Research applications-PCR has been applied to many areas of research in molecular genetics 4.4.2. Medical and diagnostic applications 4.4.3. PCR applied to the fingerprinting 4.4.4. Acellular cloning 4.4.5. PCR use in antibiotic resistance 4.4.6. PCR use in dentistry 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 Chapter 14: Protein sequence analysis 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 Chapter 15: Computational strategies and tools for protein tertiary structure prediction 1. Introduction 2. Homology modeling 3. Fold recognition/threading 4. Ab initio protein structure prediction 5. Hybrid methods and current trend in protein structure prediction 6. Conclusion References Chapter 16: Docking strategies 1. Introduction 2. Scoring functions 3. Protein-ligand docking strategies 4. Protein-protein docking strategies 5. Protein-nucleic acid docking strategies 6. 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 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 and Rank 7. Future prospects 8. Conclusion References Chapter 17: A beginners guide to measuring binding affinity during biomolecular interactions 1. Introduction 2. An introduction to major methods of detecting protein-ligand complexes 2.1. Fluorescence assays 2.1.1. An elementary explanation of the fluorescence phenomenon 2.1.1.1. Fluorescence intensity 2.1.1.2. Fluorescence polarization 2.2. Differential scanning fluorimetry (DSF) 2.3. Isothermal titration calorimetry 2.4. Surface plasmon resonance 2.5. Nuclear magnetic resonance spectroscopy 2.5.1. Heteronuclear single quantum correlation: A protein-focused NMR method 2.5.2. Ligand-focused NMR methods 2.6. Frontal affinity chromatography 3. Conclusion References Part IV: Industrially important enzymes Chapter 18: Enzymes and their significance in the industrial bioprocesses 1. Introduction 2. Enzymes in industries: The sources 3. Production of industrial enzymes 3.1. Selection of strain 3.2. Strain improvement 3.2.1. Mutation 3.2.2. Recombinant DNA technology 3.2.3. Protein engineering 3.3. Processes for enzyme production in industry 3.3.1. Submerged fermentation 3.3.2. Solid-state fermentation 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 Chapter 19: Enzyme kinetics: Industrially important enzymes 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 Chapter 20: Industrial enzymes: Basic information, assay, and applications 1. Introduction 2. Different important industrial enzymes 3. Cellulase 3.1. Sources 3.2. Application 3.3. Assay of cellulase activity 3.3.1. Cellulase assay [15] Materials Procedure 3.3.2. Filter paper assay for saccharifying cellulase (FPU assay) [16] Materials Procedure 3.3.3. Carboxymethyl cellulose (CMC) assay for endoglucanase [17] Materials Procedure 3.3.4. Endoglucanase (HEC assay) [18] Materials Procedure 4. α-Amylase 4.1. Sources 4.2. Application 4.3. Assay of amylase activity 4.3.1. DNSA method [24] Materials Procedure 4.3.2. Preparation of standard curve of glucose 4.3.3. Dextrinizing activity by decrease in starch iodine color intensity Materials Procedure 4.3.4. Radial diffusion or cup plate method Materials Procedure 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 5.3.1. Materials 5.3.2. Procedure 5.3.3. GOx activity by enzymatic reduction of benzoquinone to hydroquinone by spectrophotometric methods of Ciucu and Pat ... Procedure 5.3.4. The GOx assay by titration methods Materials Procedure 6. Invertase 6.1. Sources 6.2. Applications 6.3. Assay of invertase activity 6.3.1. Saccharolytic methods Materials Procedure 6.3.2. Colorimetric methods Materials Procedure 7. Lipase 7.1. Sources 7.2. Applications 7.3. Assay of lipase activity 7.3.1. Olive oil hydrolysis method Materials Procedure 7.3.2. Lipase activity using para-nitro phenyl palmitate (pNPP) as the substrate Materials Procedure 8. Protease 8.1. Sources 8.2. Applications 8.3. Assay of protease activity 8.3.1. Proteolytic enzyme assay Materials Procedure 9. Conclusions and future prospects References Part V: Techniques in process development Chapter 21: Fundamentals of fermentation technology 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 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 4.4.1. Bacteria 4.4.2. Yeast 4.4.3. Molds 4.4.4. Enzymes 4.5. Nutritional requirements 4.6. Hydrogen ion concentration (pH) 4.7. Inhibitors 4.8. Types of reactors 4.8.1. Stirred tank reactors 4.8.2. Continuous stirred tank reactor 4.8.3. Airlift bioreactors 4.8.4. Fluidized bed reactors 4.8.5. Packed bed columns 4.8.6. Membrane bioreactors 4.8.7. Submerged fermentation 4.8.8. Solid-state fermentation 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 Chapter 22: Sterilization in bioprocesses 1. Introduction 2. Principles of sterilization 3. Overview of fermentation process 4. Sterilization in industrial fermentation 5. Different methods of sterilization 6. Which article should be sterilized? 7. Sterilization of culture media 7.1. Heat sterilization 7.1.1. Time 7.1.2. Temperature 7.1.3. Moisture 7.1.4. Direct heat contact 7.1.5. Air removal 7.2. Batch sterilization 7.2.1. Different methods for batch sterilization 7.2.2. Major merits of batch sterilization technique over continuous sterilization 7.2.3. Major demerits of batch sterilization method Degradation of culture medium Large energy and time consumption 7.3. Continuous sterilization 7.3.1. The design of continuous sterilization processes 7.3.2. Advantages of continuous sterilization method over batch sterilization 7.4. Other physical methods 7.4.1. Filter sterilization There are a couple of limitations of filtration method 7.4.2. Sterilization of air 8. Conclusion References Chapter 23: Scale-up: Lab to commercial scale 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 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 Chapter 24: Bioprocess: Control, management, and biosafety issues 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 2.2.1. The processing of a KB control system 2.3. Bioprocess monitoring using electrochemical techniques 2.3.1. Chronoamperometry 2.3.2. Hydrodynamic chronoamperometry 2.3.3. Cyclic voltammetry 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 3.2.1. Biosafety level 1 3.2.2. Biosafety level 2 3.2.3. Biosafety level 3 3.2.4. Biosafety level 4 3.3. Biosafety perspective of India 3.3.1. A check on transgenic research trials 3.3.2. Institutional framework 3.4. Outcome result and risk approach 4. Conclusion References Chapter 25: Demonstration and industrial scale-up 1. Introduction 2. A common skeleton for bioprocess development at scale 2.1. Fermentation 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 Chapter 26: Downstream processing of biotechnology products 1. Introduction 2. Precipitation 2.1. Solubility and stability of proteins 2.2. Methods of precipitation 2.2.1. Ammonium sulfate precipitation 2.2.2. Salting out 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 6.3. Affinity chromatography 6.3.1. Applications 6.4. Expanded bed chromatography 6.4.1. Principle 6.4.2. Advantages 6.4.3. Applications 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 Chapter 27: Waste management and environment 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 5.1.1. Storage, collection, and transportation of solid waste from bioprocessing industry 5.1.2. Segregation of hazardous and nonhazardous solid waste from bioprocessing industry 5.1.3. Techniques for managing non-hazardous solid waste from bioprocessing industry 5.1.4. Different nonhazardous waste valorization techniques Pyrolysis Gasification Anaerobic digestion Fermentation Dark fermentation Syngas fermentation 5.1.5. Techniques for managing hazardous solid waste from bioprocessing industry 5.2. Liquid effluent management 5.2.1. Coagulation treatment 5.2.2. Electrocoagulation treatment 5.2.3. Biological treatment 5.2.4. Adsorption 5.2.5. Microbial fuel cell 5.2.6. Anaerobic digestion 5.3. Gaseous waste management 5.3.1. Settling chamber 5.3.2. Fabric filters 5.3.3. Electrostatic precipitators 5.3.4. Absorption 5.3.5. Adsorption 5.3.6. Syngas fermentation 6. Future prospects 7. Conclusion References Part VI: Feasibility, marketing and biobusiness Chapter 28: Biobusiness opportunities 1. Introduction 2. Emerging biobusiness opportunities 2.1. Agriclinics and centers 2.2. Vegetable processing plant 2.3. Biodiesel production 2.4. Biopesticide manufacturing 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 Chapter 29: Feasibility analysis and business plan 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 3.1.1. Tangible or intangible costs and benefits 3.1.2. Direct or indirect costs and benefits 3.1.3. Fixed or variable 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 5. Business plan: Sample 6. Conclusion References Chapter 30: Patenting, IPR, and regulatory issues 1. Introduction 2. Types of intellectual property rights (IPRs) 2.1. Patents 2.1.1. Types of 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 References Chapter 31: Commercialization and technology transfers of bioprocess 1. Introduction 2. Scale-up 3. Low-cost bioprocess development 4. Quality control in bioprocess development 5. Role of RandD in technology development 6. Bioreactor design development and challenges 7. 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 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 Chapter 32: Bioproduct marketing strategies 1. Introduction 2. Development of marketing of bioproducts 3. Classification of markets 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 Back Cover