Basic Industrial Biotechnology (2012) PDF

Basic Industrial Biotechnology This page intentionally left blank Basic Industrial Biotechnology S.M. Reddy Professor, Department of Biotechnology St. Mar...

Basic Industrial Biotechnology This page intentionally left blank Basic Industrial Biotechnology S.M. Reddy Professor, Department of Biotechnology St. Martin’s Engineering College Secunderabad, (A.P.) S. Ram Reddy Professor, Department of Microbiology Kakatiya University Warangal, (A.P.) G. Narendra Babu Former Associate Professor, SRR Govt. College Karimnagar, (A.P.) Copyright © 2012, New Age International (P) Ltd., Publishers Published by New Age International (P) Ltd., Publishers All rights reserved. No part of this ebook may be reproduced in any form, by photostat, microfilm, xerography, or any other means, or incorporated into any information retrieval system, electronic or mechanical, without the written permission of the publisher. All inquiries should be emailed to [email protected]  ISBN (13) : 978-81-224-3489-7 PUBLISHING FOR ONE WORLD NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS 4835/24, Ansari Road, Daryaganj, New Delhi - 110002 Visit us at www.newagepublishers.com Preface Developments in the area of life sciences such as recombinant DNA technology and human genome project have changed our basic concepts which has profound influence on the quality of life. It is likely that such changes will be accelerated in the future and such advances probably rely on basic knowledge along with its transformation into products that can be produced eco friendly as well as in cheapest possible manner. The fundamentals of biotechnology remains strong, the production of goods and services that are needed without risk involvement that too at economic price. Biotechnology is not mere recombinant of DNA and cloning, but production of more prosaic materials like organic acids, amino acids, beverages, fermented foods, antibiotics, biosurfactants, polysaccharides and the like. The main aim of this discipline is to provide clear technology for 21st century which will sustain the growth and development all over the world to improve the quality of life. It is likely to influence the health care, foods supply and environment. In short no aspect of our life will remain unaffected. The main aim of this book is, to provide an overview of many of the fundamental aspects that underpin all biotechnology and provide examples of how these principles are put into operation starting from substrate through final product. Since the biotechnology is huge multidisciplinary activity, we restricted ourselves to provide an mainstream account of the current state of industrial bioprocesses which may provide the reader with insight, inspiration and instruction in skills and art of the subject. We also hope that it will provide some understanding of promise, limits of biotechnology to policy makers, regulators and corporate decisions. Precise care has been taken while writing the book in simple and lucid language, keeping in view the standard of graduate and postgraduate students of Indian universities in the discipline of basic biotechnology, engineering, pharmaceutics and chemical technology. An attempt has also been made to cover the syllabi of as many universities as possible including JNTU, Hyderabad. For the last fifteen years we have offered a course on applied microbiology and industrial microbiology to the students of Kakatiya University, Warangal and Jawaharlal Nehru Technological University, Hyderabad, which provided an inspiration to develop much of the vi Preface material herein. We thank all these students profusely. The task of this magnitude needs help of many, either directly or indirectly are worthy of our thanks. We also thank those who graciously permitted us to use published materials. We thank the Chairman Sri M Laxman Reddy and management of St. Martin’s Engineering College, Secunderabad and the authorities of Kakatiya University, for kind encouragement and facilities. S. M. REDDY S. RAM REDDY G. NARENDRA BABU Contents Contents Preface v 1. History of Industrial Microbiology 1–6 1.1 Alcohol Fermentation Period (Before 1900) 2 1.2 Antibiotic Period (1900–1940) 3 1.3 Single Cell Protein Period (1940–1964) 5 1.4 Metabolite Production Period (1964–1979) 5 1.5 Biotechnological Period (1980 Onwards) 5 Review Questions 6 Further Reading 6 2. Fermentation Process 7–106 2.1 Design and Basic Functions of a Fermenter 12 2.2 Fermentation Processes 18 2.3 Types of Fermentations 20 2.4 Screening 32 2.5 Preservation of Industrially useful Organisms 37 2.6 Fermentation Medium 41 2.7 Optimization of Medium Components 50 2.8 Media Sterilization 52 2.9 Inoculum Preparation 53 2.10 Scale up of Fermentation 58 2.11 Downstream Process 60 2.12 Biological Assays 69 2.13 Containment and Environmental Safety 86 2.14 Fermentation Economics 88 viii Contents 2.15 Computer Applications in Fermentation 89 2.16 Strain Improvement in Industrial Microorganisms 90 Review Questions 105 Further Reading 106 3. Anaerobic Fermentations 107–122 3.1 Acetone–Butanol Fermentation 107 3.2 Ethyl Alcohol 113 3.3 2, 3–Butanediol 121 Review Questions 122 Further Reading 122 4. Organic Acids 123–141 4.1 Citric Acid 123 4.2 Lactic Acid 128 4.3 Acetic Acid 133 4.4 Gluconic Acid 137 Review Questions 140 Further Reading 141 5. Amino Acids 142–159 5.1 L-lysine 146 5.2 L-glutamic Acid 151 5.3 L-aspartic Acid 155 5.4 L-phenylalanine 156 Review Questions 158 Further Reading 159 6. Antibiotics 160–197 6.1 Penicillin 163 6.2 Cephalosporins 173 6.3 Streptomycin 176 6.4 Tetracyclines 179 6.5 Erythromycin 184 6.6 Chloramphenicol 187 6.7 Fusidic acid 188 6.8 Griseofulvin 189 6.9 Bacitracins 189 6.10 Nisin 193 6.11 Interferons 195 Review Questions 197 Further Reading 197 Contents ix 7. Vitamins 198–211 7.1 Cyanocobalamin (Vitamin B12) 198 7.2 Vitamin A (β-carotene) 204 7.3 Riboflavin 208 Review Questions 211 Further Reading 211 8. Enzymes 212–231 8.1 Amylases 216 8.2 Proteases 220 8.3 Pectinases 223 8.4 Lipases 223 8.5 Cellulases 225 8.6 Glucose Isomerase 227 Review Questions 231 Further Reading 231 9. Beverages 232–252 9.1 Beer 233 9.2 Wine 244 Review Questions 251 Further Reading 252 10. Microbial Polysaccharides 253–267 10.1 Xanthan 253 10.2 Pullulan 260 10.3 Dextran 260 10.4 Cyclodextrins 261 10.5 Gellan 263 10.6 Welan 264 10.7 Curdlan 264 10.8 Polyhydroxybutyrate 264 Review Questions 267 Further Reading 267 11. Hybridoma Technology 268–274 11.1 β-lymphocytes 268 11.2 Monoclonal Antibodies 272 Review Questions 274 Further Reading 274 12. Bioleaching 275–279 12.1 Mechanism of Bio-leaching 275 12.2 Bioleaching Organisms 276  x Contents 12.3 Commercial Processes 276 12.4 Copper Leaching 277 12.5 Uranium Leaching 278 Review Questions 279 Further Reading 279 13. Biosensors 280–285 Review Questions 285 Further Reading 285 14. Biosurfactants 286–301 14.1 Classification of Biosurfactants 287 14.2 Enzyme Synthesized Biosurfactants 288 14.3 Microbial Biosurfactants 289 Review Questions 300 Further Reading 300 15. Cell and Tissue Culture 302–329 15.1 Plant Cell and Tissue Culture 302 15.2 Organ Culture 309 15.3 Animal Cell and Tissue Culture 320 Review Questions 327 Further Reading 329 16. Single Cell Protein 330–347 16.1 Why Microorganisms as a Source of Protein? 331 16.2 Microorganisms 332 16.3 Raw Materials 336 16.4 SCP from Hydrocarbons 337 16.5 Mixed Cultures 338 16.6 Steps in SCP Production 338 16.7 Nutritional Status of SCP 342 16.8 Downstream Process 345 16.9 Conclusion 346 Review Questions 346 Further Reading 347 17. Biotransformation 348–361 17.1 Microorganisms 352 17.2 Isolated Enzymes 352 17.3 Other Biocatalysts 353 17.4 Types of Reactions 353 17.5 Methods of Biotransformations 354 Review Questions 360 Further Reading 360 Contents xi 18. Biopesticides 362–396 18.1 Microbial Insecticides 362 18.2 Biological Control of Plant Diseases 383 Review Questions 394 Further Reading 395 19. Vaccines 397–406 19.1 Synthetic Vaccines 402 19.2 Recombinant Subunit Vaccines 402 19.3 Genetically Altered Live Vaccines 403 19.4 Vectored Vaccines 403 19.5 DNA Vaccines 403 19.6 Plant And Plant Viruses Based Vaccines 404 19.7 Vaccines Against Bacteria 404 19.8 Future 405 Review Questions 405 Further Reading 406 20. Biofertilizers 407–420 20.1 What are Biofertilizers? 409 20.2 Potential Organisms for Biofertilizers 410 Review Questions 419 Further Reading 420 21. Mushroom Cultivation 421–435 21.1 Importance of Mushroom Cultivation 421 21.2 Classification of Edible Mushrooms 422 21.3 General Steps in Mushroom Cultivation 424 21.4 Mushroom Cultivation in India 426 21.5 Pests And Diseases of Mushrooms 431 21.6 Canning of Mushrooms 432 21.7 Nutritional and Medicinal Aspects of Mushrooms 432 21.8 Future of Mushroom Cultivation 434 Review Questions 434 Further Reading 435 22. Intellectual Property Rights and Patents 436–446 22.1 Requirements for Patentability 437 22.2 Types of Patents 439 22.3 Composition of Patent 439 22.4 Procedure For Obtaining a Patent 440 22.5 Subject Matter and Characteristics of Patent on Microbial Process or Products 442 22.6 Patents Involving Microorganisms 442 xii Contents 22.7 Cost of Patent 444 22.8 Patent in Different Countries 445 22.9 Prospects 445 Review Questions 445 Further Reading 446 Index 447-458  1 History of Industrial Microbiology Industrial microbiology came into existence, primarily, based on a naturally occurring microbiological process called fermentation. There are many evidences which clearly shows that ancient man knew fermentation process and practiced it more as an art rather than as a science. Early fermentation process practiced by man included the leavening of bread, retting of flax, preparation of vinegar from wine, production of various alcoholic beverages like beer, wine, mead and the production of various fermented foods and milk. Due to invention of microscope, discovery of microorganisms and understanding of their metabolic processes, lead to clear understanding of the fermentation, which paved the way for the development of Industrial Microbiology. The history of industrial microbiology can be divided into five phases, which are précised in table 1.1 Phase I up to 1900 Alcohol fermentation period, Phase II 1900-1940 Antibiotic period, Phase III 1940-1964 Single cell protein period, Phase IV 1964-1979 Metabolite production period, and Phase V 1979 onward Biotechnology period. Table 1.1: The phases in the history of Industrial Microbiology Phas Main Fermenters Process Culture Qual Pilot Strain e products control method ity plant selection contr facilitie ol s I Alcohol Wooden Use of Batch Prac Nil Pure Peri upto 1500 thermo tically yeast od barrels meters, nil culture befo capacity hydrome used at re ter and some of 1900 heat the exchangers brewerie s Vinegar Barrels-shall --- Batch Practi Nil Process ow cally nil inoculat trays-trickle ed with filters good vinegar contd... AVINASH/14/04.12/PRINT OUT 2 Basic Industrial Biotechnology Phas Main Fermenters Process Culture Qual Pilot Strain e products control method ity plant selection contr facilitie ol s Bakers yeast, Steel vessels pH Batch Practi Nil Pure glycerol, upto 200 m3 electrodes and cally nil cultures citric acid, for acetone with fed-batc used lactic acid / off-line h and butanol. Air control. systems acetone/ sprayers Temperatu butanol used for re control bakers yeast. II Penicillin, Mechanical Steriliza Batch Very Beco Mutatio Peri streptomy stirring used ble pH and impor mes ns and od cin other in small and fed-batc tant comm selection bet antibiotics vessels, oxygen h on program ween mechanicall electrodes common me 1900- y aerated essential 1940 vessels III Gibberelli Vessels Use of Contin Very Beco Mutation Peri ns, amino operated control uous impor mes and od acids, aseptically, loops culture tant comm selection bet nucleotides, true which introduc on program ween enzymes, fermentation were later ed for me 1940- transformatio s compu brewing essential 1964 ns terised and some primary metabolite s IV Single cell Pressure Use of Continuo Very Very Genetic Peri protein cycle and computer us impor impor engineer od using pressure jet linked culture tant tant ing of bet hydrocarbo vessels control with producer ween ns and developed loops medium strain 1964- other feed to recycle attempted 1979 stocks overcome gas and heat exchange problems  V Production Fermenters Control Batch, Very Very Introduc 1979- of developed and fed impor impor tion of onwa heterogeno in phase 3 sensors batch tant tant foreign rd us proteins and 4. developed or genes by Animal cell in phases continuo into microbial reactors 3 and 4 us microbia and developed fermenta l and animal tion animal cells; develop cells. Monoclonal ed for In vitro antibodies animal recombi produced cell nant by animal processes DNA cells techniqu es used in the improve ment of phase 3 products 1.1 ALCOHOL FERMENTATION PERIOD (BEFORE 1900) The period before 1900 is marked by the production of primarily alcohol, vinegar and beer, although without the knowledge of biochemical processes involved in it. Though beer, which History of Industrial Microbiology 3 represents the phase-I in fermentation process, was produced by ancient Egyptians, large scale brewing in large wooden vats of 1500-barrel capacity was started in the early 1700. An attempt was also made for process control by the use of thermometers and heat exchangers in these early breweries. In the middle of 18th century, the chemist Liebig considered fermentation purely as a chemical process. He believed fermentation as a disintegration process in which molecules present in the starter substance like starch or sugar underwent certain changes resulting in the production of alcohol. Other eminent chemists of this period like Berzelius (1779–1848) and Bertholet (1827–1907) have also supported this view. Cagniard Latour, Schwan and Kutzilog while working independently concluded that alcoholic fermentation occurs due to action of yeast which is an unicellular fungus. But, it was Louis Pasteur who eventually convinced the scientific world that the fermentation is a biological process. By conducting series of experiments, Louis Pasteur conveniently proved that yeast is required for conversion of sugars into alcohol. In 1857, he discovered the association of different organisms other than yeasts in the conversion of sugars into lactic acid. These observations led Pasteur to conclude that different kinds of organisms are required for different fermentations. While working on butyric acid fermentation in 1861, Pasteur made another important discovery that the fermentation process can proceed in the absence of oxygen. The rod shaped organisms responsible for butyric acid fermentation, remains active in the absence of oxygen. This organism was later on identified as butyric acid bacterium. This observation subsequently lead to the emergence of a new concept of anaerobic microorganisms and a classification of three organisms broadly into two categories, viz., aerobic and anaerobic microorganisms. During this period, wine Industry in France was incurring heavy losses due to soaring of wine. Pasteur was requested by the Government of France to study this problem. After careful study, he reported that the soaring of wine was due to the growth of other unwanted microorganisms, other than yeast, which invaded the wine and changed its chemical and physical properties leading to soaring. He showed that these unwanted organisms could be eliminated from the wine by partially sterilizing the juice from which wine is produced, below the boiling point. This process is now called as Pasteurization. Pasteurization kills all the bacteria but does not alter the desirable qualities of juice. This proposition of Pasteur saved the wine industry of France from heavy losses. Later on Pasteur has also studied the fermentation of acetic acid and beer. He disproved the concept of chemical basis of fermentation.  During the late 19th century Hansen, working at Carlsberg Brewery, developed methods for production of pure cultures of yeast and techniques for production of starter cultures. Thus, by the end of nineteenth century, the concept of involvement of microorganisms in fermentation process and its control were well established in brewing industry. 1.2 ANTIBIOTIC PERIOD (1900–1940) Important advances made in the progress of industrial microbiology were the development of techniques for the mass production of bakers yeast and solvent fermentations. However, the growth of yeast cells in alcoholic fermentation was controlled by the addition of Wort periodically in small amounts. This technique is now called as fed batch culture and is widely used in the fermentation industry specially to avoid conditions of oxygen limitation. The aeration of early yeast cultures was also improved by the introduction of air through sparging tubes. 4 Basic Industrial Biotechnology The other advancement during this period was the development of acetonebutanol fermentation by Weisman, which was considered to be truly aseptic and anaerobic fermentation. The techniques developed for the production of these organic solvents were major advances in fermentation technology, which led to the successful introduction of aseptic aerobic processes, which facilitated in the production of glycerol, citric acid and lactic acid. Another remarkable milestone in the industrial microbiology was the large-scale production of an antibiotic called penicillin, which was in great demand to save lives of thousands of wounded soldiers of Second World War. The production of penicillin is an aerobic process which is carried out by submerged culture technique under aseptic conditions. The inherent problems of contamination, requirement of large amount of liquid medium, sparging the culture with large volume of sterile air, mixing of highly viscous broth were solved. The technology established for penicillin fermentation paved the way for the development of a wide range of new processes such as production of other antibiotics, vitamins, amino acids, gibberellins, enzymes and steroid transformations. At about the same time Dubos at Rockfeller Institute, discovered a series of microbial products which showed antimicrobial properties and hence useful in treating certain human diseases. Waksman, a soil microbiologist, and his associates have discovered many antibiotics produced by species of Streptomyces, soil inhabiting, which is now widely used (table 1.2). Table 1.2: List of antibiotics and the year of their discovery Name of the antibiotic Name of the Year of Producing organism discoverer discovery Penicillin Alexander Fleming 1929 Penicillium Chrysogenum Tyrothricin – 1939 Bacillus Griseofulvin – 1939 Penicillium griseofulvum Streptomycin S.A. Waksman et al. 1943 Bacillus licheniformis Bacitracin Johnson et al. 1945 Streptomyces griseus Chloramphenicol Ehrlich 1947 St. Venezuelae Polymyxin – 1947 Bacillus polymyxa Chlortetracycline Duggar 1948 St. aureofacieus Cephalosporin, C, N, P Brolzu 1948 Cephalosporium acremonium Neomycin Waksman et al. 1949 St. fradiae Oxytetracycline Finley et al. 1950 St. rimosus Nystatin – 1950 St. noursei  Erythromycin Clark 1952 St. erythreus Novobiocin – 1955 St. niveus Kanamycin – 1957 St. kanamyceticus Fusidic Acid – 1960 Furidium calcineurin Ampicillin – 1961 Semi synthetic Cephalothin – 1962 Semi synthetic Lincomycin – 1962 St. lincolensis Gentamycin – 1963 Micromonospora purpurea Carbenicillin – 1964 Semi synthetic Cephalexin – 1967 Semi synthetic Clindamycin – 1968 Semi synthetic History of Industrial Microbiology 5 1.3 SINGLE CELL PROTEIN PERIOD (1940–1964) This period is marked by the production of proteinaceous food from the microbial biomass. As the cost of the resultant product was very low there was a need for large-scale production of microbial biomass. This led to the development of largest mechanically stirred fermenters ranging from 80,000 to 1,50,000 liters or even more in diameter, which were to be operated continuously for several days, if they were to be economical. Thus, a new fermentation process called continuous culture fermentation came into existence. The most long-lived continuous culture fermentation was the ICI Pruteen animal feed process employing the culture of Methylophillus methylotrophus. 1.4 METABOLITE PRODUCTION PERIOD (1964–1979) During this period, new microbial processes for the production of amino acids and 51- nuclosides as flavour augmenters were developed in Japan. Numerous processes for enzyme production, which were required for industrial, analytical and medical purposes, were perfected. Techniques of immobilization of enzymes and cells were also developed. Commercial production of microbial biopolymers such as Xanthan and dextran, which are used as food additives, had been also started during this period. Other processes that were developed during this period includes the use of microorganisms for tertiary oil recovery. 1.5 BIOTECHNOLOGICAL PERIOD (1980 ONWARDS) Rapid strides in industrial microbiology have taken place since 1980, primarily because of development of new technique like genetic engineering and hybridoma technique. By genetic engineering it was made possible to in vitro genetic manipulations which enabled the expression of human and mammalian genes in microorganisms so thereby facilitating large scale production of human proteins which could be used therapeutically. The first such product is the human insulin used for treating the ever growing disease, diabetes. This was followed by the production of human growth hormone, erythropoietin and myeloid colony stimulating factor (CSFs), which control the production of blood cells by stimulating the proliferation, Erythro-poietin used in the treatment of renal failures, anemia and platelet deficiency associated with cancer, gametocyte colony stimulating factor (GCSF) used in cancer treatment and several growth factors used in wound healing processes. The hybridoma technique, which is employed for the production of monoclonal antibodies which aid in medical diagnosis and therapeutics, is also developed during this period. Perfection of production of microbial secondary metabolites related fermentation processes and their large-scale production is the other major development of this period. Some of such secondary metabolites released into the market includes: 1. Cyclosporine, an immunoregulant used to control rejection of transplanted organs. 2. Imipenem, a modified carbapenem used as a broad-spectrum antibiotic. 3. Lovastatin, a drug used for reducing blood cholesterol levels. 4. Ivermectin, an antiparasitic drug used to prevent African River Blindness disease. This brief account of history of development of industrial microbiology justifies the statement of Foster (1949), “Never underestimate the power of microbes”. 6 Basic Industrial Biotechnology REVIEW QUESTIONS I. Essay Type Questions 1. Trace the history of use of microorganisms in industry. 2. Discuss the role of microorganisms in food industry. 3. Discuss milestones in the development of industrial microbiology. II. Write short notes on: (a) Antibiotic era (b) Alcoholic beverage period (c) Microbial metabolites era (d) Biotechnology era (e) Single cell protein concept (f) Monoclonal antibody era (g) Pasteurization (h) cyclosporin (i) lovastatin FURTHER READING 1. Bader, F.G. (1992). Evolution in fermentation facility design from antibiotics to recombinant proteins in Harnessing Biotechnology for the 21st century (eds. Ladisch, M.R. and Bose, A.) American Chemical Society, Washington DC pp. 228–231. 2. Bushell, M.E. (1998). Application of the principles of industrial microbiology to biotechnology (ed. Wiseman, A.) Chapman and Hall, New York pp. 5–43. 3. Rehm, H.J. and Reed, G. (1993), Biotechnology (2nd edition) Vol. 1–12, VCH, Weinheim. 2 Fermentation Process Fermentation term for the first time was coined by Louis Pasteur for a phenomenon of bubbling of sugar solution. Later on, it has been applied for the phenomenon of production of different chemicals involving microorganisms. Presently, the term is used solely to any phenomenon involving microorganisms. Many products are made by large-scale fermentation including amino acids, enzymes, organic acids, vitamins, antibiotics, solvents and fuels. The typical fermentation process is depicted in Fig. 2.1.  Biomass cultureShake flask Production Culture fermenter fluid Cell separation Cell-free supernatant Stock Seed fermenter Product extraction Medium sterilization Medium raw purification Medium Product materials packaging formulation Effluent treatment Product Fig. 2.1: A schematic representation of a typical fermentation process The advantages in producing materials by fermentation are as follows: 1. Complex molecules such as antibiotics, enzymes and vitamins are impossible to produce chemically. 2. Optically active compounds such as amino acids and organic acids are difficult to prepare chemically. 8 Basic Industrial Biotechnology 3. Though some of the products that can be economically derived by chemical processes, but for food purpose they are better produced by fermentation such as beverages, ethanol and vinegar (acetic acid). 4. Fermentation usually uses renewable feed stocks instead of petrochemicals. 5. Reaction conditions are mild, in aqueous media and most reaction steps occur in one vessel. 6. Byproducts of fermentation are usually chemicals. The cell mass and other major by products are highly nutritious and can be used in animal feeds. However, it is beset with some drawbacks, which are as follows: 1. The products are made in complex solutions in low concentrations as compared to chemically derived compounds. 2. It is difficult and expensive to purify the product. 3. Microbial processes are much slower than chemical processes, increasing the fixed cost of the process. 4. Microbial processes, are subjected to contamination by competiting microorganisms, requires the sterilization of the raw materials and the containment of the process to avoid contamination. 5. Most microorganisms do not tolerate wide variation in temperature, pH and are also sensitive to upsets in the oxygen and nutrient levels. Such upsets not only slow the process, but fatal to microorganism. Thus careful control of pH, nutrients, air and agitation require close monitoring and control. 6. Although nontoxic, waste products have high BOD and requires extensive sewage treatment. Though microorganism belonging to bacteria, fungi and yeasts are extensively used in these fermentation, few fermentations are also based on algae, plants and animal cells. Several cellular activities contribute to fermentation products such as: 1. Primary metabolites: Ethanol, lactic acid and acetic acid. 2. Energy storage compounds: Glycerol, polymers and polysaccharides. 3. Proteins: SCP, enzymes of both extra and intracellular nature and foreign protein. 4.  Intermediate metabolites: Amino acids, citric acid, vitamins and malic acid. 5. Secondary metabolites: Antibiotics. 6. Whole cell products: SCP, bakers yeast, brewers yeast, bioinsecticides. Some of the products such as ethanol, lactic acid and cell mass products are generally growth associated, while secondary metabolites, energy storage compounds, and polymers are non-growth associated. Other products, such as protein depends on the cellular or metabolic function. Unlike primary metabolites which are essential for growth and reproduction, secondary metabolites are not essential for the growth and development of reproducing organism and are produced only in luxuriant conditions (Bu Lock, 1961). The secondary metabolites are basically are: Fermentation Process 9 1. Secondary metabolites are produced only by few organisms. 2. Secondary metabolites are needed depending on environmental conditions. 3. Secondary metabolites are produced as a group of closely related structures. 4. Some organisms forms a variety of different classes of substances such as secondary metabolites. 5. The regulation of biosynthesis of secondary metabolites differs significantly from that of primary metabolites. 6. Secondary metabolites are mostly produced in iodophase (Fig. 2.3) Origin and production of different secondary metabolites are depicted in Fig. 2.2 and 2.2 a. Fermentative products are in use by man since ancient times. Fermentation of grains or fruit produce, bread, beer and wine that retained much of the nutrition of raw materials, while keeping the product from spoiling. The natural yeasts that caused fermentation added some vitamins and other nutrients to the bread or beverage. Lactic acid producing bacteria ferment milk to yogurt and cheese and extend the life of milk products. Other food products such as pickles, vegetables and the fermentation of tea leaves and coffee beans were preserved or enhanced in flavor by fermentation. Cell walls histidine Tetrose-P 6-P Triose-P nucleotides Storage DNA RNA ATP Polysaccharides Nucleotides, Sugar Storage lipids Pentose-P Glucose deoxynucleotides, etc. Glycerol Membrane lipids ADP Porphyrins etc. -hydroxybenzoate P-enolpyruvate Folic acid Alanine + Valine, leucine NAD etc. Serine Glycine Pyruvate Respiratory quinones Fatty acids, lipids, Phenylalanine, PHB, polyketides tyrosine, Cysteine, methionine Acetyl-CoA Mevalonate, steroids, tryptophan, Purines, carotenoids Purines, pp Oxaloacetate Citrate P-glycerate pyrimidines -aminobenzoate, Succinate 2-oxoglutarate Glutamate, glutamine Arginine, pyrimidines Asparatate Threonine, isoleucine, methionine, lysine Folic acid Porphyrins Heme proline Cytochromes Chlorophyll Vitamin B12 Fig. 2.2: Primary metabolites giving rise to variety of cell substances Fermentation was an art until the second half of the 19th century. A batch was begun with either a starter, a small portion of previous culture, or with culture residing in the products or vessel. Pasteur (1775) made it clear that fermentation needs, heat treatment to improve storage quality and thus formed the basis for sterilization of medium. Emil Christian Hansen (1883) used for the first time pure culture of yeast for production of yeast in Denmark. During 1920–30 10 Basic Industrial Biotechnology the emphasis in fermentation shifted to organic acids primarily lactic acid and citric acid. The discovery of penicillin in 1929 and commercialized in 1942, gave a boost to fermentation industry and led to the development of big fermenters and submerged cultivation. Success of penicillin inspired pharmaceutical companies to launch massive efforts to discover and develop many other antibiotics. In 1960s amino acid fermentations were developed in Japan. Commercial production of enzymes for use in industrial process began on a large scale in 1970. The discovery of the tools of genetic engineering expanded the possibilities for products made by fermentation in situ, and the first genetically engineered fermentation product was developed and commercialized in 1977. The historical events developed in the progress of fermentations are précised in table 2.1. Pyruvate Citrate/itaconate CO2 Acetyl-CoA Poly b-hydroxy butyrate ×3 Polyketides Mevalonate (C )6 CO2 Quinones Isoprene units (C )5 ×2 Terpenes C10 Sterols C15 Gibberellins Carotenoids C20 Fatty acids (oils & fats) Fig. 2.2(a): Production of secondary metabolites Biomass, nutrient and nutrient metabolite concentration Secondary Tropophase Iodophase metabolite Biomass Time Limiting Fig. 2.3: The growth phases of biomass production and secondary metabolite production Fermentation Process 11 Table 2.1: Historical events in the progress of fermentation  ! " # % $ ! & * " + ' ( " ) , & -./ ,0 1 % 0 + ) (0 %" % 1 - 2 * 1 4 &5 1 1 0 1 3 % 2 6 % % ) 4 # 8 7 7 * # #1 6 (0 Fermentation may be aerobic if it is operated in the presence of oxygen, while it may be anaerobic if carried out in the absence of oxygen. Anaerobic fermentations can be carried out either by use of fresh medium, covered with an inert gas such as nitrogen or argon or accumulation of CO2 or foam (Fig. 2.4). 5 4 2 6 7 3 1 Fig. 2.4: Anaerobic fermenter 12 Basic Industrial Biotechnology The fermentation is called batch fermentation when it is operated for a definite period. On the other hand, fermentation which is operated for a indefinite period it is called continuous fermentation. Some of the organisms are sensitive to substrate concentration and they are inhibited when the substratum is in high concentration. Under such conditions, fermentation can be carried by addition of substrates in installments and the process is called Fed batch fermentation. Fermentations can be carried out under non-aseptic conditions where the risk of contamination is not a major concern. However, fermenters must be designed for prolonged aseptic operation. The design rules for an aseptic bioreactor demand that there is no direct contact between the sterile and non-sterile sections to eliminate microbial contamination. Similarly, fermentation based on number of organisms involved can be classified into simple fermentation when only one organism is involved to produce a product from substratum. On the other hand, in some fermentations two organisms are involved in order to get a fermentation product from a substratum. The product of first phase of fermentation serves as substratum for second phase in order to yield desired product. In this type of fermentation, two organisms may grow simultaneously and product is formed instantly. Commercial growth of lichens involving algae and fungi is a good example for simultaneous fermentation. Production of glutamic acid from glucose firstly gets oxidized to ketoglutaric acid, which inturn get aminated to produce glutamic acid and production of lactic acid from glucose by yeast and Lactobacillus lactis, production of β-carotene jointly by (+) and (–) strains of either choaenophoracucurbitarum or Blakesleea trispora are three very good examples. On the other hand, the two organisms involved in a fermentation are separated widely in time and space, such fermentation is called successive fermentation. For example, production of acetic acid from glucose. First glucose is acted by yeast to produce ethyl alcohol, which is oxidized to acetic acid by Acetobacter aceti. Similarly production of lysine from glycerol. Glycerol is fermented to Diaminopimelic acid (DAPA) by an auxotrophic mutant of E. coli which gets aminated to form L-Lysine by Aerobacter aerogenes. When more than two organisms are involved in a fermentation it is called as mixed fermentation or multiple fermentation. In this fermentation, the substratum is heterogeneous and organisms with different potentialities of producing enzymes are involved in the fermentation. For instance, degradation of municipal wastes and decomposition of dead plants and animals can be taken as mixed or multiple fermentation. Similarly, remediation of waste water comes under this fermentation. 2.1 DESIGN AND BASIC FUNCTIONS OF A FERMENTER A process in which a chemical product of human utility is produced involving microorganisms is called as fermentation. The vessel in which it is carried out is known as fermenter. An ideal fermenter should provide congenial environmental conditions, which promote the optimum growth of an organism and produce maximum product. Fermenter plays a critical role in the product yield. Hence design of a fermenter is important in the process of fermentation. The fermenter may be simple for fermentation products, which does not require aseptic, conditions, while fermenter requiring an aseptic conditions have to be designed to prevent interference by the contaminating organisms.  Fermenter supports best possible growth, biosynthetic conditions and ease of manipulation for all operations associated with the use of it. For the better production of a desired product an ideal fermenter should have following provisions: Fermentation Process 13 1. Fermenter body 2. Agitator 3. Coil 4. Gas outlet 5. Inoculation port 6. Thermometer 7. pH electrode (i) Characteristic features of a good fermenter: 1. It must be strong enough to withstand pressure of large volume of aqueous medium. 2. It must not corrode and contribute toxic ions to the growing microorganism. 3. Make provision for control or prevention of growth of contaminating microorganisms. 4. Provision for rapid incorporation of sterile air into the medium. 5. Carbon dioxide released during fermentation must be flushed out. 6. Stirrer must be available to mix the medium and microorganisms to facilitate the availability of nutrients and oxygen. 7. Intermittent addition of antifoaming agent. 8. Provision for controlling temperature. 9. Aseptic withdrawal of culture sample during fermentation and introduction of inoculum at the initiation of fermentation. 10. Determination of the pH and its adjust mentill, if required. 11. Some means of sterilization of medium and addition of antifoaming agent. 12. Air filter. 13. Drain in the bottom. 14. Access to the inside of the fermenter to clean it. (ii) Fermentation vessel: Fermenters used in microbial fermentation represent a wide range of devices from a simple type of tube aeration system fermenter operating on the air lift system or the deep jet principle or devices with rotary stirrers in which the air is sucked in or distributed under pressure into the stirrer space. There are wide variety of designs of fermenter (bioreactor) available. Selection of a fermenter design for a particular process depends on a variety of factors such as mass transfer considerations, mixing, sheer sensitivity, broth viscosity, oxygen demand, reliability of operations, sterilization considerations, the cost of construction and operation. Stirred tank reactors use sparged air and submerged impellers to aerate and mix the broth. They are versatile and are specially adapted to highly aerobic cultures and highly viscous fermentations. Even in this, there are many variations in design such as the style, number and placement of impellers, the height to diameter ratio, the number and placement of coils or baffles that affect the mixing characteristics of the vessels. The main drawbacks are high-energy input and the use of rotating scale on the agitator shaft which may cause contamination risk. Airlift fermenters (Fig. 2.5) mix broth with air from the sparger. Some designs have an internal shaft tube to direct the flow of fluid. Most airlift designs have a much greater height to diameter ratio than stirred tank vessels to improve oxygen transfer. The mixing is not as good as in a stirred tank but the energy input and shear forces are much lower, thus, useful for shear sensitive cultures or in processes where the energy cost of agitation is a significant factor. Ability to clean the vessel and maintenance cost are important factors for the selection of a bioreactor. Some reactor designs have excellent characteristics but in the pilot plant are not good choice for larger scale operation due to mechanical complexity that causes sterility and maintenance 14 Basic Industrial Biotechnology problem on scale up. Most large-scale airlift fermenters are used for plant effluent treatment production or for baker’s yeast or for fungal fermentations where the size of the mycelial pellets is controlled by shear forces. Gas outlet  Draft tube Lower S.G. liquid rises Higher S.G. liquid rises Air in via sparger Steel base Fig. 2.5: A diagram illustrating the principle of an airlift fermenter Fermenters with mechanical stirrers are used to mix the reactant mixture and they are called stirred tank fermenters (Fig. 2.6a). Stirrer Riser Air Downcc Air (a) Stirred-tank (b) Air lift Air (c) Packed bed (d) Bubble column Fig. 2.6: Stirred tank fermenters Fermentation Process 15 Fermenter with a draft tube is a hollow perforated tube that improves circulation and oxygen transfer. The air is introduced from the bottom of the fermenter that lifts the draft tube and it is known as Airlift Fermenter (Fig. 2.6b). The fermenters can also fluidize its bed where the microbial cells are immobilized on small particles. These particles move along with the fluid and as a result, nutrient easily stick to it that enable high rate of oxygen and nutrient transfer to cells. On the other hand, flocculated or packed bed reactor (fermenter) contains larger particles which immobilize cells and cannot move along with the liquids (Fig. 2.6c). The reactor can be operated in either upflow or downflow mode, that is the liquid containing the substrate can be introduced either at the top or the bottom of the reactor. These type of fermenters are employed in sewage treatment where cells are immobilized by flocculation. They may also be used for bioconversion of small molecule. Bubble column fermenter (reactor) is another type of reactor in which the agitation and aeration are provided by a bottom sparger. To ensure even agitation, the sparger nozzles must be distributed uniformly over the cross-section of the bottom. Either a ring with regularly spaced holes, a small number of parallel pipes or star like arrangement of pipes is used (Fig. 2.6d).  Fig. 2.7: Laboratory fermenter According to the size they may be classified as laboratory fermenter (Fig. 2.7), 500 ml to 50 liters in volume, pilot plant with 50 to 500 liters in volume and production fermenter with 500 liters and above one lakh liters (Fig. 2.8). The fermenter shape may vary from cylindrical to spherical, to tubular usually with a D-shaped bottom. It is closed at the top and bottom. The material with which a fermenter is constructed vary according to the type of fermentation process. For example, fermentation of alcohol and lactic acid is carried out in a wooden fermenter, where sterilization is not necessary or where there is no chance of corrosion of inner lining of the fermenter. However, present day fermenters are constructed with inner surface lined with stainless steel or copper or iron or glass, which are chemically inert. Normally fermenters upto 1000 litres capacity have an external jacket and larger vessels have internal coils. Both, provide a mechanism for vessel sterilization and temperature control during the fermentation. 16 Basic Industrial Biotechnology Pressure indicator Pump Acid-base reservoir Steam pH recorder and control Catalyst or nutrient addition Motor Exhaust line Steam recorder Air filter Sample Air flow line recorder and control Cooling water in Impeller Cooling water out Air supply Temp. Steam Harvest line Fig. 2.8: Typical fermenter (stirred tanker fermenter) 2.1.1 Provision for control of microbial growth: Since most industrial fermentations utilize pure cultures, fermenters should be designed in such a way that promotes luxuriant growth of microbe but prevent the growth of contaminating microorganisms. 2.1.2 Provision for incorporation of sterile air (oxygen): Most aerobic fermentation processes requires oxygen supply, which is called as aeration. Aeration is done by passing sterile air under pressure into the fermenter. The required air is sterilized by passing it through a  sterile filter consisting of glass wool or some other finely powdered material that help in trapping microorganisms present in the air. The sterile air bubbled into the liquid medium through a sparger in order to make oxygen distributes uniformly in the medium. 2.1.3 A device for removal of CO2: During fermentation process carbon dioxide and hydrogen gases are liberated which collect in the head space of the fermenter. The fermenter should be provided with a device to release these gases outside aseptically. 2.1.4 An impeller: An impeller, a rotating device, is generally provided to most of the fermenters, which accomplishes vigorous stirring and agitation of the medium. The rotation is carried out either by indirect or direct methods. In indirect method, the impeller is mounted on a shaft, which is driven by an electric motor fitted at the top of the fermenter. In direct method, impeller action is varied by using different impeller blades and is driven by a magnetic coupling fitted to a motor which is mounted beneath the fermenter. The impeller blades are arranged at different heights to achieve vigorous stirring and agitation of the medium. 2.1.5 A device for addition of antifoam agent: Aeration and agitation of the liquid medium causes the production of foam. Media with high levels of proteins or peptides cause more foam than with pure sugars and inorganic salts. Proteolytic bacteria that degrade proteins into peptides and amino acids also produce more foam. Appearance of foam leads to problems like contamination of the medium and impediment of aeration. Fermentation Process 17 The formation of foam is undesirable, can be prevented by the addition of antifoam agents. An antifoam agent lowers surface tension of the foam and thereby it collapses, which leads to the disappearance of foam. Antifoam agents may be added to the medium either manually or mechanically. Manual addition requires the presence of some device in the fermenter to add antifoam agent aseptically, whenever needed. In mechanical addition, which is done automatically, an electrical sensing mechanism is provided at the top of the fermenter. It consists of two electrodes projecting into the headspace of the fermenter. They are connected to a pump of antifoam reservoir. When foam builds up in the headspace and touches the electrodes, current flows between the electrodes and activates the pump for addition of antifoam. When foam collapses the electrodes get disconnected and addition of antifoam ceases. 2.1.6 A device for temperature control: Microorganisms widely differ in their temperature dependence for growth. However, they grow well at optimum fermentation temperature, which may be below or above ambient temperature. During fermentation lot of temperature is generated due to metabolic activities of microorganism, which leads to a rise in the temperature in the fermenter. For maintaining optimum temperature in the fermenter, one of the following devices is provided (Fig. 2.9). Temperature probe Water out Controller/computer Cooling jacket Fermenter Cooling water in Valve Fig. 2.9: A scheme for controlling fermenter temperature 1. Sparging cold water on the fermenter, 2. By circulating cold water through the jacketed walls of fermenter, or 3. Through coils arranged along the inside walls of the fermenter. 2.1.7 A device for addition or withdrawal of inoculum: The fermenter should also be provided with a device to introduce inoculum at the beginning of the fermentation, and its withdrawal aseptically during fermentation. 2.1.8 A provision for pH adjustment: pH, which plays an important role in the growth and metabolism of microbes, influences fermentation process. A mechanism for determining pH of the fermentation broth intermittently and adjusting the values is often required. This is usually accomplished by withdrawing a sample from the fermenter for pH measurement, followed by addition of alkali or acid to the fermentation medium to adjust pH. 18 Basic Industrial Biotechnology 2.1.9 Seed tanks: Inoculum of 1-10% is required to inoculate production tanks to reduce incubation period. They are also called as inoculum tanks. They are generally small sized fermenters in which inoculum is produced under controlled conditions. 2.1.10 Medium preparation vessel: Fermentation requires additional vessels for the preparation of medium. Required nutrients for the medium is transferred to the fermenter from these vessels. 2.1.11 Sterilization of the medium: Most of the fermentations require pure culture and needs sterilized medium. For this purpose, the medium is passed through retention tubes and heat exchangers before passing into the large, empty and sterilized fermentation tank. The retention tubes contain steam waters jet that inject high pressure steam into the medium to sterilize it as it passes through the pipes and the rate of passage is adjusted in such a way that there will be complete sterilization. The heat exchanger, which consists of a pipe containing the medium within a second pipe containing cool water moving in the opposite direction, cool the medium before it is passed into the fermenter. After entry into the fermenter the medium is diluted with sterile water. 2.1.12 Device for withdrawal of used medium: There must be a device at the bottom of the fermenter or some mechanism may be provided for removing the completed fermentation broth from the tank. The fermenter should be accessable for cleaning after fermentation is completed. Fermentation system must be efficiently controlled in order to optimize productivity, product yield and ensure reproducibility. The key physical and chemical parameters involved largely depend on the bioreactor, its mode of operation and the microorganism being used. They are primarily aeration, mixing, temperature, pH and foam control. The control and maintenance at optimum levels inside the reactor is mediated by sensors (electrodes) along with compatible control systems and data logging. 2.2 FERMENTATION PROCESSES Fermentation process can be conveniently divided into six stages regardless of the type of process. They are: 1. The formulation media used for the growth of the microorganism to be employed as inoculum and also in the production of fermentation products. 2. The sterilization of the medium, fermenter and other associated equipment. 3. The preparation of adequate quantities of pure culture that is to be inoculated into the fermenter. 4. The creation of optimum conditions in the fermenter for optimum growth of the organism and for optimum output of the desired product. 5. The extraction of the product and its purification. 6. The disposal of effluents generated during fermentation. The inter relationships among these six phases are diagrammatically illustrated in Fig. 2.10. This process varies with the type of organism used and product to be produced. The entire process can be discussed under two headings. Initial isolation Upstream processes Upstream processes Strain improvement Microorgranism  Cooling/heating Production strain Fermentation Process 19 Constraints: nutritional requirements, metabolic controls, shear sensitivity, temperature optima, morphology, O and CO effects and requirements, 22 genetic stability, metabolic by-products, viscosity Fermentation raw materials effects. Sources of carbon, nitrogen, phosphorous Starter culture and sulphur, minor elements, trace elements, propagation growth factors, water etc. (availability, cost, stability, and pretreatment and sterilization requirements) Media development Propagation mediumMaintenance medium Production medium Supported or suspended growth, Fermenter type, stirring mechanism, geometry, mode of operation, instrumentation and +/– Oxygen pH control Antifoam processes Cell separation Downstream Influenced by product centrifugation or ex situ DSP filtration Processes automation Downstream in situ DSP Biomass waste: Fermentation debris or ultrafiltration if product is extracellular Harvested cells Spent medium Intracellular Extracellular product concentration and or stability. Other Periplasmic product Concentration step Primary recovery considerations are yield at each step, Cell disruption process costs and purity requirements Cell Centrifugation  extract Inclusion bodies Medium concentrate Cell-free Effluent Dialysis, precipitation, partition, Finished product chromatographic steps, ultrafiltration, Product distillation etc. purification Crystallization, drying, lyophilization, Finishing process sterile filtration, packaging etc. Fig. 2.10: The upstream and downstream process of a typical fermentation process (a) Upstream process: It includes selection of organism and medium, medium sterilization, inoculation and ends with monitoring of fermentation process and product formation. This involves selection of microorganism. The selection of 20 Basic Industrial Biotechnology microorganisms for fermentation should be critically done. At first it should have potential to produce particular substance in an economic amounts. It should be non pathogenic and non-hazardous. Further it should be amenable to growth in a fermenter and produce the product in good amounts. (b) Downstream process: It includes the product separation and purification and effluent treatment. 2.3 TYPES OF FERMENTATIONS The vessel in which fermentation is carried out is called fermenter. The yield of the product is atleast partly dependent on the type of fermenter. Generally, fermenters are designed to provide best possible growth and biosynthetic conditions and ease of manipulations for all operations associated with the use of fermenters. A good fermenter is that which fulfills the following characteristics. It should provide control and observation of many facets of microbial growth and biosynthesis. Fermentation processes can be classified into the following three categories. They are:- 1. Batch fermentation 2. Continuous culture fermentation 3. Fed batch fermentation The choice of the operation depends largely upon the organism and the type of product being produced. 2.3.1 Batch fermentation: A batch fermentation is a closed culture system, because initial and limited amount of sterilized nutrient medium is introduced into the fermenter. The medium is inoculated with a suitable microorganism and incubated for a definite period for fermentation to proceed under optimal physiological conditions. Oxygen in the form of air, an antifoam agent and acid or base, to control the pH, are being added during the course of fermentation process (Fig. 2.11). Substrate Initial concentration Concentration Batch fermenter (BF) Time Substrate Fig. 2.11: A typical batch fermenter  During the course of incubation, the cells of the microorganism undergo multiplication and pass through different phases of growth and metabolism due to which there will be change in Fermentation Process 21 the composition of culture medium, the biomass and metabolites. The fermentation is run for a definite period or until the nutrients are exhausted. The culture broth is harvested and the product is separated. Batch fermentation may be used to produce biomass, primary metabolites and secondary metabolites under cultural conditions supporting the fastest growth rate and maximum growth would be used for biomass production. The exponential phase of growth should be prolonged to get optimum yield of primary metabolite, while it should be reduced to get optimum yield of secondary metabolites. The used medium along with cells of microorganism and the product is drawn out from the fermenter. When the desired product is formed in optimum quantities, the product is separated from the microorganism and purified later on. It has both advantages and disadvantages which are detailed below. (i) Merits: (a) The possibility of contamination and mutation is very less. (b) Simplicity of operation and reduced risk of contamination. (ii) Demerits: (a) For every fermentation process, the fermenter and other equipment are to be cleaned and sterilized. (b) Only fraction of each batch fermentation cycle is productive. (c) It is useful in fermentation with high yield per unit substratum and cultures that can tolerate initial high substrate concentration. (d) It can be run in repeated mode with small portion of the previous batch left in the fermenter for inoculum. (e) Use of fermenter is increased by eliminating turn round time or down time. (f) Running costs are greater for preparing and maintaining stock cultures. (g) Increased, frequency of sterilization may also cause greater stress on instrumentation and probes. (h) Fresh sterilized medium and pure culture are to be made for every fermentation process. (i) Yield of the desired product may also vary. (j) There will be a non-productive period of shutdown between one batch productive fermentation to the other. (k) More personal are required. 2.3.2 Continuous fermentation: It is a closed system fermentation, run for indefinite period. In this method, fresh nutrient medium is added continuously or intermittently to the fermenter and equivalent amount of used medium with microorganisms is withdrawn continuously or intermittently for the recovery of cells or fermentation products (Fig. 2.12). As a result, volume of the medium and concentration of nutrients at optimum level are being maintained. This has been operated in an automatic manner. The continuous fermenter has its maximum use that take long time to reach high productivity, reduces down time and lowers the operating costs. 22 Basic Industrial Biotechnology Pump Air in Air out Sterile air filter Overflow weir Nutrient reservoir  reservoir Fermenter Harvest Fig. 2.12: Continuous fermentater In continuous mode, starting medium and inoculum are added to the fermenter. After the culture is grown the fermenter is fed with nutrients and broth is withdrawn at the same rate maintaining a constant volume of broth in the fermenter. In continuous mode with cell cycle, the cell mass is returned to the fermenter using micro filtrations with bacteria or screens with fungal mycelium. A continuous fermentation is generally carried out in the following ways: (a) Single stage fermentation (b) Recycle fermentation (c) Multiple stage fermentation (a) Single stage fermentation: In this process, a single fermenter is inoculated and the nutrient medium and culture are kept in continuous operation by balancing the input and output of nutrient medium and harvested culture, respectively. (b) Recycle fermentation: In this method, a portion of the medium is withdrawn and added to the culture vessel. Thus, the culture is recycled to the fermentation vessel. This method is generally adopted in the hydrocarbon fermentation process. The recycling of cells provides a higher population of cells in the fermenter which results in greater productivity of the desired product. (c) Multiple stage fermentation: In this process, two or more fermenters are employed simultaneously and the fermentation is operated in a sequence. Different phases of fermentation process like growth phase and synthetic phase are carried out in different fermenters. Generally, growth phase is allowed in the first fermenter, synthetic phase in the second and subsequent fermenters. This process is adapted Fermentation Process 23 particularly to those fermentations in which growth and synthetic activities of the microorganisms are not simultaneous. Synthesis is not growth related but occurs when cell multiplication rate has slowed down. The process of continuous fermentation is monitored either by microbial growth activity or by product formation and these methods are called: (i) Turbidostat method, and (ii) Chemostat method. (i) Turbidostat Method: In this method the total cell content is kept constant by measuring the culture turbidity at a regular interval of fermentation process. By turbidity measurment it is possible to the fermenter to regulate both the nutrient feed rate and the culture withdrawal rate. Fermentation, in which this method is employed, must be carried out at a low maximum cell population which leads to the usage of less amount of substrate and wastage of greater amount of substrate as unused and residual medium, which is removed from the fermenter along with the harvested culture (Fig. 2.13). 1 3 2 4  2. electrodes for drop counting 3. air inlet 4. medium and air outlet 5. magnet 6 6. wiper 5 7. inspection port for automatic turbidity 8 measurement 8. spiral wire for culture heating 7 1. medium outlet Fig. 2.13: Turbidostat (ii) Chemostat Method: In this method nutrient feed rate and harvest culture withdrawal rate are maintained at constant value. This is achieved by controlling the growth rate of the microorganism by adjusting the concentration of any one of the chemicals of the medium, like carbon source, nitrogen source, salts, O2 etc. which acts as a growth limiting factor. Apart from the above chemicals, sometimes the concentration of the toxic product generated in the fermentation process, the pH values and even temperature also act as growth limiting factors. This method is 24 Basic Industrial Biotechnology employed more often than turbidostat method because of fewer mechanical problems and presence of less amount of unused medium in the harvested culture (Fig. 2.14). However, continuous fermentations have certain advantages and limitations which are as follows: 3 1 5 2 6 7 9 4 8 Fig. 2.14: Chemostat 1. Air inlet 2. Mariotte’s bottle 3. Capillary for medium inlet 4. Culture vessel 5. Inoculation port 6. Air inlet 7. Air outlet 8. Overflow capillary 9. Sampling tube (i) Merits: 1. The fermenter is continuously used with little or no shutdown time. 2. Only little quantity of initial inoculum is needed and there is no need of additional inoculum.  3. It facilitates maximum and continuous production of the desired product. 4. There is optimum utilization of even slow utilizable substances like hydrocarbons. (ii) Demerits: 1. Possibility of contamination and mutation because of prolonged incubation and continuous fermentation, are more. 2. Possibility of wastage of nutrient medium because of continuous withdrawal for product isolation. 3. The process becomes more complex and difficult to accomplish when the desired products are antibiotics rather than a microbial cells. 4. Lack of knowledge of dynamic aspects of growth and synthesis of product by microorganism used in fermentation. (iii) Applications: Continuous culture fermentation has been used for the production of single cell protein, antibiotics, organic solvents, starter cultures etc. (table 2.2). Fermentation Process 25 Table 2.2: Chemical products produced in continuous fermentation ! + + ) % ; ) $ 1 " % 1 * + Pilot plants or production plants have been installed for production of beer, fodder yeast, vinegar, baker’s yeast. A wide variety of microorganisms are used for this type of fermentation (table 2.3). Table 2.3: Microorganisms used in continuous fermentation ! )  + 3 ) " 7 < 2.3.3 Fed batch fermentation: It is a modification to the batch fermentation. In this process substrate is added periodically in instalments as the fermentation progresses, due to which the substratum is always at an optimal concentration. This is essential as some secondary metabolites are subjected to catabolite repression by high concentration of either glucose, or other carbohydrate or nitrogen compounds present in the medium. For this reason, the critical elements of the nutrient medium are added in low amount in the beginning of the fermentation and these substrates continue to be added in small doses during the production phase. This method is generally employed for the production of substances such as penicillin. Yoshida (1973) introduced this term for the first time for feeding the substrates to the medium as the nutrients are exhausted, so as to maintain the nutrients at an optimum level. The fed-batch fermentation may be of three types: (i) Variable volume fed batch culture. The same medium is added resulting in an increase in volume. (ii) Fixed volume fed batch culture. A very concentrated solution of the limiting substrate is added at a very little amount resulting in an insignificant increase in the volume of medium. 26 Basic Industrial Biotechnology (iii) Cyclic fed batch culture. As it is not possible to measure the substrate concentration by following direct methods during fermentation, which is necessary for controlling the feeding process, generally indirect methods are employed. For example, in the production of organic acids, the pH value may be used to determine the rate of glucose utilization. (i) Advantages: 1. Production of high cell densities due to extension of working time (particularly growth associated products). 2. Controlled conditions in the provision of substrates during fermentation, particularly regarding the concentration of specific substrates for e.g. the carbon source. 3. Control over the production of, by products or catabolite repression, effects due to limited provision of substrates solely required for product formation. 4. The mode of operation can overcome and control deviations in the organism’s growth pattern as found in batch fermentation. 5. Allows the replacement of water loss, by evaporation. 6. Alternative mode of operation for fermentations dealing with toxic substances or low solubility compounds. 7. Increase of antibiotic marked plasmid stability by producing the correspondent antibiotic during the time span of the fermentation. 8. No additional special piece of equipment is required as compared with the batch fermentation. 9. It is an effective method for the production of certain chemicals, which are produced at optimum level when the medium is exhausted like penicillin. (ii) Disadvantages: 1. It is not possible to measure the concentration of feeding substrate by following direct methods like chromatography. 2. It requires precious analysis of the microorganism. Its requirements and the under standing of its physiology with productivity is essential. 3. It requires a substantial amount of operator skill for the set up of fermentation and development of the process. 4. In a cyclic fed batch culture, care should be taken in the design of the process to ensure that toxins do not accumulate to inhibitory levels and that nutrients other than those incorporated into the fed medium become limited also, if many cycles are run. The accumulation of non-producing or low producing variants may result. 5. The quantities of components to control must be above the detection limits of the available measuring equipment. Fed-batch with recycle of cells can also be used for specific purpose such as ethanol fermentation and waste water treatment. At present following products are being produced under fed batch culture. 1. Production of baker’s yeast. 2. Penicillin production. 3. Production of Thiostrepton by Streptomyces laurentii 4. Production of industrial enzymes, histidine, glutathione (Brevibacterium flavum), Lysine (Corynebacterium glutamicum) Fermentation Process 27 (iii) Applications: 1. It facilitates in avoidance of repressive effect. 2. It has control over organisms growth rate and O2 requirement. 3. In maintaining concentration of both the biomass and non-limiting nutrient substrates constant. 4. Production phase may be extended under controlled conditions and overcome problems associated with the use of repressive rapidly metabolized substrates. 5. Shift in growth rate may provide an opportunity to optimum product synthesis. 6. It facilitates to overcome viscosity problems or its toxicity at higher concentration. 2.3.4 Anaerobic fermentation: A fermentation process carried out in the absence of oxygen is called as anaerobic fermentation. There are two types of anaerobic microorganisms viz, obligate anaerobic microorganisms and facultative anaerobic microorganisms. The former like Clostridium sp. cannot withstand oxygen or remain active only in the absence of oxygen. They remain active in the absence of oxygen and produce optimum amount of the desired product. The facultative anaerobes like lactic acid bacteria are able to withstand small amount of oxygen. However, certain organisms like yeast require an initial aeration to build up high cell yield before anaerobic conditions are created. Anaerobic conditions in the fermenter are created either by withdrawing the oxygen present in the head space by an exhaust pump and pumping some inert gases like nitrogen, argon etc. or by flushing it out, by the emergence of certain gases like carbon dioxide or hydrogen (Fig. 2.4). Stationary medium and viscous medium also creates anaerobic conditions. Sometimes in order to create anaerobic condition, medium is inoculated at the bottom of the fermenter soon after sterilization. (i) Merits: 1. Production of economically valuables byproducts like carbon dioxide and hydrogen gas during anaerobic fermentation, which may fetch some profits to the manufacturers. (ii) Demerits: 1. Manufacturers may have to spend more money in providing extra provisions to the fermenter like exhaust pump in order to enforce anaerobic conditions. 2. It requires special media like viscous media whose preparation requires certain costly  chemicals. 2.3.5 Aerobic fermentation: A fermentation process carried out in the presence of oxygen is called as aerobic fermentation. In most of the commercial processes and majority of the products of human utility are produced by this type of fermentation. Fermentation can be surface culture or static and submerged. 2.3.6 Surface fermentations: are those where the substratum may be solid or liquid. The organism grows on the substratum and draws the nutrients from the substratum. These types of fermentations are desirable where the products are based on sporulation. But it has several disadvantages such as it exposes the organism to unequal conditions, both oxygen and nutrients. 28 Basic Industrial Biotechnology Table 2.4: History and development of solid-state fermentation " #$ + + ' ) )1 ># ' ) +? & + 3 $ @1 B ) 8 8 8 D & +./ 1 2./ 1 $ E1 * ) $ 1 F $) )8 1 )8 D - %B) " $$ ) 7 8'/ ! & 3 1 $ 81 1 & - %  $ " $ 1 8 1 $ 1 $$ 8B ' ' ) 1 $ %%3 1 ( 1 $ 1 $ %%38 1 @1 1 G ! + ) $ 7 1 8 ) 0 $ $ ) 8 $ 8 1 1 ) D ! + 1 G $ 0 8 , 0 8 0 8 ) 8 7 8 ) ' 8 1 8 1 1 8 1 8 8 8 0 8 8 8 $ 8 8 1 8 0 8 $ ) D 8 1 D  ! 8 β) 8 6 8 8 0 8 1 ) 8 ) 8 0 8 β0 8 α $ 8 1 0 8 6 1 0 8 0 8 8 1 0 8 1 > 8 ' ?8 1 8 α) 8 β) 8 α 8 β 8 ) 8 ) 8 8 8 8 $ 1 D " ! 8 $ 8 8 0 8 ) D ! ) 8 1 ) 8 8 0 ) 8 ) 8 8 1 8 G + 8 + -8 $ 8 8 8 8 ) 8 $ 8 1 @ D 2.3.7 Submerged fermentations: are those in which the nutrient substratum is liquid and the organism grows inside the substratum. The culture conditions are made uniform with the help of spargers and impeller blades. Most of the industrial fermentations are of this type. The substratum which is in a liquid state and such medium is also called as broth.  Fermentation Process 29 2.3.8 Solid substrate/state fermentation: Solid state (substratum) fermentation (SSF) is generally defined as the growth of the microorganism on moist solid materials in the absence or near the absence of free water. In recent years SSF has shown much promise in the developme