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National Institute of Science Communication Dr K.S. Krishnan Marg New Delhi 110 012,lndia

Friendly Fermentation ].G. Shewale

© National Institute of Science Communication First Edition: July 1998 ISBN: 81-7236-185-8

Foundations of Biotechnology Series Book No. 2 Series Editor Volume Editor Cover Design lllustrations

Production

Printing

S.KNag Srilekha Bhattacharya and Dr Sukanya Datta Pradip Banerjee Sushila Vohra, Neeru Vijan, J.M.L. Luthra, Malkhan Singh, Harjeet Singh and Yogesh Kumar Shiv Kumar Marhkan, Rohini Raina, Dharmender Mohan, Ashok Kalra, Neeta Sahney and Suresh Kumar Sharma S. Bhushan, S.C. Mamgain, G.C. Pore!, Tika Ram, Rajbir Singh, Rattan Lal and Om Pal

Designed, Printed and Published by National Institute of Science Communication (CSIR) under the project 'Dissemination of Biotechnology Information' sponsored by Department of Biotechnology (Govt. of India)

Price: Rs. 30/-

Foreword The knowledge of biotechnology has equipped man to modify the microbe, be it a harmless or a deadly one compelling it to yield products useful to mankind. Fermentation, the age old process of making bread and wine, is now gaining teeth by the introduction of biotechnology. The range of products obtained by fermentation spans the spectrum from curd to in vitro synthesized insulin. Proposals for using various microbes as source of food and in cutting down pollution are also coming up. The National Institute of Science Communication (NI~ COM) is bringing out popular science books under a new series entitled 'Foundations of Biotechnology' as a part of the project on 'Dissemination of Biotechnological Information' sponsored by the Department of Biotechnology CDBn, Govt. Of India. This venture is yet another step taken by the Institute to make both students and laymen understand the science underlying the wonders achieved by applying hi-tech methods. Attractively illustrated and written in extremely simple and lucid style, these books would certainly help in p~rcolating the awareness of biotechnology down to the school level. By introducing the vast subject of biotechnology especially to children of classes VII to X, it is hoped that many would be inspired to take up this subject for an advanced study as it is the time for them to decide upon their career options. Keeping up with its major mandate of disseminating scientific information to large masses, NISCOM has undertaken this very important venture of popularizing the basic concepts essential to the understanding of sophisticated biotechniques. I am confident that these books would enhance the reader's curiosity to know more"about this interesting multidisciplinary subject, so

important from the view point of excellence in biology and its relevance to human kind.

M~~ (Manju Sharma) Secretary to the Govt. of India Deptt. of Biotechnology

Preface Fermentation processes yield products useful to all of us in everyday life. The bread we eat, medicinal products we take and many beverages that we drink are all results of fermentation through the application of friendly microorganisms or microbes. Fermentation dates back to times immemorial. It predates our knowledge of microbes. Though fermentation has been practised for ages, rapid scientific and technological advancements have come about only in the last century. Fermentation is a multidisciplinary technology wherein knowledge of biology, chemistry, and physics, is involved in one way or the other. Needless to say that advancements in these areas have brought fermentation to the forefront. The know-how is now so advanced that it is possible to manipulate the transfer of genes to evolve newer microbes that produce the desired product. The purpose of writing this book is to share some basic knowledge about fermentation processes and products with young students. We owe a lot to microbes for our good living. If the excitement of progress in this area would inspire some of the readers to takeup research as a career, this book would have served its purpose.

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Acknowledgement I profusely thank Mr S.K Nag, Chief Investigator, DBT Project and Head, Popular Science Division of NISCO M for extending an invitation to write this book. I appreciate his suggestions and efforts in bringing out this book in presentable form. Thanks are also due to Dr Sukanya Datta and Ms Srilekha Bhattacharya for editing the book under the guidance of Mr Nag. I sincerely thank Dr S.R Naik, General Manager, R & D; Mr M.C. Abraham, Managing Director, Hindustan Antibiotics limited for providing the photographs and for constant encouragement. Dr C. Siva Raman, FNA and Prof S.R Tophkhane have read the manuscript. Their critical comments and suggestions have made the book scientific enough for the students. I thank them for their concern. - I thank Arona Deshpande and also the brilliant artists headed by Mr P. Banerjee at NISCOM who have created the pictures to keep up the spirits of the readers. I also thank all my colleages at HAL,and the production and printing staff of NISCOM who have contributed to this book.

Dr J,G. Shewale

rmented ation Microbes Protocol Products oking Forward ossary esent

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mouthwatering eatables such as fluffy bread, de~rmentation. wordchocolate, is familiar. of licious cheese, The yummy tastyPlenty sour yogurt (curd), which we enjoy eating, are all gifts of fermentation. A wide variety of fermentatively obtained foodstuffs are available nowadays. These are the outcome of large scale industrialization of fermentation, particularly in developed countries, and are no longer limited to the home as was the case in the past Of course, some products such as curd and idli are still produced in homes. The term fermentation is derived from the Latin verb fervere which means 'to boil'. It describes the emission of carbon dioxide due to the action of yeasts on fruits or malted grain. Thus, fermentation is a process in which chemical changes are brought about in organic compounds through the activities of enzymes secreted by or present in microorganisms or microbes. In other words, specific chemical transformations are brought about in the presence of microbes. Fermenta-

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Friendly Fermentation

tion processes are thus the result of a sequence of chemical changes since all biological systems obey the laws of chemistry. Fermentation existed even in prehistoric days. It may never be known who first obsetved the phenomenon of fermentation. Primitive man who obsetved that natural changes improved the quality of stored food must have at some point of time woken up to the reality of fermentation. This must have been a process of painstaking trial and error. Microbes are the earliest of the living forms that evolved on the planet Earth an estimated 2000 million years ago. Microbial contamination of food material generally makes it inedible because of foul odour and unpalatable taste. Consumption of such spoiled food results in sickness and even death. However, in exceptional case, the food material becomes more appetizing. Indeed, it is the obsetvation of such desirable changes brought about by microbial intetvention that has led to today's fermentation industry. Fermentation is the first biotechnological process man developed. Fermented food is acceptable to us because the nutritive value of food is retained or improved. The physical and chemical characteristics of the food are altered during fermentation. But with correct microbial intetvention food value is not lost and the food is not spoiled. As a result, fermentation has been and is still the most important method of presetving food or improving its nutritive value. Primitive man knew the methods to prepare alcohol from cereal grains and fruits. What he did not know

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were the details of the biological changes brought about during fermentation. Nonetheless, without even being aware of the role of microbes, he improved the fermentation processes to obtain alcoholic beverages with pleasant flavours. He learnt all this through painstaking experience. The history of many fermented products dates back to ancient times: Chinese and Indian records go back to 3000 B.C., Greek to 1550 B.C. and Roman to 750 B.C. Bread was baked probably as far back as 7000 B.C. The Egyptians discovered that if dough was allowed to cure for several hours, it expanded when baked, resulting in spongy light loaves. Bread was a staple food of the ancient Egyptians and was often given in lieu of wages. The documentation of the science of microbiology and fermentation, however, came only about few hundred years ago with the invention of the microscope. This instrument literally introduced man to a strange, new world. The hobby of the Dutch linen draper Antony von Leeuwenhoeck was to build microscopes. His first mi-

Ancient Egyptian painting depicting wine production

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Friendly Fermentation

Leeuwenhoeck with his microscope (inset) and the _microscopic animals and plants observed by him

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croscope was a simple one, with lenses of short focal length which he ground in his spare time. With it he obsetved single celled organisms in a drop of pond water. In 1676, he introduced the world of micro organisms to man. The first person to suggest the role of microscopic organisms in fermentation was the French scientist L.]. Themard in 1803. His legacy was carried forward by the eminent French scientist Louis Pasteur who contributed to the significant progress in the knowledge of fermentation in the 1850s. He described bacteria and

Louis Pasteur

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Friendly Fermentation

yeasts at the physiological level, introduced aseptic methods and defined nutrient requirement of the microbes. With the turn of the century, the knowledge of fermentation made rapid progress.

Omnipresent Microbes which are visible to us. But in reality there are a birds and beasts are living of seemingly invisible livingbeings forms existing on this earth. These minute living entities are called microorganisms or microbes. They are not visible because of their extremely small size. The term microorganisms was coined by a combination of the word "micro" which comes from the Greek word mikros meaning small and the word "organism" meaning individual living forms. Microorganisms are complete and independent living entities. Microorganisms are generally unicellular and visible only under a microscope. Microorganisms are omnipresent, i.e. they are present everywhere. They are present in water, soil and air. They are also present in and on the bodies of plants and animals.

Plants, insects, large number

Microbes are very minute, such is their size that it is impossible to see them with unaided eyes. For this purpose, today we have different types of powerful microscopes. The compound microscope and the elec-

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Amoeba

Relative sizes of microbes as compared to hydrogen atom and haemoglobin molecule

tron microscopes are the instruments that empower us to 'see' the usually invisible microbes. Since microbes are so small we need to use a special scale to measure them. Microbes are measured employing a special unit called micron. One micron is one millionth of a metre! Microorganisms differ widely in size and shape. Variations in shape, cell structure, physiology and other biochemical characteristics form the criteria for the classification of microorganisms. They are grouped into five types: protozoa, algae, fungi, bacteria and viruses.

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Omnipresent Microbes

Protozoa derive their name from the Greek words protos, meaning 'first' andzoion, meaning 'living being'. They are the smallest type of animal life, being made up of a single cell. They are different from bacteria in that they have at least one, well-defined nucleus. Protozoa are oval or cylindrical in shape and are found in stagnant water or in mud. They are capable of locomotion. Amoeba and Paramecium are common examples. Algae are simple, photosynthetic plants that grow in water or in damp places. They may be unicellular or multicellular and exist in a variety of shapes. Some algae may even be large enough to be seen, as are the giant k~lps or seaweeds. Algae have the ability to photosynthesize due to the presence of the photosyilthetic pigment, chlorophyll.

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Diagrammatic representation of (a) Amoeba and (b) Paramecium

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Friendly Fermentation

Fungi are a low form of plant life that lack chlorophyll. Fungi occur everywhere in nature and grow in damp environment A piece of bread, cooked food or even raw vegetable when kept unattended in moist air can support the growth of fungi. Fungal growth is also seen on the walls in rainy season. Fungi may exist in unicellular or in multicellular form. Yeasts are unicellular forms of fungi. Multicellular fungi are commonly termed molds. The multicellular structure of fungi is called mycelium. Bacteria, like fungi occur everywhere in nature. The size of bacterial cells is much smaller than the algal or fungal cells. Structurally, bacterial cells contain cytoplasmic and nuclear matter in a dispersed state. They do not have a discrete nucleus. Some bacterial cells may have locomotory flagella, or fimbriae. Althoughlher:e are thousands of different species of bacteria, the individual bacterium bears anyone of the three forms of shapes spherical or ellipsoidal, cylindrical or rod shaped and spiral or helical. Vrruses are the smallest entities among the microorganisms and are visible only under the electron microscope. Vrruses do not have a cellular structure. A virus has a nucleic acid core enveloped by a coat of protein. They cannot exist independently, and hence must attach to living cells, transfer their genetic material into the host cell and grow in a parasitic manner. Obviously, viruses grow only in specific host cells. They multiply by diverting the host cell machinery for their replication. Vrruses have the unique distinction of hav-

Omnipresent Microbes

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Friendly Fermentation

a

Three basic kinds of bacteria (a) Spherical (b) Cylindrical (c) Spiral

ing either DNA or RNA as their genetic material,...as------there are DNA virus as well as RNA-virus. RNA virus are called retrovirus. Some virus have even been ob- ~ tained in the crystalline form. They seem to inhabit the twilight zone between the living and non-living. All living things on earth have a unique biological name which is generally composed of two words. The first denotes the genus and the latter, the species. Rana tigrina is the biological name of frog, and Bos taurus, that of cow. Mangifera indica is the scientific name of mango and Cocos nucifera that of coconut. Microorganisms too are no exceptions. There is a proper system

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Omnipresent Microbes

nvelope with surface projections

Vaccinia virus

Orf Virus Mumps virus

Tipula iridescent virus T -even bacteriophage

Herpes virus

Influenza virus

Different types of virus; Generalized features of virus {inset}

for classifying them after studying their physical, biochemical and genetic characteristics. The nomenclature of microorganisms also includes a generic and a specific name. For example Aspergillus niger, Fusarium solani, Bacillus subtilis, Candida utilis. In all cases, the first name indicates the genus, and the second identifies the species. At times, characterizing a micro organ-

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Friendly Fermentation

ism up to species level is not possible. But it is mandatory to characterize the microbe atleast up to genus level. As a result, sometimes nomenclature may mean using a name such as Bacillus species. This means that the bacterium is a member of the genus Bacillus. It is said that microorganisms are man's best friends as well as his worst enemies. Man's enemies are the microorganisms that cause diseases and spoilage of food. Infections by protozoa, bacteria and viruses are widely prevalent Malaria and amoebiasis (dysentery) are common diseases caused by protozoa. There are many bacterial ailments. Tuberculosis, cholera and typhoid are the more frequently encountered bacterial diseases of man. COmnion cold, measles, chicken pox, infective hepatitis Gaundice) are manifestation of viral infections. Fungal diseases are mainly restricted to skin infections such as ringworm. However, all microorganisms are not our enemies. In fact, very few microorganism are pathogenic. Most are our friends. They give us good food, medicines to combat diseases and useful chemicals. Industrial fermentation is the story of the friendly microorganisms roped in to serve the human cause. Microorganisms have contributed to make our life more healthy, comfortable and our food more tasty. Among the microorganisms, yeasts and bacteria are widely used in fermentation. like all our living brethren, microbes too need energy for survival, metabolic functions and reproduction.

Omnipresent Microbes

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Thus, they either take up nutrients directly or after external enzymatic simplification of the food material. The ingested nutrients are converted into energy and cell constituents. These metabolic functions though apparently simple, encompass several complex chemical reactions that are continuously and simultaneously carried out in the individual cell.

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Friendly Fermentation

The molecules that specifically catalyse various chemical reactions in the cells are the enzymes. Enzymes enhance the rate of chemical reactions at ambient temperatures without being themselves consumed and hence are bio-catalysts. Microbial cells contain several enzymes. The levels of production of these enzymes are dependent on the environment in which the cell grows. Thus, it is possible to either increase, decrease or even initiate the formation of a microbial enzyme by altering the environmental conditions. If one can manipulate an enzyme, then, one can control the reaction catalysed by that enzyme, so that a desired product is obtained. So it will not be inappropriate to treat the microbial cell as a bank of enzymes or catalysts. Further, the bank is flexible. The rapid rate of multiplication of microorganisms has enabled us to use them in fermentation to achieve highly specific chemical changes. Microorganisms possess tremendous potential. They perform many chemical reactions easily under favourable conditions and in an environmentally friendly way' whereas an organic chemist has to struggle for days to achieve the same result. Doctor, engineer, teacher, carpenter, tailor are specialists in their respective professions. Man achieves these specializations through an extensive special training. Specialization also exist among microorganisms. Saccharomyces cerevisiae produces alcohol, Aspergillus niger produces citric acid, Penicillium chrysogenum produces penicillin, Bacillus subtilis produces proteases. Scientists are aware of these microbial specializations. Much of research is targeted at finding new species of

Omnipresent Microbes

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microbes that show a tendency to produce chemicals that we need. A lot of attention is paid to selecting those variants, that produce more of a chemical or produce the chemicals in which we are particularly interested. These are then carefully nurtured in special nutrient media that help them survive under laboratory conditions. The industrially developed culture is often termed 'strain' emphasizing the special properties of that microorganism, e.g. Penicillium chrysogenum produces penicillin. But not all Penicillium chrysogenum cultures that are isolated produce penicillin in high Yields. Isolates or strains that Yield penicillin in large quantity are selected, maintained and developed for use in industry.

Heat loving bacterium and its habitat

Like other plants and animals, microbes exist in a variety of habitats. Sometimes they grow in the ordinary environment and are called mesophiles. Microbes

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Friendly Fermentation

that display a love for the extremes of environmental conditions are called Extremophiles. There are many types of extremophiles and each is given a name. For example, microbes which grow at high temperatures are called thermophiles. Most microorganisms currently used in commercial fermentation are mesophiles. Many chemical conversions that require harsh conditions of temperature, pressure, acidity or alkalinity cannot be performed by using mesophilic microorganisms or even the enzymes produced by them. It is now evident that a variety of microorganisms grow in extreme environments that exist in the biosphere. The extreme environments include high temperature (geothermal marine sediments), low temperature (Antarctic sea water), high pressure (deep sea hydrothermal vent), high acidity (acid mine drainage), high alkalinity (sewage sludge) and high salt (hypersaline water). The enzymes catalysing the metabolic reactions for the survival and growth of these organisms must work and remain effective under the respective extreme environment, after all harsh or not, it is 'home' for the microbe. Thus, extremophiles would certainly be a better choice for the chemical industry than mesophiles. The metabolic activity of a microbial cell is determined by its enzymes. SYnthesis of the enzyme is in turn controlled by the genes of the microorganisms. It is now possible to alter the genes of a microorganism. The method is termed genetic engineering. Byadopting this methodology, a gene from one organism can be transferred to another. A genetically engineered microorganism is so designed that it may express the

Omnipresent Microbes

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desired change or synthesize the product in high Yields. The gene for chymosin, an enzyme present in the stomach of calves which is used in cheese making can be transferred from its original mammalian source to a bacterium (Escherichia colt) or a fungus (Aspergillus niger). This permits the production of chymosin by fermentation thus sparing the calves' life. The microorganisms, Erwinia herbicola and Corynebacterium species were used to convert glucose to vitamin C. Now, the corresponding genes from Corynebacterium species have been transferred to Erwinia herbicola and the production of vitamin C precursor is achieved in one step. Genetically engineered microorganisms hold great promise. for the fermentation industry in the future.

lermentntion Protocol

propagation of microorganism and its application the cultivation and in due course. The process for making of idli, or yogurt has probably been seen by all. For making curd, a spoon of curd is added to warm milk which is kept covered overnight. Similarly, for making idli, batter rice and black gram (urad daf) are soaked in water for some time, then coarsly ground and kept overnight at room temperature. These are common examples of fermentation. Aged curd develops a sour taste which is due to accumulation of a chemical called lactic acid. Nice fluffy idlies are obtained if the soaked idli mix is allowed to remain so for only a certain period. Thus, some controls are required. Even with all advancements in science, it is said that "Fermentation is often more of an art than a science". Household fermentations are relatively simple because control of the fermentation process is easy. We must thank our ancestors who discovered and developed

F1rmentation a process for to obtain the is desired product

Fermentation Protocol

./ Fermentation is a multistep process

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Friendly Fermentation

such simple but useful processes through trial and error and careful observations. Large-scale or industrial fermentation, on the other hand has a complex protocol. It starts with the isolation of microorganisms. The yeast, fungus or bacterium which yields the product of interest is isolated either from soil, air, water or sources such as plant or animal material. Generally, more than one microorganism are obtained at the first attempt. In the next step, the microbes collected are grown in the laboratory maintaining stringent control and taking utmost care to avoid contamination of the specimens obtained from different sources, thus pure cultures are raised. Once the desired pure culture is obtained, it is cultured in its pure form. Isolated cultures are often deposited in culture collections or culture banks. Culture collection centres preserve and maintain thousands of microorganisms in this way. One can obtain a culture from such culture collections instead of going through the tedious process of strain isolation. The physical and physico-chemical properties of the microbial strain chosen for fermentation playa crucial role in process design and engineering of the fermentation plant. Therefore, the isolation, preservation and improvement of the strain are of fundamental importance. The ve-sselin which fermentation is performed is called a fermentor. The size of fermentors varies from 1 litre (laboratory fermentor) to 250,000litres (indus-

Fermentation Protocol

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trial fermentor). Some proteins are produced in 50 to 50,000 litre fermentors whereas, chemicals, antibiotics, and Baker's yeast are usually produced in 100,000 to 250,000 litre fermentors. Smaller fermentors are used for research purpose. Stages in the development of a fermentation process 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Isolation and choice of microorganism. Preservation and maintenance of the microorganism. Improvement of productivity of the microorganism. Formulation of media for all stages, i.e. preservation, seed and production. Optimization of seed quality. Growth of the microorganism and product formation during the fermentation. Monitoring and control of fermentation. Laboratory analysis. Isolation of the product Effluent treatment.

Components of a fermentation process 1. Preparation of the medium. 2. Sterilisation of the medium, fermentor and other equipments. 3. Propagation of pure and active culture (seed)/Inoculation or seeding the medium. 4. Culturing of the microorganism in the fermentor. 5. Isolation of the product. 6. Effluent treatment.

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Friendly Fermentation

Fermentation is a multistep process. The culture is first grown in a test tube or petriplate~ The growth is transferred to a conical flask (500ml to 3litre) containing nutrient media so that a teeming population ofthe desired microbe is obtained. The cultivation of microbes is performed by shaking the flask containing them on a platform called shaker. The growth from the flask is then transferred to a seed fermentor containing seed medium. The cell or spore suspension which is transferred is termed inoculum and the operation is called inoculation. Once the seed is ready, it is transferred to the production fermentor containing production media Production fermentor is a big vessel. The vessel is provided with agitator for mixing the contents. The temperature is kept steady by the circulation of hot and cold water through coils and the vessel has inlets for air, inoculation and addition of feeds. Valves for sampling and withdrawal of fermented broth are also fitted in it During fermentation, various parameters such as pH (ameasure ofacidity or alkalinity), oxygen level, rate of air flow, and vessel pressure are controlled. Thus, individual sockets for mounting of the appropriate probes are also provided. After the fermentation is over the products are isolated. Roti, vegetables, meat, sweets, milk, and raw salad, for dinner followed by fruits sounds good to us. Such a wholesome diet provides us carbohydrates, proteins, fats, vitamins, minerals and other necessary nutrients to keep us healthy. Microm:ganisms too are living cells. Obviously, they need energy to survive, grow and multiply. The energy is provided by various organic and inorganic constituents. The nutrition of micro organ-

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Fermentation Protocol

Stock culture

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Fermentation protocol

Product

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Friendly Fermentation

A balanced diet keeps the doctor away

Fermentation

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isms depends on carbon and nitrogen sources, minerals, oxygen, vitamins and other necessary growth factors. Combination of these materials constitutes their growth media. Sugars such as glucose, lactose, sucrose are common carbon sources. Cheaper carbon sources include starch, com steep liquor, molasses. Soyabean meal, cotton seed meal, peanut meal serve as organic nitrogen sources. Inorganic nitrogen sources are ammonium salts or nitrates. In general, mineral requirement is met by salts of sodium, potassium, magnesium, phosphates, and chlorides. Trace elements include cobalt, manganese and iron. At times, it is essential to supplement the medium with vitamins, especially in cases where the microorganisms are unable to sYnthesize them. The dietary requirement of microorganisms can vary widely. Moreover, the nutrient requirement at different stages of growth also differs. Thus, composition of media for maintenance, germination, seed preparation and production stage are different. The dietary requirement varies even during the different stages of fermentation. The availability of appropriate nutrients in correct amounts at all stages of fermentation is crucial for high yield of product. The most important step taken prior to fermentation is the formulation of the nutrient media in which the microbes will grow. If any of the constituent reagents is present in excess or in lesser amount than what is required, the entire process gets hindered, largely reducing the total yield, e.g. excess feeding of glucose to

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Friendly Fermentation

penicillin producing fungus will result in high cell mass but low product formation. In addition to the usual media constituents, certain compounds called effectors may "switch on" or "switch off' the product sYnthesis. The former are called inducers and the latter repressors. Some compounds may enhance the sYnthesis of a product For example, the addition of starch to the medium induces the formation of amylase (starch degrading enzyme) by Aspergillus niger whereas presence of glucose in addition to starch represses the formation of amylase. Some organisms have unique adaptive properties. If they are fed polysaccharides, then polysaccharide degrading enzymes are secreted by the microbes, and if supplied with protein the same organism produces protein degrading enzyme, e.g. Sclerotium rolfsii produces amylase when starch is the carbon source and cellulase (cellulose degrading enzyme) when cellulose is the carbon source in the medium. This is mainly due to the pressures for survival as they have to adapt themselves to whatever food source they get This type of behaviour is exploited to design the composition of medium to obtain therlesired product. In other words, the conditions are so adjusted that the microorganisms are compelled to make the product we want. Oxygen is a primary requirement for aerobic micro.organisms. This is provided by bubbling of sterile air through an in-built structure called sparger. Due to agitation and presence of proteinaceous material, foaming is (li common phenomenon. Some times foaming is

Fermentation Protocol

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also observed due to secretion of certain compound~ (metabolites) or excessive lysis (death) of the microorganism. Oils are the normal antifoam agents used to control foaming. Environmental conditions affect our lives. In summer, we use cotton clothing and air coolers. On the other hand, in winter, we keep ourselves warm by using woollen clothing and room heaters. Similarly, for the growth of each microorganism the environmental conditions are different. Temperature, pH, amount of avail-

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Friendly Fermentation

Microbes differ in their choice of habitat

Fermentation Protocol

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able oxygen are crucial requirements. Industrial fermentations are usually performed at temperatures between 20° to 40°C. The optimum pH of the medium is determined by both the microorganism and the product, for example, fermentation of Aspergillus niger for citric acid production is carried out under highly acidic conditions while fermentation of Bacillus subtilis for alkaline protease production is carried out in highly alkaline conditions. Agitation is necessary for the mixing of media and microbial cells and for the distribution of air. The levels of carbon, nitrogen and fat in the medium are maintained by addition of nutrients from time to time to standardized optimal levels. An important factor in fermentation is the maintenance of sterility. Sterile environment here means a state where microorganisms except the seeded one are absent. Microorganisms other than the one being inoculated are termed contaminants and the system is said to be contaminated if not sterile any longer. Once contamination occurs, the contaminant microorganism consumes nutrients meant for the cultured microbe, produces unwanted products, disturbs the control parameters, degrades the main product and affects the process of recovery of product. All these results in economic loss. Therefore, contamination must be avoided if commercial fermentation is to succeed. There is one sure way of ensuring sterility. !tis heat. The medium is prepared in the fermentor and sterilized

a; e,

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Friendly Fermentation

Parameters to be controlled during the fermentation Parameter

probe Foam

Foam sensor Tacometer Rotameter Measurement Thermometer Circulation of Airflow Addition of feeds Addition of acid or Outlet valve Means ofdevice control Dissolv;ed oxygen Antifoam agents Pressure gauge Speed of agitator Laboratory Sparging analysis sterile pH electrode

levels medium aIr alkali in water hot/cold

by heating with steam at 121°C for 20 to 30 minutes to kill the microorganisms present in the medium. Thermolabile compounds or compounds that are heat-sensitive, such as vitamins are sterilized by filtration through a microfilter which is capable of arresting any particle up to 0.2 micron in size. Once the medium in the fermentor is sterilized, stringent controls are exercised so that no other microorganism enters the vessel. The seed culture of the microorganism is first tested for purity and transferred to the fermentor. Water, air, nutrients and other additives are common sources of contamination. To avoid contamination, the fermentor is kept under slight pressure, sterile air is bubbled, feeds and other necessary additions are also sterilized. Inoculation and sampling of the cultures are done after sterilization of the pathway with steam.

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Fermentation Protocol

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From the account of these operations, it is easily comprehended that fermentation is a complex process, involving lot of controls, analyses, recording of data and monitoring of parameters. In true sense one has to study and understand the microorganism, its metabolism and the means to make it yield high levels of the product. Tireless, and round the clock attention is required. Some bacterial fermentations take 16 to 18 hours whereas some fungal fermentation may take even 5 to 7 days. With developments in computers, automated fermentors have now been developed. One can take advantage of the decision-making ability of the computers to maintain various parameters. Broadly, fermentation can be of two types, depending on the nature of the process. Fermentations using microorganism which do not require oxygen are called anaerobic fermentation and those requiring oxygen are called aerobic fermentation. An example of anaerobic fermentation is the production of methane (biogas). Production of antibiotics, single cell protein and amino acids are examples of aerobic fermentation. Fermentation may also be classified as solid state or submerged type. In solid state fermentation, the microorganisms are grown in trays and on agricultural residues like wheat bran, rice bran, or rice straw mixed with nutrient medium. The medium is kept moist but excess watering is avoided as it hinders the growth of the microbes. The microorganisms grow on the surface, e.g. mushroom cultivation. In submerged fermentation, the nutrient medium is either suspended or dissolved in water and microorganisms grow as a suspension. Depending

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Friendly Fermentation

on feeding needs, submerged fermentation is further subdivided into three classes: batch, fed batch and continuous. In batch fermentation, all nutrients are added at the beginning and the fermented broth is harvested at the end of fermentation. In fed batch fermentation, nutrients are added in lots at varying stages of fermentation but the fermented broth is harvested at the end of fermentation. In continuous fermentation, as the name indicates, there is continuous feeding of nutrients and continuous withdrawal of fermented broth. All living-beings und('rgo certain fixed and specific stages in their ::fe cycles. An organism is born, then it matures, in time it ages and dies. Microorganisms also

Growth curve

Fermentation Protocol

35

exhibit a comparable life cycle. The life cycle of a microorganism is divided into four main phases. After inoculation to the fresh medium, there is a brief period when growth is almost absent, this stage is aptly named lag phase. Lag phase is followed by active growth phase or exponential phase when the microorganism multiplies very rapidly thereby increasing the total cell mass. Once the growth reaches the pinnacle it remains stagnant for some time. This phase is called stationary phase or saturation phase when the multiplication ability of cells is reduced or lost. At the end, the cells lyse resulting in -reduction of total cell mass. This is known as the lYticphase. The growth and reproduction of a microorganism are the result of a series of chemical reactions that occur in an orderly manner. The nutrients are taken 'up by the microbes and then subjected to sequential chemical alterations. The process is called metabolism. The nutrient is said to be metabolized and the sequences of the reactions constitute metabolic pathways. The product obtained at the end of a metabolic pathway, is the metabolite. The metabolic pathways whjch lead to the conversion of nutrients into cell constituents are termed anabolic pathways and those which degrade organic substances to produce energy for cell functions are termed catabolic pathways. The compounds that are formed during the different stages of metabolic reactions of anabolism and catabolism are called intermediates. A precursor molecule is the starting compound which is m.etabolised to generate the end-product. Precursors can-be synthesized within the

-

36

-- -----

-- -- - - -- - -- - -- - - - -.-

- -

-- - - ---

--- ----

Friendly Fermentation

cell or supplied in the medium. Thus, metabolism of a microorganism is manipulated during the process of fermentation to give high yield of desired product. Growth and reproduction are the primary metabolic functions of microbial cells. The product produced during the primary metabolism are termed primary products of metabolism or primary metabolites and accumulation of such products is parallel to growth. Some products, for example, antibiotics start accumulating a bit late in the exponential phase of growth and much of it is accumulated during the stationary phase. Such products are termed as secondary products of metabolism or secondary metabolites. The aim of industrial fermentation is to obtain the commercially useful products in a form that can be sold. Therefore, isolation of product forms an integral part of the fermentation industry. Fermentation products are of three types: the biomass itself, the product accumulated inside the cells, i.e. intracellular products and the products secreted outside the cells which are present in the fermented broth, i.e. extracellular products. Baker's yeast, Brewer's yeast and single cell protein are examples where the biomass or cells of microorganisms are isolated as product. The cells are separated either by filtration or by centrifugation. The intracellular products are first extracted in solution by disruption of cells by either grinding, pressure pressing or by using special chemicals. The products are then recovered from the broken cells. The intracellular product present in the extract or the extracellular

37

Fermentation Protocol

Crystalisation

Drying

Isolation of fermentation products

38

Friendly Fermentation

product present in the fermented broth are isolated by a series of steps which include concentration, purification and crystallization. Each one of these steps in itself can be a multi step operation. The product crystals are dried, blended with other ingredients and packed.

lcrmcntcd Products

chemical route and the fermentation route for

'TIere has always a competition between the preparation of a been product. Besides the technical

advantages, economic aspects influence our decision when forced to choose between the two. The success of the fermentation industry in the production of various chemicals is due to the ability of selected microorganism to consistently give high yield of the desired product in a reasonable time from cheap and readily available raw materials. A microorganism should have certain characteristics for its application in industrial fermentation. It should be non-pathogenic, have the ability to grow rapidly on suitable nutrients and should possess high levels of the enzymes required for rapid formation of the product. Productivity in terms of the product formed in a given volume of fermentor should economically be the same or higher than the chemical route. Finally, it is no secret that products obtained from

40

Friendly Fermentation

__I1-/

\

\

I

1

I fermentation processes are purer and chemically active. Industrial fermentation processes were applied initially in the production offood and alcoholic beverages. Today these have been extended to many other products. The list of fermentation products is very long and is growing day by day. The value of fermentation products also seems to be growing by leaps and bounds. It has been estimated that in the year 1990 fermentation products worth 30 x 109 U.S. Dollars were produced worldwide. Antibiotics contribute a major share to this value. Other principal product categories are enzymes, organic acids, Baker's yeast, ethanol, vitamins, and

Fermented Products

41

steroid hormones. It is impossible to describe or to even merely list all the fermentation products in each category. Hence, only a few of them are dealt here to get an idea of the impact made by fermentation on the quality of our life. Food is a basic requirement for human beings. The use of fermented food or rather the use of microorganisms for food processing is a long story dating from ancient days. Over the ages each civilization has developed a line of fermented food items. The variation is because of diversity in raw materials, knowledge, perception, taste, and eating habits. Food was not thought to be a marketable item in ancient days and as a result traditional fermented food and beverages are household fermentations. Even today the microbiology and biochemistry of some household fermentations are poorly understood. Nevertheless, some of the traditional products have now become industrial products as a result of modemisation and greater demand. A good example of a fermentation product that has successfully bridged the gap from the kitchen to the supermarket is the soya sauce. The appearance of a nice :fluffyloaf of bread, spongy cakes, soft rolls or crisp cookies on the bakery shelf is no doubt very tempting. Microorganisms have a great role to play in the making of these sumptuous bakery items. The production of baked items, by and large, call for a general protocol of preparation of raw materials, dough formation, dough processing, baking and packing. The spongy nature of bakery products is a result of fermentation. The dough is prepared by mixing the ingredients.

42

Friendly Fermentation

Traditional fermented food items Product

Substrate

Microorganism

Dhokla

Bengal gram and wheat

Unknown

Idli

Black gram and rice

Bacteria and Yeast

Jalebi

Wheat flour

Yeast

Papadam

Black gram

Yeast

Indian

Oriental Oapan, China, Philippines, Indonesia) Soy sauce

Soy bean and wheat

Fungi, Yeast, Bacteria

Tempeh

S0Ybean Fish

Fungi Unknown

Vegetables

Bacteria

Sorghum Cassava

Yeast

Wheat meal and yogurt Maize

Bacteria Yeast, Bacteria

Taro corns

Yeast, Bacteria

Maize

Fungi, Yeast, Bacteria

Maize

Fungi, Yeast, Bacteria

Bagoong Kimchi South Africa Maerissa Banku

Maize

Other Tarhana (furkey) Kanga-kopuwari (New Zealand) Poi (Hawaii) Chicha (peru) Pozol (Mexico)

Fermented Products

43

Bake a cake and much more

The mixture is then processed by fermentation. This operation is called leavening. Only in exceptional cases, e.g. when a high concentration of sugar is present, is the leavening process done non-fermentatively. The yeast used for dough fermentation is Saccharomyces cerevisiae, popularly known as Baker's yeast. Baker's yeast is available either as yeast cake, yeast cream or active dry Baker's yeast. It is mixed with

44

Friendly Fermentation

dough and the dough is incubated at temperatures, between 28° to 32°C. It is left for 4 to 12 hours during which period the pH ranges between 4-5. The concentration of the yeast during leavening is between 1to 6 per cent depending upon the type of :flour and the product desired. Similarly, the fermentation temperature, pH and the time too varies with the raw materials and the type of product. Higher concentration of yeast is always avoided as it gives undesirable results. During the leavening process, the yeast breaks down the carbohydrates in the dough and generates carbon dioxide which more than doubles the size of the loaf giving the spongy structure. Other products of fermentation contribute to the flavour of baked items. The processing of dough can also be accelerated by the addition of enzymes such as amylase which degrades the starch present in the flour to glucose. Glucose is then consumed more easily by the yeast A mixture of yeast and bacteria (lactobacillus) is also used specially when we want to develop a sour taste. Baker's yeast is produced fermentatively on a very largescale and sold to the bakers. The cheese industry deserves special mention here. Cheese is prepared by using an enzyme called chymosin or rennin. Rennin coagulates casein the constituent protein in milk. The process of cheese making is a multistep one. The microbial fermentation is encouraged in two steps; culturing of milk with bacteria and ripening of cheese. During the culturing, the lactic acid bacteria convert lactose (milk sugar) to lactic acid. This conversion has an impact on the gross composition of cheese. The introduction of microorganism during ripening step has many advantages. It enhances :flavour

_

-

..

------ - -

---

--------

- -----

--

-

Fermented Products

45

Say cheese!

due to metabolites produced by the microorganisms. The carbon dioxide produced by the microbes is trapped in soft cheese and gives the typical eyed structure. Soft centred cheese is a result of microbial degradation of casein proteins at the centre of the cheese and blue cheese is the result of microbial growth on the surface of the cheese. The different types of cheeses produced by utilising different microbes and fermentation processes are Cheedar, Gouda, Blue, Cottage Cream, Swiss and Mozzarella.

46

Friendly Fermentation

Pretreatment of milk J,

Milk culturing with bacteria J,

Clotting of milk by rennin J,

Collection of raw cheese J,

Fortification of cheese .1

Ripening of cheese J,

Packing More than 30 major fermented dairy foods are prepared in different parts of the world today. The very familiar names are yogurt, sour cream, cultured buttermilk, acidiphilos milk and shrikhand. Yogurt is a very popular fermented dairy product It is a semisolid product resulting from fermentation of heat-treated milk. The fermentation is performed by two bacteria (namely Lactobacillus delibrueckii and Streptococcus salivarius). In household fermentation the taste of yogurt varies due to variation in both milk quality and type of cultures used. However, commercial production involves stringent control of conditions at all stages so that an uniform texture and taste is obtained repeatedly. The lingering soft and sweet taste of chocolate is yet another gift of fermentation. Chocolate in any form, be

Fermented Products

47

it chocolate milk, chocolate ice-cream, chocolate bars, chocolate cookies, or cocoa drinks is liked by all age groups. People like chocolate not for its nutritional value but for its flavour. Cocoa and chocolate are produced from cocoa beans, the seeds of the plant Theobroma cocoa. The seeds of the ripe fruits are subjected to fermentation followed by drying. The operation is called curing of cocoa seeds. Fermentation is carried out primarily to induce biochemical changes within the seeds which leads to the formation of precursor of chocolate aroma, flavour and colour. Another reason, though secondary, is to get rid of the pulp surrounding the ~eed. In fact, untreated seeds do not develop the chocolate flavour when processed into chocolate. The cured seeds are called beans and they develop rich colour and flavour upon roasting. The fermentation is carried out in boxes or in heaps covered with banana leaves. Microorganisms in the environment contaminate the pulp during handling and convert the sugars present in the pulp. The fermentation process goes on for about 4 to 6 days. Pickling of vegetables thereby preventing spoilage on one hand and making the vegetable tastier on the other, is an example of microbial intervention that is profitable to us. Pickles of mango, chilli and lemon, are known to us. Many of us may have raided mother's pickle-jars as children. But not many of us realize how fermentation helps pickling. Pickling involves anaerobic fermentation using the naturally occurring bacteria. Some enzymes present in the fruit and vegetable help to provide nuttients to the fermenting bacteria. Pickling

48

Friendly Fermentation

MOTHE.RS' QUALiTY PRODUC

PIC- k.L-E.

Making pickles has become an industry throughout the world

of cucumbers and cabbage is a big industry in the western world. The product of cabbage fermentation is Sauerkraut. Kimchi is a Korean product produced by fermentation . of either radish, cucumber, cabbage or green onlOns. Lactic acid producing bacteria grow in salted vegetables and produce organic acids which lower the pH

Fermented Products

49

of the mixture. The combined action of the salt and the acid lowers the activity of the enzymes that are responsible for the breakdown of the vegetable tissue. At the same time it inhibits changes in the tissue preventing growth of undesired microorganisms. Microbes have proved their expertise in producing gums too. Gums are the sticky materials that give viscous solution when dissolved in water. Chemically gums are polysaccharides. Sometimes microorganisms secrete such polysaccharides outside the cell and then, these are termed exopolysaccharides. Microbes secrete these compounds either for protection by forming capsules around the cells or as a reserve food material. Various bacterial, fungal and yeast cultures are developed for this fermentation. Xanthan gum pullulan, and Baker's yeast glycan are two examples of widely used microbial gums. Xanthan gum is a popular microbial gum. This gum is widely used as a thickening agent and in the preparation of meat like gels in food industry, as lubricant in drilling oil wells, in textile printing and dyeing, ceramic glaze manufacture, polishing agent and rust-curing agent. Down the ages, a good deal of man's food has come through microbial agency. Bread, cheese, alcoholic beverages, cocoa, pickles, soyasauce and vinegar to name a few. These are products that are enjoyed as a result of microbial activity. However, there are some instances where the producer microbe is consumed together with the product. Curd, is a good example of this.

50

Friendly Fermentation

'"

C.UIt.:D

All these and more

It will not be irrelevant to cite here the alarming situation posed by human population explosion. Another decade of sustained growth rate would no doubt force us to envisage a future where starvation would be a stark reality. The grave problem cannot be fully met by increasing areas under cultivation, improved Yield, better pest control and storage. Studies indicate that microbial biomass could have a fair chance to bridge the shortfall in food production and thus mitigate the

Fermented Products

51

food crisis to some extent. A close look will tell us how. Microorganisms such as algae, bacteria, yeast and fungi have been considered as source of protein. Culture of microorganisms for food started at the end of First WorId War as a result of food crises and reached a peak in the 1970s. The term "single cell protein" (SCP) was coined to describe the microbes as protein sources. Various microorganisms are grown on different substrates for SCPoThe choice hinges mainly on the availability of raw materials, nutrient value of the cells, contents of essential amino acids and toxicity of the microorganism. They have not been widely accepted due to taste and food preferences of consumers. Maximum attention has been paid to yeast as a source of microbial food. They are not toxic and are non-pathogenic and hence best acc~pted by consumers. Surplus brewer's yeast finds use as animal feedstuff and for therapeutic purposes. Their generation time (time required for doubling of the cell population) is 2 to 5 hours. Their protein contents is slightly low (about 60%)but they contain essential amino acids and are rich in B-group vitamins. Bacteria yield highest biomass due to their short generation time (about 1 hour) compared to other microorganisms. The protein content is also high, it has been found that several bacterial species contain all the essential amino acids necessary for us. Research has also revealed that symbiotic culture of yeasts and bacteria give improved crops of microbial food. However, bacteria are not popular as protein source because of their pathogenic nature. Their small size causes diffi-

52

Friendly Fermentation

SPirulina tablets are now available in medical shops

culties in harvesting. But even then, various bacterial species are grown on bagasse and methanol and the biomass is used to produce animal feed. Algae have many positive points for being considered as a candidate for biomass production. Algae use atmospheric carbon dioxide as the carbon source, they

Fermented Products

53

can be grown in ponds or lakes, and are easy to recover. :md have low cost of production. Extensive research has been carried out on various algal species. The most promising ofthem are ChIorella and Spirulina. Both contain amino acids and vitamins of interest to us. Spirulina tablets are now available and are ideal for patients suffering from vitamin deficiency and for those requiring protein supplements. Scientists speculate that ChIorella, could be grown under controlled conditions in spaceships and thus could not only generate edible biomass but also oxygen. In theory at least, it is easy to vote for ChIorella as a candidate that can make the spacecraft self-sufficient in food and oxygen. Of course, the theory needs proper evaluation before it can be recommended as standard practice. But the theory itself is enough to highlight the potential microbes have. Fungi are not very popular as biomass because of their long generation time (5 to 12 hours), low protein content (15%)and because their cell walls are difficult to break. The last criterion makes it difficult for us to harvest chemicals inside the fungal cells. "Health is wealth". The age old axiom still holds true. Microbes are ubiquitous. A lot of them are not friendly to us, as soon as they find a susceptible living host they attack and diseases break out. Few such diseases are tuberculosis, throat infection, jaundice, influenza, and amoebiasis. These diseases are caused by our microbial enemies. But friendly microbes have stretched out their helping hands, so to say, in rescue and have given

54

Friendly Fermentation

us medicines to fight the ailments. There are some diseases that are not caused by microbes. These are manifested due to certain congenital abnormalities or physiological dysfunctions. Diabetes, arthritis and hypertension are examples of this type of disease. Interestingly enough, microbes provide a cure for these too. Medicines are classified according to their biological activities. Antibiotics are compounds that kill bacteria, antifungals kill fungi, antiparasitic agents kill parasites, anticancer agents are used in treatment of cancer and insecticides kill insects. Among these, antibiotics have highest market value. Pencillin was discovered by Alexander Fleming in 1928, and its clinical application was the result of the pioneering work by E. B. Chain and H.W. Florey in early 1940s. Selman Waksman discovered streptomycin in 1943. We have been very lucky that these antibiotics discovered quite early turned out to be powerful life saving drugs with low mammalian toxicity. Mer the discovery of these two antibiotics, thousands of other antibiotics have been obtained but only a few hundred have found medical application . . The antibacterial action of an antibiotic depends on its chemical structure. By altering its chemical structure, one can improve its antibacterial activity. This concept is successfully exploited to produce more potent antibiotics. Such antibiotics are called semisynthetic antibiotics since chemical SYnthesis is only a part of the total process. For example, pencillin is a fermentatively produced antibiotic and ampicillin is a semisYnthetic penicillin derived from pencillin.

Fermented Products

55

(a)

(b)

(c) (a) Alexander Fleming (b) Howard W Florey (c) Ernst B. Chain

56

Friendly Fermentation

But, antibiotics are only one part of the story. Steroids tell another tale of microbial versatility. Steroids are complex chemical substance found in plants and animals. In 1949 it was demonstrated that cortisone, a steroid, produces dramatic effects in treatment of rheumatoid arthritis. This discovery paved the way for extensive investigations ofvarious steroids as therapeutic agents. Today a large number of steroid hormones have been identified as valuable therapeutic agents in the treatment of arthritis, rheumatism, leukemia, hemolytic anemias and many other diseases. In the early 1950s it was discovered that certain fungi can cause chemical changes in steroidal substance obtained from plants or animals and thereby transform them to therapeutically active steroids. To transform steroids a microorganism known to be capable of producing the designed chemical change is grown in suitable medium under optimum conditions. Mer adequate growth has occurred, the steroid is added in the culture, and the designed chemical transformation occurs. The steroid so formed is then recovered for purification and may be processed for the market. Other health care products include antifungals, insecticides, antiparasitic agents, alkaloids, vaccines and insect toxins. They are used widely in treating various health related problem which are very frequently confronted by us. Commercially useful enzymes are yet another useful gift of microbial fermentation. Enzymes are powerful catalysts, they can accelerate the rate of reaction over a million times. Enzymes have become very handy in

Fermented Products

57

Some antibiotics produced by fermentation Compound

Producing organism

Disease controlled

Antibacterial antibiotics Penicillin G Streptomycin Tetracycline Chloramphenicol Erythromycin Rifampicin Gentamycin

Penicillium chrysogenum Streptomyces griseus Streptomyces aureofaciens Streptomyces venezuelae

Pneumonia, Meningitis, Syphilis Tuberculosis Cholera, Plague

Streptomyces erythraeus Streptomyces mediterranei

Bronchitis

Micromonospora purpurea

Typhoid

Tuberculosis, Leprosy Urinary tract infection

Antifungal antibiotics Griseofulvin Nystatin Hamycin

Penicillium griseofulvin Streptomyces noursei Streptomyces p imp rina

Ring worm infection Candida infection Candida infection

many industries because of their unique properties of specificity, ability to perform the reaction under normal conditions of temperature, pressure and pH, and minimum by-products formation. In the old days, enzymes were obtained from plant and animal tissues. But such sources could not meet increasing demands. The ability of microorganisms to synthesize enzymes and cultivation of microorganisms on large-scale has made fermentation the method of choice for production of enzymes on a large-scale. Many yeasts, fungi and bac-

58

Friendly Fermentation

Fermented Products

59

teria are used for production of various enzymes. In order to improve the microbial strain, genetic engineering is employed to transfer a gene responsible for synthesis of a desired enzyme, from one microorganism to another or from animals to microorganisms. The aim of using such a technique is to obtain the desired enzyme in greater quantities and at a lower cost. Each enzyme catalyses a specific reaction. This property finds many applications, for example, amylases convert starch to glucose. This ability of amylases has enabled their use in production of glucose from starch. Bakeries use it for the hydrolysis ofgrain starch to enhance leavening, while the textile industry uses it for the removal of starch during the desizing process. It is used in detergents to aid the removal of starchy stains from clothes. It is used in animal feed, and as a digestive aid. Similarly, proteases hydrolyse proteins to give simpler smaller fragments (polypeptides) and even amino acids. Proteases active under alkaline conditions are used widely in detergents for removal of organic stains from clothes. In fact detergent enzymes constitute about 40 per cent of the total market value of enzymes. Polysaccharide degrading enzymes such as amylases and cellulases are used in breweries and wineries to accelerate fermentation. Candies are sweet, and this sweetness is courtesy fructose which is twice as sweet as glucose. Glucose obtained from starch can be converted to a syrup containing equal quantities offructose and glucose by glucose isomerase. This syrup is used to prepare candies and as a substitute for cane sugar.

60

Friendly Fermentation

Many other useful products such as cheese, ice-cream, coffee and fruit juice require processing with enzymes obtained by microbial fermentation. Enzymes have clinical applications too. Enzymes catalyse various metabolic reactions in living cells. Therefore, it would not be surprising to know that enzymes are used for treatment of various bodily disorders. Many digestive enzyme are secreted by the pancreas. Some children and elderly people suffer from deficiency of such enzymes which results in incomplete digestion of food. Fermentatively produced enzymes such as amylase and lipase, which degrade starch and fats, respectively, are used as digestive aids. Other examples of fermentatively produced enzymes include streptokinase used for dissolution of blood clots in the circulatory system, L-asparginase as an anticancer agent and lysozyme as healing agent. Besides the use of enzymes as medicines, some fermentatively produced enzymes are used in industries for production of drugs or drug intermediates, e.g. Escherichia coli produces penicillin acylase which is used in preparation of ampicillin. Another important application of enzymes is in diagnostic aids. Estimation of glucose in urine and in blood which is particularly significant in the diagnosis of diabetes and its control is done very rapidly by using the enzyme glucose oxidase which in turn is usually produced by fermentation. Microorganisms play an important role in the life of plants too. The story is again of friendship and enemity .. Plants suffer from various diseases caused by plant pathogenic microorganisms. Nevertheless, the story of

61

Fermented Products

Some useful fennentative enzymes Enzyme Amylase Protease Cellulase Pectinase Rennin (Chymosin) Lactase lipase Glucose Isomerase

Function

ApPlication

Hydrolysis of starch to Bakery, Detergent, glucose Textile, Brewing, Glucose production. Hydrolysis of proteins Detergent, Silk, Wine, to amino acids Leather, Coffee Hydrolysis of cellulose Textile, Brewing, to glucose Detergent Fruit juice, Wine Hydrolysis of pectin Cheese limited hydrolysis of casem Hydrolysis of lactose Baby food, Ice-cream Hydrolysis of fats Detergent Conversion of glucose Confectionary to fructose

positive co-operation is equally fascinating. Compost prepared from cowdung and agricultural waste by anaerobic fermentation in a covered ditch is a familiar story. Besides these, various agricultural aids are now produced by industrial fermentation. One of such products is gibberellic acid. It is a plant hormone and is used in propagation of cash crop like grapes. Gibberellic acid is produced by the fungus Gibberella jujikuroi when grown under nitrogen limited medium. Herbicides like Bialophos are obtained by fermentation of Streptomyces species. Bialophos does not have non-selective phytotoxic effects like antibiotics nor does it create environmental pollution like chemical herbicides. Yet another avenue of supporting agriculture is the use of microbial biomass or fermented broth, (after removal of product) as a fertilizer. If the mycelia or broth is from antibiotic fermentation then there is the added advantage of pro-

62

Friendly Fennentation

tection from certain diseases due to residual levels of antibiotics. The emphasis has, no doubt, been on several commercially valuable, fermentatively obtained products. In addition to these, the microbial biomass itself has found use as a bio-catalyst in various chemical reactions. This is another novel and innovative application. Microbial cells contain many enzymes that catalyse chemical reactions. Thus, the cells can be used as a

----

-----~-

Fermented Products

---------~------

---

--~----- - -.

63

single source for performing many chemical reactions. Such catalytic transformations are termed biotransformations. Use of microbial cells as a catalyst has an added advantage. Factors necessary for enzymatic reactions and extended stability of some enzymes are inherently present in the bio-system. Biotransformation is exploited for production of useful compounds. For example, the production of vitamin C from glucose by the strictly chemical route is a six step process. Strains of Erwinia herbicola can enable conversion of glucose into vitamin C in two steps. An important advance in enzyme technology has enabled scientists to increase the product yields manifold. This involves immobilization of the enzymes. Immobilization is a process in which microbial cells or the enzymes produced by them are encased within solid support materials. The advantage of this process is that the enzymes are not lost with every batch processed but may be retrieved for re-use. Immobilized cells are used both as bio-catalysts and in continuous fermentation. Last but not the least is, alcoholic fermentation. This is fermentation of course, but the end-product here is alcohol. Alcoholic fermentation is the cornerstone of all 'spirited' beverages. Alcoholic drinks are intoxicating. Consumption of alcoholic drinks has been a much debated issue over ages. Though the adverse effects of too much alcohol on the human body are well-established, ,theproduction and consumption of alcoholic beverages have been increasing. In almost all civilisations, the history of microorganisms converges at a common point - alcoholic beverages. It is said that wine is as old as human

64

FriendlyFennentation

civilisation. Today we have modem well-established large-scale and small-scale breweries and distilleries. Wine making or brewing at home is still practiced though it is not too common. The basic concept in alcoholic fermentation is the conversion of sugary and starchy raw materials into alcohol and carbon dioxide by using yeast such as Saccharomyces cerevisiae. There are more than a thousand strains of yeast, each with different characteristics. The breweries maintain and protect their special strains. Brewer's yeast is also available for household fermentation. The variations in alcoholic drinks depend on the fermentation process that the raw material undergoes, yeast strain, and control of production of metabolites that give colour, flavour and taste. Production of various organic compounds are monitored and controlled to avoid unwanted taste and flavour. Agricultural products such as barley, malt, hops, rice and fruits are used for production of alcoholic drinks. Beer is produced from malt, and barley. White wine is produced from crushed grapes after removing the skin an~ other insoluble parts ..Red wine is made from crushed purple grapes. The characteristic red colour is due to the extraction of pigments from the grape by the alcohol formed. Champagne is sparkling wine containing dissolved carbon dioxide. Fruit wines are made from a wide range offruits such as pomegranate, pit fruits, and berries. Brandy is the distillate of wine, while whisky is the distillate from fermented mash of grains and rum is the distillate from fermented sugarcane molasses.

Fermented Products

65

• Filter

Filter

Wine making

66

Friendly Fermentation

The monitoring of fermentation conditions, incorporation of other nutrients, operational steps, distillation protocol and ageing process varies from one type of alcoholic drink to another. Microbial enzymes such a~ amylase, cellulase and hemicellulase are used to accelerate fermentation and also to degrade the residual carbohydrates after fermentation. The latter makes for easier filtration.

1110

D

Brazilians opt for 'Gasohol' (gasoline + alcohol)

Fermented Products

67

Alcohol as a beverage is well-known but the role of alcohol as a chemical is often overlooked. Alcohol is used in many industries as a solvent. Distilled pure alcohol (ethanol) is also used as a chemical feedstock. For this purpose alcohol is obtained by fermentation of cheap raw materials like molasses. The alcohol obtained at the end of fermentation is then separated by distillation. Alcohol has been used in Brazil as automobile fuel instead of petrol. This is possible as Brazil has sufficient land and water resources for extensive cultivation of sugarcane. Besides alcohol, various chemicals such as amino acids, organic acid, nucleotides, lipids, fatty acids, vitamins, glycerol and vaccines are also produced by fermentation.

.(po!dnu lofwnfd

'TIe

from its modest and obscure beginning. It has has comeorigins a longwhen way seenfermentation many phasesindustry from primitive man used microorganisms without any systematic knowledge to this date when we know a good deal about them. The progress of the fermentation industry depends on three aspects: improvements in existing fermentation pro.cesses,. development of newer fermentation products and unrelenting exploration of newer more potent microorganisms. Increase in production at a lower cost is the moving force. Development of high performance microorganisms by selection, mutation and genetic manipulation, finer control of fermentation processes, cheaper raw materials, better fermentors, automation with the help of computers, and newer methods for isolation of products are different avenues for improvements in a fermentation process. The techniques of genetic manipulation hold promise for making microorganisms compelled to produce compounds otherwise generated only by mammalian and plant cells. Genetic engineering has been applied

Looking Forward

69

to fermentation processes to enable bacteria to use a wider variety of feedstocks, to biosynthesize new products to accumulate intermediate metabolites via blocked pathways, or to increase product yields by enhanced synthesis of special enzymes. In many commercial fermentation processes the cost of raw materi.: als is the most expensive component. Industrially useful bacteria can be modified to use cheaper feedstock by genetic manipulation. Research is being carried out to achieve better strains of microbes in laboratories spread allover the world. Scientists have relied heavily on recombinant DNA technology to obtain better breed (s) of microbes. This technology owes its origin to a key discovery - that of the wonder enzyme restriction endonuclease. This can be likened to molecular scissors with which DNA molecules can be 'cut' at certain sites. These enzymes are naturally found in many bacterial species. The original use was to cut up any parasitic viral nucleic acid entering the bacterial cells. However, in the hands of scientists, the restriction enzymes became potent tools for tailoring genetic material to suit our purposes. Another enzyme, DNA ligase helps in joining pieces of DNA Once the tool for genetic engineering became available, it became comparatively easier to get on with the job. Genes responsible"for the synthesis of the desirable enzyme or antibiotics or those governing desirable traits were identified in the donor' species. Plans could now be drawn up to transfer these genes to the recipient. Thus the entire long-drawn sequential biochemical pathway for the chemical synthesis of a compound could easily be shrunk to essentially an one-step process. For exam-

70

Friendly Fermentation Foreign DNA

Restriction enzymes cut st specific point

'/

E.coll

,,' ., •. ';"'i ~

-

,

'

..

'
t2 -

...'

4-' ·;;l.

Cells divide

'

\j

..

Genetic manipulation has empowered scientists to design microbes

pIe, the production of vitamin C initially involved a two-step fermentation of the feedstock. It also necessitated the use of two bacterial species - Erwinia herbicola and Corynebacterium. Genetic engineers could, however, de~ign a shortcut. They 'borrowed' the gene responsible for the production of vitamin C from one of the two species and introduced it into the other species.

Looking Forward

71

The second species now had both the genes and thus was an unique microbe. It could carry out single-handedly (so to say) the job that used to be a two-step process involving two microbial species. Such is the potential of genetic engineering. It is providential that Nature has provided scientists not only with the 'molecular scissors' but also with the means to 'ferry' genes out of the host cell and into the recipient. For this, scientists use plasmids. Plasmids are circular extra chromosomal DNA molecules of small size (inthe average range of5-10kilobases compared with the size ofan average chromosome which may be several thousand kilobases). They are found in many bacteria, and sometimes there may be more than one plasmid in a single bacteria. Several of the bacterial plasmid carry genes for drug resistance. Plasmids which can best serve the role of a molecular taxi is one that is small in size, exist in high copy number per cell and contain genes of drug resistance. Vrruses offer another 'ready- to-use' natural route to ferry DNA segments. In nature viruses readily enter host cells and the viral genomes easily integrate, with the host's genetic material. Thus, the virus was almost perfect in its role as a molecular ferry which scientists could manipulate to carry a genetic cargo of their choice. The only factor that scientists must guard against is the inherent pathogenicity of viruses. Essentially, the plasmid or viral DNA is cleaved with restriction endonucleases at a site where the cut will not hinder normal functioning. The piece of foreign DNA, also cut by the same enzyme, is then inserted into the viral (or plasmid) DNA. The ends are complementaty and they pair up easily.

72

Friendly Fermentation

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by the enzyme DNAligase. This results inthe formatio~ These are of annealed a vehicle DNA whIch now contains a piece of foreign DNAas an integral part ofitself. Ithasnowayof discriminating against the inserted foreign DNA since it recognises it as part of itself. When this DNAenters a host cell it reproduces just the way it would have done normally. The foreign DNA, which by itself

able tonotsurvive, let would have been alone reproduce in the Cleave as you please host cell thus gets a free ride and an assured chance of survival. Scientists get a transgenic organism. Fermentative production of insulin used in treatment of diabetes and of chymosin used in cheese production are recent successes. Transgenic microbes have helped the pharmaceutical industry tremendously. Just one example will help to highlight the point. Insulin, the hormone that keeps blood sugar under control in normal people, is used as a medicine by those suffering from diabetes mellitus. This is a condition characII

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LookIng Forward

73

terized by the presence of sugar in abnormal amounts in the blood and urine. In the early days of the pharmaceutical industry, insulin was sourced from the pancreas of freshly slaughtered cattle. The amount obtained was quite meagre and there was considerable unwillingness among the recipients to using animalsourced insulin. Scientists therefore needed a new, cheap and plentiful source of insulin and were compelled to find an alternative. Luckily enough, by then, the concept of recombinant DNA technology had gained ground. Scientists chose a normal mouse and also a bacterial species. These were to be the donor and the recipient respectively. Mice produce insulin and thus, obviously possess the gene responsible for its sYnthesis. The next step was time-consuming. They had to identify the gene responsible for insulin sYnthesis from amongst the thousands of other genes in the genome. Then, they had to selectively remove the gene of choice and clone it. The plasmid DNA had to be readied to accept a copy of the mouse gene. Once the mouse gene for insulin was attached to the plasmid DNA the result was a chimeric DNA The chimeric DNA was part mouse and part plasmid in origin. The chimeric DNA was then introduced into the bacteria. This is when the fun began. Each time the bacteria divided, so did the chimeric plasmid. Very soon the scientists had a colony ofgenetically engineered bacteria, each carrying a copy of the mouse gene for insulin. Each time the foreign genes expressed, they helped produce insulin. In effect, scientists had a factory producing insulin inside the test-

74

Friendly Fermentation

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Looking Forward

75

tube. Furthermore, this insulin was free of the stigma of animal sacrifice. It also did not need to be tediously purified and was copiously available. A similar protocol using the human gene for insulin was also successfully demonstrated. The product available is marketed as 'Humulin' (Le.Human Insulin). A similar success story is that of chymosin production which previously used to be obtained from calves. The use of recombinant DNA technology has made the in vitro production of enzyme easy. Extremophiles are another group of microorganisms which have been exploited only to a limited extent in industrial fermentations. Fermentation, bioconversion and enzymatic catalysis constitute the answer to the current pollution crisis in the world. Municipal and industrial waste water treatment by employing genetically engineered bacteria has been proposed recently. Toxic and undesirable metals can now be sequestrated from waste water by the bacteria skilled in absorbing metals on the cell surface or by intracellular uptake. Metallothioneins are peptides that are produced in response to increased metal concentration in body. Human metallothionein has been cloned into E. coli and it has been proposed that these engineered bacteria be used as an immobilized cell system for removing metals from waste water. Recombinant DNA technology has helped in engineering bacterial species which would flocculate better thereby aiding in removal of more biomass for disposal or recycling purposes.

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Friendly Fermentation

The benefits offered by the lowly microorganisms to man are many. Modem developments are indeed spectacular and exciting. Initiating and sustaining friendship with microorganisms is the most natural way of living on this earth.

Glossary Centrifugation - Separating molecules by size or density using forces generated by a spinning motor. Clone - Individuals having exactly the same genetic makeup. Cure - A crucial step in preparation of fermentatively derived food during which the impregnated microbes are allowed to work on aiding the development of characteristic flavour and taste of the food stuff. Express - Refers to the manifestation of the characters controlled by the introduced genes following recombination DNA technology in a living cell. In vitro - Literally 'in glass'. It refers to any biological experiment carried out outside the body. Non-pathogenic - Inability to produce diseases. The term can be applied to bacteria, fungi, viruses and other microbes. Phytotoxic - Anything that is poisonous to plants. The term consist of two parts. The prefix 'phyto' denotes plants and 'toxic' means pOIsonous. Polysaccharides - Carbohydrates produced by combination of many molecules of simple sugars. Proteases - An enzyme that cleaves peptide bonds which link amino acids in protein molecules. Transgenic - A plant or animal in which foreign DNA (a transgene) is stably incorporated.

I FOUNDATIONS

OF BIOTECHNOLOGY SERIES

Fermentation is a household word. Its origins are lost in the murky mist of time, but its future is as bright as the summer sun. Curds and chocolates, alcohol and medicines, the list of fermentation products is unending. We use all these all the time without any conscious thought about the underlying microbial activity that makes fermentation possible. This lucidly written and profusely illustrated book is particularly meant for school students of an impressionable age. It highlights the role our invisible allies, the microbes, play in making our lives more comfortable and, in the process, introduces the readers to the world of friendly fermentation. ABOUT THE.AUTHOR Dr Jaiprak.ash G. Shewale obtained his Ph.D. from National Chemical Laboratory, University of Pune, and Diploma in Management from All Indi.~. Management Association, New Delhi. At p'resent he is Group Manager at R&D of Hindustan Antibiotics Ltd (HAL), Pimpri. During his stay in the US, he has contributed to the deciphering of amino acid sequences of -several proteins of physiological importance. At HAL, he has played a key role in the development of immobilized penicillin G acylase and 6-APA technology and has developed other important enzymes useful in the interconversion of penicillins and cephalos- porins. Dr Shewale has over 60 publications and 11 patents to his credit. He is a fellow of Maharashtra Academy of Sciences. He is also a recipient of Meritoriallnvention Award from National Research Development Corporation, New Delhi. Besides Friendly Fermentation, he has authored another popular science book entitled 'Enzymes Everywhere'.

J88172"361853 ISBN: 81-7236-185-8