[J .G. Shewale] Friendly Fermentation(BookZZ.org)

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National Institute of Science CommunicationDr K.S. Krishnan Marg

New Delhi 110 012,lndia

Friendly Fermentation

].G. Shewale

© National Institute of Science Communication

First Edition: July 1998ISBN: 81-7236-185-8

Foundations of Biotechnology Series

Book No. 2Series EditorVolume Editor

Cover Designlllustrations

Production

Printing

S.KNag

Srilekha Bhattacharya and Dr Sukanya DattaPradip BanerjeeSushila Vohra, Neeru Vijan, J.M.L. Luthra,Malkhan Singh, Harjeet Singh and YogeshKumar

Shiv Kumar Marhkan, Rohini Raina,Dharmender Mohan, Ashok Kalra, Neeta Sahneyand Suresh Kumar Sharma

S. Bhushan, S.C. Mamgain, G.C. Pore!, Tika Ram,Rajbir Singh, Rattan Lal and Om Pal

Designed, Printed and Published byNational 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 toyield products useful to mankind.

Fermentation, the age old process of making bread and wine,is now gaining teeth by the introduction of biotechnology. Therange of products obtained by fermentation spans the spectrumfrom curd to in vitro synthesized insulin. Proposals for usingvarious microbes as source of food and in cutting down pollutionare also coming up.

The National Institute of Science Communication (NI~COM) is bringing out popular science books under a new seriesentitled 'Foundations of Biotechnology' as a part of the projecton 'Dissemination of Biotechnological Information' sponsoredby the Department of Biotechnology CDBn, Govt. Of India.

This venture is yet another step taken by the Institute tomake 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 awarenessof biotechnology down to the school level. By introducing thevast subject of biotechnology especially to children of classes VIIto X, it is hoped that many would be inspired to take up thissubject for an advanced study as it is the time for them to decideupon their career options.

Keeping up with its major mandate of disseminating scientific information to large masses, NISCOM has undertaken thisvery important venture of popularizing the basic concepts essential to the understanding of sophisticated biotechniques. I amconfident that these books would enhance the reader's curiosityto know more"about this interesting multidisciplinary subject, so

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

M~~(Manju Sharma)

Secretary to the Govt. of IndiaDeptt. of Biotechnology

PrefaceFermentation processes yield products useful to all

of us in everyday life. The bread we eat, medicinalproducts we take and many beverages that we drink areall results of fermentation through the application offriendly microorganisms or microbes. Fermentationdates back to times immemorial. It predates our knowledge of microbes.

Though fermentation has been practised for ages,rapid scientific and technological advancements havecome about only in the last century. Fermentation is amultidisciplinary technology wherein knowledge of biology, chemistry, and physics, is involved in one way orthe other. Needless to say that advancements in theseareas have brought fermentation to the forefront. Theknow-how is now so advanced that it is possible tomanipulate the transfer of genes to evolve newer microbes that produce the desired product.

The purpose of writing this book is to share somebasic knowledge about fermentation processes andproducts with young students. We owe a lot to microbesfor our good living. If the excitement of progress in thisarea would inspire some of the readers to takeup research as a career, this book would have served itspurpose.

- ------- ----- -------- - - -------

AcknowledgementI profusely thank Mr S.K Nag, Chief Investigator,

DBT Project and Head, Popular Science Division ofNISCOM for extending an invitation to write this book.I appreciate his suggestions and efforts in bringing outthis book in presentable form. Thanks are also due toDr Sukanya Datta and Ms Srilekha Bhattacharya forediting 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 photographsand for constant encouragement.

Dr C. Siva Raman, FNA and Prof S.R Tophkhanehave read the manuscript. Their critical comments andsuggestions have made the book scientific enough forthe students. I thank them for their concern.

- I thank Arona Deshpande and also the brilliant artists headed by Mr P. Banerjee at NISCOM who havecreated 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

ContentS

From Times Unknown

...1

Omnipresent Microbes

...7

Fermentation Protocol

... 20

Fermented Products

... 39

Looking Forward

... 68

Glossary

... 77

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

1rol11 ril11es

Unknown

~rmentation. The word is familiar. Plenty ofmouthwatering eatables such as fluffy bread, delicious cheese, yummy chocolate, tasty sour yo

gurt (curd), which we enjoy eating, are all gifts offermentation. A wide variety of fermentatively obtainedfoodstuffs are available nowadays. These are the outcome of large scale industrialization of fermentation,particularly in developed countries, and are no longerlimited to the home as was the case in the past Ofcourse, some products such as curd and idli are stillproduced in homes.

The term fermentation is derived from the Latinverb fervere which means 'to boil'. It describes theemission of carbon dioxide due to the action of yeastson fruits or malted grain. Thus, fermentation is a process in which chemical changes are brought about inorganic compounds through the activities of enzymessecreted by or present in microorganisms or microbes.In other words, specific chemical transformations arebrought about in the presence of microbes. Fermenta-

2 Friendly Fermentation

tion processes are thus the result of a sequence ofchemical changes since all biological systems obey thelaws of chemistry.

Fermentation existed even in prehistoric days. Itmay never be known who first obsetved the phenomenon of fermentation. Primitive man who obsetved thatnatural changes improved the quality of stored foodmust have at some point of time woken up to the realityof fermentation. This must have been a process ofpainstaking trial and error.

Microbes are the earliest of the living forms thatevolved on the planet Earth an estimated 2000 millionyears ago.

Microbial contamination of food material generallymakes it inedible because of foul odour and unpalatabletaste. Consumption of such spoiled food results in sickness and even death. However, in exceptional case, thefood material becomes more appetizing. Indeed, it isthe obsetvation of such desirable changes broughtabout by microbial intetvention that has led to today'sfermentation industry.

Fermentation is the first biotechnological processman developed. Fermented food is acceptable to usbecause the nutritive value of food is retained or improved. The physical and chemical characteristics ofthe food are altered during fermentation. But with correct microbial intetvention food value is not lost and thefood is not spoiled. As a result, fermentation has beenand is still the most important method of presetvingfood or improving its nutritive value.

Primitive man knew the methods to prepare alcoholfrom cereal grains and fruits. What he did not know

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

From Times Unknown 3

were the details of the biological changes broughtabout during fermentation. Nonetheless, without evenbeing aware of the role of microbes, he improved thefermentation processes to obtain alcoholic beverageswith pleasant flavours. He learnt all this through painstaking experience.

The history of many fermented products dates backto ancient times: Chinese and Indian records go backto 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. TheEgyptians discovered that ifdough was allowed to curefor several hours, it expanded when baked, resulting inspongy light loaves. Bread was a staple food of theancient Egyptians and was often given in lieu of wages.

The documentation of the science of microbiologyand 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 vonLeeuwenhoeck was to build microscopes. His first mi-

Ancient Egyptian painting depicting wine production


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

From Tunes Unknown 5

croscope was a simple one, with lenses of short focallength which he ground in his spare time. With it heobsetved single celled organisms in a drop of pondwater. In 1676, he introduced the world of micro organisms to man.

The first person to suggest the role of microscopicorganisms in fermentation was the French scientist L.].Themard in 1803. His legacy was carried forward bythe eminent French scientist Louis Pasteur who contributed to the significant progress in the knowledge offermentation in the 1850s. He described bacteria and

Louis Pasteur


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

OmnipresentMicrobes

Plants, insects, birds and beasts are living beingswhich are visible to us. But in reality there are alarge number of seemingly invisible living forms

existing on this earth. These minute living entities arecalled microorganisms or microbes. They are not visible because of their extremely small size. The termmicroorganisms was coined by a combination of theword "micro" which comes from the Greek word mikrosmeaning small and the word "organism" meaning individual living forms. Microorganisms are complete andindependent living entities. Microorganisms are generally unicellular and visible only under a microscope.Microorganisms are omnipresent, i.e. they are presenteverywhere. They are present in water, soil and air.They are also present in and on the bodies of plants andanimals.

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

8

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Hemoglobin I

Hydrogen atom molecule :t8

Polio virus


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•.•.•...., ....•}yiJ'?~:·t, ...•~..".

Amoeba

Relative sizes of microbes as compared to hydrogen atom andhaemoglobin molecule

tron microscopes are the instruments that empower usto 'see' the usually invisible microbes.

Since microbes are so small we need to use a specialscale to measure them. Microbes are measured employing a special unit called micron. One micron is onemillionth of a metre! Microorganisms differ widely insize and shape. Variations in shape, cell structure,physiology and other biochemical characteristics formthe criteria for the classification of microorganisms.They are grouped into five types: protozoa, algae, fungi,bacteria and viruses.

Omnipresent Microbes 9

Protozoa derive their name from the Greek wordsprotos, meaning 'first' andzoion, meaning 'living being'.They are the smallest type of animal life, being madeup of a single cell. They are different from bacteria inthat they have at least one, well-defined nucleus. Protozoa are oval or cylindrical in shape and are found instagnant water or in mud. They are capable of locomotion. Amoeba and Paramecium are common examples.

( b )

Algae are simple, photosynthetic plants that grow inwater or in damp places. They may be unicellular ormulticellular and exist in a variety of shapes. Somealgae may even be large enough to be seen, as are thegiant k~lps or seaweeds. Algae have the ability to photosynthesize due to the presence of the photosyiltheticpigment, chlorophyll.

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


Fungi are a low form of plant life that lack chlorophyll. Fungi occur everywhere in nature and grow indamp environment A piece of bread, cooked food oreven raw vegetable when kept unattended in moist aircan support the growth of fungi. Fungal growth is alsoseen on the walls in rainy season. Fungi may exist inunicellular or in multicellular form. Yeasts are unicellular forms of fungi. Multicellular fungi are commonlytermed molds. The multicellular structure of fungi iscalled mycelium.

Bacteria, like fungi occur everywhere in nature. Thesize of bacterial cells is much smaller than the algal orfungal cells. Structurally, bacterial cells contain cytoplasmic and nuclear matter in a dispersed state. Theydo not have a discrete nucleus. Some bacterial cellsmay have locomotory flagella, or fimbriae.

Althoughlher:e are thousands of different species ofbacteria, the individual bacterium bears anyone of thethree 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. Avirus has a nucleic acid core enveloped by a coat ofprotein. They cannot exist independently, and hencemust 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. Theymultiply by diverting the host cell machinery for theirreplication. Vrruses have the unique distinction of hav-


12

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 virusare called retrovirus. Some virus have even been ob- ~

tained in the crystalline form. They seem to inhabit thetwilight zone between the living and non-living.

All living things on earth have a unique biologicalname which is generally composed of two words. Thefirst denotes the genus and the latter, the species. Ranatigrina is the biological name of frog, and Bos taurus,that of cow. Mangifera indica is the scientific name ofmango and Cocos nucifera that of coconut. Microorganisms too are no exceptions. There is a proper system


nvelope with

surface projections

Vaccinia virusOrf Virus

Mumps virus

T-even bacteriophageTipula iridescent virus

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 aspecific name. For example Aspergillus niger, Fusariumsolani, Bacillus subtilis, Candida utilis. In all cases, thefirst name indicates the genus, and the second identifies the species. At times, characterizing a micro organ-


ism up to species level is not possible. But it is mandatory to characterize the microbe atleast up to genuslevel. As a result, sometimes nomenclature may meanusing a name such as Bacillus species. This means thatthe bacterium is a member of the genus Bacillus.

It is said that microorganisms are man's best friendsas well as his worst enemies. Man's enemies are themicroorganisms that cause diseases and spoilage offood. Infections by protozoa, bacteria and viruses arewidely prevalent Malaria and amoebiasis (dysentery)are common diseases caused by protozoa. There aremany bacterial ailments. Tuberculosis, cholera and typhoid are the more frequently encountered bacterialdiseases of man. COmnion cold, measles, chicken pox,infective hepatitis Gaundice) are manifestation of viralinfections. Fungal diseases are mainly restricted to skininfections such as ringworm.

However, all microorganisms are not our enemies.In fact, very few microorganism are pathogenic. Mostare our friends. They give us good food, medicines tocombat diseases and useful chemicals. Industrial fermentation is the story of the friendly microorganismsroped in to serve the human cause. Microorganismshave contributed to make our life more healthy, comfortable and our food more tasty. Among the microorganisms, yeasts and bacteria are widely used infermentation.

like all our living brethren, microbes too need energy for survival, metabolic functions and reproduction.


Thus, they either take up nutrients directly or afterexternal enzymatic simplification of the food material.The ingested nutrients are converted into energy andcell constituents. These metabolic functions thoughapparently simple, encompass several complex chemical reactions that are continuously and simultaneouslycarried out in the individual cell.


The molecules that specifically catalyse variouschemical reactions in the cells are the enzymes. Enzymes enhance the rate of chemical reactions at ambient temperatures without being themselves consumedand hence are bio-catalysts. Microbial cells containseveral enzymes. The levels of production of theseenzymes are dependent on the environment in whichthe cell grows. Thus, it is possible to either increase,decrease or even initiate the formation of a microbialenzyme by altering the environmental conditions. Ifone can manipulate an enzyme, then, one can controlthe reaction catalysed by that enzyme, so that a desiredproduct is obtained. So it will not be inappropriate totreat the microbial cell as a bank of enzymes or catalysts. Further, the bank is flexible. The rapid rate ofmultiplication of microorganisms has enabled us to usethem in fermentation to achieve highly specific chemical changes. Microorganisms possess tremendous potential. They perform many chemical reactions easilyunder favourable conditions and in an environmentallyfriendly 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 achievesthese specializations through an extensive special training. Specialization also exist among microorganisms.Saccharomyces cerevisiae produces alcohol, Aspergillusniger 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


microbes that show a tendency to produce chemicalsthat we need. A lot of attention is paid to selecting thosevariants, that produce more of a chemical or producethe chemicals in which we are particularly interested.These are then carefully nurtured in special nutrientmedia that help them survive under laboratory conditions. The industrially developed culture is oftentermed 'strain' emphasizing the special properties ofthat microorganism, e.g. Penicillium chrysogenum produces penicillin. But not all Penicillium chrysogenumcultures that are isolated produce penicillin in highYields. Isolates or strains that Yield penicillin in largequantity are selected, maintained and developed for usein industry.

Heat loving bacterium and its habitat

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


that display a love for the extremes of environmentalconditions are called Extremophiles. There are manytypes of extremophiles and each is given a name. Forexample, microbes which grow at high temperaturesare called thermophiles. Most microorganisms currently used in commercial fermentation are mesophiles. Many chemical conversions that require harshconditions of temperature, pressure, acidity or alkalinity cannot be performed by using mesophilic microorganisms or even the enzymes produced by them. It isnow evident that a variety of microorganisms grow inextreme environments that exist in the biosphere. Theextreme environments include high temperature (geothermal marine sediments), low temperature (Antarctic sea water), high pressure (deep sea hydrothermalvent), high acidity (acid mine drainage), high alkalinity(sewage sludge) and high salt (hypersaline water). Theenzymes catalysing the metabolic reactions for the survival and growth of these organisms must work andremain effective under the respective extreme environment, after all harsh or not, it is 'home' for the microbe.Thus, extremophiles would certainly be a better choicefor the chemical industry than mesophiles.

The metabolic activity of a microbial cell is determined by its enzymes. SYnthesis of the enzyme is inturn controlled by the genes of the microorganisms. Itis now possible to alter the genes of a microorganism.The method is termed genetic engineering. Byadopting this methodology, a gene from one organism canbe transferred to another. A genetically engineeredmicroorganism is so designed that it may express the


desired change or synthesize the product in highYields. The gene for chymosin, an enzyme present inthe stomach of calves which is used in cheese makingcan be transferred from its original mammalian sourceto a bacterium (Escherichia colt) or a fungus (Aspergillus niger). This permits the production of chymosin byfermentation 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 theproduction of vitamin C precursor is achieved in onestep. Genetically engineered microorganisms holdgreat promise. for the fermentation industry in the future.

lermentntion

Protocol

F1rmentationis a process for the cultivation andpropagation of microorganism and its applicationto obtain the desired product in due course. The

process for making of idli, or yogurt has probably beenseen by all. For making curd, a spoon of curd is addedto warm milk which is kept covered overnight. Similarly, for making idli, batter rice and black gram (uraddaf) are soaked in water for some time, then coarslyground and kept overnight at room temperature. Theseare common examples of fermentation. Aged curd develops a sour taste which is due to accumulation of achemical called lactic acid. Nice fluffy idlies are obtained if the soaked idli mix is allowed to remain so foronly 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 becausecontrol of the fermentation process is easy. We mustthank our ancestors who discovered and developed


./

21

Fermentation is a multistep process


such simple but useful processes through trial anderror and careful observations.

Large-scale or industrial fermentation, on theother hand has a complex protocol. It starts with theisolation of microorganisms. The yeast, fungus orbacterium which yields the product of interest isisolated either from soil, air, water or sources suchas plant or animal material. Generally, more than onemicroorganism are obtained at the first attempt. Inthe next step, the microbes collected are grown in thelaboratory maintaining stringent control and takingutmost care to avoid contamination of the specimensobtained from different sources, thus pure culturesare raised.

Once the desired pure culture is obtained, it iscultured in its pure form. Isolated cultures are oftendeposited in culture collections or culture banks.Culture collection centres preserve and maintainthousands of microorganisms in this way. One canobtain a culture from such culture collections insteadof going through the tedious process of strain isolation. The physical and physico-chemical properties ofthe microbial strain chosen for fermentation playacrucial role in process design and engineering of thefermentation plant. Therefore, the isolation, preservation and improvement of the strain are of fundamental importance.

The ve-sselin which fermentation is performed iscalled a fermentor. The size of fermentors varies from1 litre (laboratory fermentor) to 250,000litres (indus-

Fermentation Protocol 23

trial fermentor). Some proteins are produced in 50 to50,000 litre fermentors whereas, chemicals, antibiotics,and Baker's yeast are usually produced in 100,000 to250,000 litre fermentors. Smaller fermentors are usedfor research purpose.

Stages in the development of a fermentation process1. Isolation and choice of microorganism.

2. Preservation and maintenance of the microorganism.

3. Improvement of productivity of the microorganism.

4. Formulation of media for all stages, i.e. preservation,seed and production.

5. Optimization of seed quality.

6. Growth of the microorganism and product formationduring the fermentation.

7. Monitoring and control of fermentation.

8. Laboratory analysis.

9. Isolation of the product10. 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)/Inocula-tion or seeding the medium.

4. Culturing of the microorganism in the fermentor.

5. Isolation of the product.

6. Effluent treatment.

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


Fermentation is a multistep process. The culture isfirst grown in a test tube or petriplate~ The growth istransferred to a conical flask (500ml to 3litre) containingnutrient media so that a teeming population ofthe desiredmicrobe 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 thentransferred to a seed fermentor containing seed medium.The cell or spore suspension which is transferred istermed inoculum and the operation is called inoculation.Once the seed is ready, it is transferred to the productionfermentor containing production media Production fermentor is a big vessel. The vessel is provided with agitatorfor mixing the contents. The temperature is kept steadyby the circulation of hot and cold water through coils andthe vessel has inlets for air, inoculation and addition offeeds. Valves for sampling and withdrawal of fermentedbroth are also fitted in it During fermentation, variousparameters such as pH (ameasure ofacidityor alkalinity),oxygen level, rate of air flow, and vessel pressure arecontrolled. Thus, individual sockets for mounting of theappropriate 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 awholesome diet provides us carbohydrates, proteins,fats, vitamins, minerals and other necessary nutrientsto 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 andinorganic constituents. The nutrition of micro organ-


Stock culture

Shaker

Agitator.

~

Seed fermentor

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Production fermentorProduct

Fermentation protocol


A balanced diet keeps the doctor away


isms depends on carbon and nitrogen sources, minerals, oxygen, vitamins and other necessary growth factors. Combination of these materials constitutes theirgrowth media.

Sugars such as glucose, lactose, sucrose are common carbon sources. Cheaper carbon sources includestarch, 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 requirementis 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 caseswhere the microorganisms are unable to sYnthesizethem.

The dietary requirement of microorganisms canvary widely. Moreover, the nutrient requirement atdifferent stages of growth also differs. Thus, composition of media for maintenance, germination, seedpreparation and production stage are different. Thedietary requirement varies even during the differentstages of fermentation. The availability of appropriatenutrients in correct amounts at all stages of fermentation is crucial for high yield of product.

The most important step taken prior to fermentationis the formulation of the nutrient media in which themicrobes will grow. If any of the constituent reagentsis present in excess or in lesser amount than what isrequired, the entire process gets hindered, largely reducing the total yield, e.g. excess feeding of glucose to

-------- ------- ---- ------ --------- -- - -- ---------- ------ --------


penicillin producing fungus will result in high cell massbut low product formation. In addition to the usualmedia constituents, certain compounds called effectorsmay "switch on" or "switch off' the product sYnthesis.

The former are called inducers and the latter re

pressors. Some compounds may enhance the sYnthesisof a product For example, the addition of starch to themedium induces the formation of amylase (starch degrading enzyme) by Aspergillus niger whereas presence of glucose in addition to starch represses theformation of amylase. Some organisms have uniqueadaptive properties. If they are fed polysaccharides,then polysaccharide degrading enzymes are secretedby the microbes, and if supplied with protein the sameorganism produces protein degrading enzyme, e.g.Sclerotium rolfsii produces amylase when starch is thecarbon source and cellulase (cellulose degrading enzyme) when cellulose is the carbon source in the medium. This is mainly due to the pressures for survivalas they have to adapt themselves to whatever foodsource they get This type of behaviour is exploited todesign the composition of medium to obtain therlesiredproduct. In other words, the conditions are so adjustedthat the microorganisms are compelled to make theproduct we want.

Oxygen is a primary requirement for aerobic micro.organisms. This is provided by bubbling of sterile airthrough an in-built structure called sparger. Due toagitation and presence of proteinaceous material, foaming is (li common phenomenon. Some times foaming is


also observed due to secretion of certain compound~(metabolites) or excessive lysis (death) of the microorganism. Oils are the normal antifoam agents used tocontrol foaming.

Environmental conditions affect our lives. In sum

mer, we use cotton clothing and air coolers. On theother hand, in winter, we keep ourselves warm by usingwoollen clothing and room heaters. Similarly, for thegrowth of each microorganism the environmental conditions are different. Temperature, pH, amount of avail-


Microbes differ in their choice of habitat


able oxygen are crucial requirements. Industrial fermentations are usually performed at temperatures between 20° to 40°C. The optimum pH of the medium isdetermined by both the microorganism and the product, for example, fermentation of Aspergillus niger forcitric acid production is carried out under highly acidicconditions while fermentation of Bacillus subtilis foralkaline protease production is carried out in highlyalkaline conditions.

Agitation is necessary for the mixing of media andmicrobial cells and for the distribution of air. The levelsof carbon, nitrogen and fat in the medium are maintained by addition of nutrients from time to time tostandardized optimal levels.

An important factor in fermentation is the maintenance of sterility. Sterile environment here means astate where microorganisms except the seeded one areabsent. Microorganisms other than the one being inoculated are termed contaminants and the system issaid to be contaminated if not sterile any longer. Oncecontamination occurs, the contaminant microorganismconsumes nutrients meant for the cultured microbe,produces unwanted products, disturbs the control parameters, degrades the main product and affects theprocess of recovery of product. All these results ineconomic loss. Therefore, contamination must beavoided 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


Parameters to be controlled during the fermentation

Parameter Measurement deviceMeans of control

Temperature

ThermometerCirculation ofhot/cold waterPressure

Pressure gaugeOutlet valve

pHpH electrodeAddition of acid or

alkaliAirRotameterSparging of sterile

aIrAgitationTacometerSpeed of agitator

OxygenDissolv;ed oxygenAirflow

probe FoamFoam sensorAntifoam agents

Carbohydrate,Laboratory analysisAddition of feeds

fat, ammonia; levels inmedium

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

~- -- - - - - - - - - - - - - ..- - -~ -- - .


From the account of these operations, it is easilycomprehended that fermentation is a complex process,involving lot of controls, analyses, recording of data andmonitoring of parameters. In true sense one has tostudy and understand the microorganism, its metabolism and the means to make it yield high levels of theproduct. Tireless, and round the clock attention is required. Some bacterial fermentations take 16 to 18hours whereas some fungal fermentation may takeeven 5 to 7 days. With developments in computers,automated fermentors have now been developed. Onecan take advantage of the decision-making ability of thecomputers to maintain various parameters.

Broadly, fermentation can be of two types, depending on the nature of the process. Fermentations usingmicroorganism which do not require oxygen are calledanaerobic fermentation and those requiring oxygen arecalled aerobic fermentation. An example of anaerobicfermentation is the production of methane (biogas).Production of antibiotics, single cell protein and aminoacids are examples of aerobic fermentation. Fermentation may also be classified as solid state or submergedtype. In solid state fermentation, the microorganismsare grown in trays and on agricultural residues likewheat bran, rice bran, or rice straw mixed with nutrientmedium. 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 waterand microorganisms grow as a suspension. Depending


on feeding needs, submerged fermentation is furthersubdivided into three classes: batch, fed batch andcontinuous. In batch fermentation, all nutrients areadded at the beginning and the fermented broth isharvested at the end of fermentation. In fed batch fer

mentation, nutrients are added in lots at varying stagesof fermentation but the fermented broth is harvested atthe end of fermentation. In continuous fermentation, asthe name indicates, there is continuous feeding of nutrients and continuous withdrawal of fermented broth.

All living-beings und('rgo certain fixed and specificstages in their ::fe cycles. An organism is born, then itmatures, in time it ages and dies. Microorganisms also

Growth curve


exhibit a comparable life cycle. The life cycle of amicroorganism is divided into four main phases. Afterinoculation to the fresh medium, there is a brief periodwhen growth is almost absent, this stage is aptly namedlag phase. Lag phase is followed by active growth phaseor 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 stationaryphase or saturation phase when the multiplication ability of cells is reduced or lost. At the end, the cells lyseresulting in -reduction of total cell mass. This is knownas the lYticphase.

The growth and reproduction of a microorganismare the result of a series of chemical reactions thatoccur in an orderly manner. The nutrients are taken 'upby the microbes and then subjected to sequentialchemical 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 metabolicpathway, is the metabolite. The metabolic pathwayswhjch lead to the conversion of nutrients into cell constituents are termed anabolic pathways and thosewhich degrade organic substances to produce energyfor cell functions are termed catabolic pathways. Thecompounds that are formed during the different stagesof metabolic reactions of anabolism and catabolism arecalled intermediates. A precursor molecule is the starting compound which is m.etabolised to generate theend-product. Precursors can-be synthesized within the

36

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


cell or supplied in the medium. Thus, metabolism of amicroorganism is manipulated during the process offermentation to give high yield of desired product.

Growth and reproduction are the primary metabolicfunctions of microbial cells. The product produced during the primary metabolism are termed primary products of metabolism or primary metabolites andaccumulation of such products is parallel to growth.Some products, for example, antibiotics start accumulating a bit late in the exponential phase of growth andmuch of it is accumulated during the stationary phase.Such products are termed as secondary products ofmetabolism or secondary metabolites.

The aim of industrial fermentation is to obtain thecommercially useful products in a form that can be sold.Therefore, isolation of product forms an integral part ofthe fermentation industry. Fermentation products areof three types: the biomass itself, the product accumulated inside the cells, i.e. intracellular products and theproducts secreted outside the cells which are presentin the fermented broth, i.e. extracellular products.Baker's yeast, Brewer's yeast and single cell protein areexamples where the biomass or cells of microorganisms are isolated as product. The cells are separatedeither by filtration or by centrifugation.

The intracellular products are first extracted in solution by disruption of cells by either grinding, pressurepressing or by using special chemicals. The productsare then recovered from the broken cells. The intracellular product present in the extract or the extracellular


Crystalisation

Drying

37

Isolation of fermentation products


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

lcrmcntcd

Products

'TIere has always been a competition between thechemical route and the fermentation route forpreparation of a product. Besides the technical

advantages, economic aspects influence our decisionwhen forced to choose between the two. The successof the fermentation industry in the production of various chemicals is due to the ability of selected microorganism to consistently give high yield of the desiredproduct in a reasonable time from cheap and readilyavailable raw materials.

A microorganism should have certain characteristics for its application in industrial fermentation. Itshould be non-pathogenic, have the ability to growrapidly on suitable nutrients and should possess highlevels of the enzymes required for rapid formation ofthe product. Productivity in terms of the productformed 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


\\1-/__I I

1

40

Ifermentation processes are purer and chemically ac-tive.

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 andis growing day by day. The value of fermentation products also seems to be growing by leaps and bounds. Ithas been estimated that in the year 1990 fermentationproducts worth 30 x 109 U.S. Dollars were producedworldwide. Antibiotics contribute a major share to thisvalue. 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 toeven merely list all the fermentation products in eachcategory. Hence, only a few of them are dealt here toget an idea of the impact made by fermentation on thequality of our life.

Food is a basic requirement for human beings. Theuse of fermented food or rather the use of microorganisms for food processing is a long story dating fromancient days. Over the ages each civilization has developed a line of fermented food items. The variation isbecause of diversity in raw materials, knowledge, perception, taste, and eating habits. Food was not thoughtto be a marketable item in ancient days and as a resulttraditional fermented food and beverages are household fermentations. Even today the microbiology andbiochemistry of some household fermentations arepoorly understood. Nevertheless, some of the traditional products have now become industrial productsas a result of modemisation and greater demand. Agood 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, spongycakes, soft rolls or crisp cookies on the bakery shelf is nodoubt very tempting. Microorganisms have a great roleto play in the making of these sumptuous bakery items.The production of baked items, by and large, call for ageneral protocol of preparation of raw materials, doughformation, dough processing, baking and packing. Thespongy nature of bakery products is a result of fermentation. The dough is prepared by mixing the ingredients.


Traditional fermented food items

Product Substrate Microorganism

Unknown

Idli

Indian

Dhokla Bengal gramand wheat

Black gram Bacteria andand rice Yeast

Jalebi Wheat flour Yeast

Papadam Black gram Yeast

Oriental Oapan, China, Philippines, Indonesia)

Soy sauce Soy bean Fungi, Yeast,and wheat Bacteria

S0Ybean Fungi

Fish Unknown

Vegetables Bacteria

Tempeh

BagoongKimchi

South Africa

Maerissa

Banku

Other

Tarhana(furkey)

Kanga-kopuwari

(New Zealand)

Poi(Hawaii)

Chicha(peru)

Pozol(Mexico)

SorghumCassava

Wheat mealand yogurtMaize

Taro corns

Maize

Maize

Yeast

Maize

Bacteria

Yeast, Bacteria

Yeast, Bacteria

Fungi, Yeast,Bacteria

Fungi, Yeast,Bacteria


Bake a cake and much more

The mixture is then processed by fermentation. Thisoperation is called leavening. Only in exceptional cases,e.g. when a high concentration of sugar is present, isthe leavening process done non-fermentatively.

The yeast used for dough fermentation is Saccharomyces cerevisiae, popularly known as Baker'syeast. 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 theyeast during leavening is between 1to 6 per cent dependingupon the type of :flour and the product desired. Similarly,the fermentation temperature, pH and the time too varieswith the raw materials and the type of product. Higherconcentration of yeast is always avoided as it gives undesirable results. During the leavening process, the yeast breaksdown the carbohydrates in the dough and generates carbondioxide 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 enzymessuch as amylase which degrades the starch present in theflour to glucose. Glucose is then consumed more easily bythe yeast A mixture of yeast and bacteria (lactobacillus) isalso 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 amultistep one. The microbial fermentation is encouraged in two steps; culturing of milk with bacteria andripening of cheese. During the culturing, the lactic acidbacteria convert lactose (milk sugar) to lactic acid. Thisconversion has an impact on the gross composition ofcheese. The introduction of microorganism during ripening step has many advantages. It enhances :flavour

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


Say cheese!

due to metabolites produced by the microorganisms.The carbon dioxide produced by the microbes istrapped 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 andblue cheese is the result of microbial growth on thesurface of the cheese. The different types of cheesesproduced by utilising different microbes and fermentation processes are Cheedar, Gouda, Blue, CottageCream, Swiss and Mozzarella.


Pretreatment of milkJ,

Milk culturing with bacteriaJ,

Clotting of milk by renninJ,

Collection of raw cheeseJ,

Fortification of cheese.1

Ripening of cheeseJ,

Packing

More than 30 major fermented dairy foods are prepared in different parts of the world today. The veryfamiliar names are yogurt, sour cream, cultured buttermilk, acidiphilos milk and shrikhand.

Yogurt is a very popular fermented dairy product Itis a semisolid product resulting from fermentation ofheat-treated milk. The fermentation is performed bytwo bacteria (namely Lactobacillus delibrueckii andStreptococcus salivarius). In household fermentationthe taste of yogurt varies due to variation in both milkquality and type of cultures used. However, commercialproduction involves stringent control of conditions atall stages so that an uniform texture and taste is obtained repeatedly.

The lingering soft and sweet taste of chocolate is yetanother gift of fermentation. Chocolate in any form, be


it chocolate milk, chocolate ice-cream, chocolate bars,chocolate cookies, or cocoa drinks is liked by all agegroups. People like chocolate not for its nutritionalvalue but for its flavour.

Cocoa and chocolate are produced from cocoabeans, the seeds of the plant Theobroma cocoa. Theseeds of the ripe fruits are subjected to fermentationfollowed by drying. The operation is called curing ofcocoa seeds. Fermentation is carried out primarily toinduce biochemical changes within the seeds whichleads 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 flavourwhen processed into chocolate. The cured seeds arecalled beans and they develop rich colour and flavourupon roasting. The fermentation is carried out in boxesor in heaps covered with banana leaves. Microorganisms in the environment contaminate the pulp duringhandling and convert the sugars present in the pulp.The fermentation process goes on for about 4 to 6 days.

Pickling of vegetables thereby preventing spoilageon one hand and making the vegetable tastier on theother, is an example of microbial intervention that isprofitable to us. Pickles of mango, chilli and lemon, areknown to us. Many of us may have raided mother'spickle-jars as children. But not many of us realize howfermentation helps pickling. Pickling involves anaerobic fermentation using the naturally occurring bacteria.Some enzymes present in the fruit and vegetable helpto provide nuttients to the fermenting bacteria. Pickling

48

QUALiTY PRODUC


MOTHE.RS'PIC- k.L-E.

Making pickles has become an industry throughout the world

of cucumbers and cabbage is a big industry in thewestern world. The product of cabbage fermentation isSauerkraut. Kimchi is a Korean product produced byfermentation of either radish, cucumber, cabbage or.green onlOns.

Lactic acid producing bacteria grow in salted vegetables and produce organic acids which lower the pH


of the mixture. The combined action of the salt and theacid lowers the activity of the enzymes that are responsible for the breakdown of the vegetable tissue. At thesame time it inhibits changes in the tissue preventinggrowth of undesired microorganisms.

Microbes have proved their expertise in producinggums too. Gums are the sticky materials that giveviscous solution when dissolved in water. Chemicallygums are polysaccharides. Sometimes microorganisms secrete such polysaccharides outside the cell andthen, these are termed exopolysaccharides. Microbessecrete these compounds either for protection by forming capsules around the cells or as a reserve foodmaterial. Various bacterial, fungal and yeast culturesare developed for this fermentation. Xanthan gum pullulan, and Baker's yeast glycan are two examples ofwidely used microbial gums. Xanthan gum is a popularmicrobial gum. This gum is widely used as a thickeningagent and in the preparation of meat like gels in foodindustry, as lubricant in drilling oil wells, in textileprinting and dyeing, ceramic glaze manufacture, polishing agent and rust-curing agent.

Down the ages, a good deal of man's food has comethrough microbial agency. Bread, cheese, alcoholicbeverages, cocoa, pickles, soyasauce and vinegar toname a few. These are products that are enjoyed as aresult of microbial activity. However, there are someinstances where the producer microbe is consumedtogether with the product. Curd, is a good example ofthis.

'"


C.UIt.:D

All these and more

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


food crisis to some extent. A close look will tell us how.Microorganisms such as algae, bacteria, yeast andfungi have been considered as source of protein. Culture of microorganisms for food started at the end ofFirst WorId War as a result of food crises and reacheda peak in the 1970s. The term "single cell protein"(SCP) was coined to describe the microbes as proteinsources. Various microorganisms are grown on different substrates for SCPoThe choice hinges mainly onthe availability of raw materials, nutrient value of thecells, contents of essential amino acids and toxicity ofthe microorganism. They have not been widely accepted due to taste and food preferences of consumers.

Maximum attention has been paid to yeast as asource of microbial food. They are not toxic and arenon-pathogenic and hence best acc~pted by consumers. Surplus brewer's yeast finds use as animal feedstuffand for therapeutic purposes. Their generation time(time required for doubling of the cell population) is 2to 5 hours. Their protein contents is slightly low (about60%)but they contain essential amino acids and are richin B-group vitamins.

Bacteria yield highest biomass due to their shortgeneration time (about 1 hour) compared to othermicroorganisms. The protein content is also high, it hasbeen found that several bacterial species contain all theessential amino acids necessary for us. Research hasalso revealed that symbiotic culture of yeasts and bacteria give improved crops of microbial food. However,bacteria are not popular as protein source because oftheir pathogenic nature. Their small size causes diffi-


SPirulina tablets are now available in medical shops

culties in harvesting. But even then, various bacterialspecies are grown on bagasse and methanol and thebiomass is used to produce animal feed.

Algae have many positive points for being considered as a candidate for biomass production. Algae useatmospheric carbon dioxide as the carbon source, they


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 variousalgal species. The most promising ofthem are ChIorellaand Spirulina. Both contain amino acids and vitaminsof interest to us. Spirulina tablets are now available andare ideal for patients suffering from vitamin deficiencyand for those requiring protein supplements. Scientistsspeculate that ChIorella, could be grown under controlled conditions in spaceships and thus could not onlygenerate edible biomass but also oxygen. In theory atleast, it is easy to vote for ChIorella as a candidate thatcan make the spacecraft self-sufficient in food and oxygen. Of course, the theory needs proper evaluationbefore it can be recommended as standard practice. Butthe theory itself is enough to highlight the potentialmicrobes have.

Fungi are not very popular as biomass because oftheir long generation time (5 to 12 hours), low proteincontent (15%)and because their cell walls are difficultto break. The last criterion makes it difficult for us toharvest chemicals inside the fungal cells.

"Health is wealth". The age old axiom still holds true.Microbes are ubiquitous. A lot of them are not friendlyto us, as soon as they find a susceptible living host theyattack and diseases break out. Few such diseases aretuberculosis, throat infection, jaundice, influenza, andamoebiasis. These diseases are caused by our microbial enemies. But friendly microbes have stretched outtheir helping hands, so to say, in rescue and have given


us medicines to fight the ailments. There are somediseases that are not caused by microbes. These aremanifested due to certain congenital abnormalities orphysiological 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 killparasites, anticancer agents are used in treatment ofcancer and insecticides kill insects. Among these, antibiotics have highest market value. Pencillin was discovered by Alexander Fleming in 1928, and its clinicalapplication was the result of the pioneering workby E. B. Chain and H.W. Florey in early 1940s. SelmanWaksman discovered streptomycin in 1943. We havebeen very lucky that these antibiotics discovered quiteearly turned out to be powerful life saving drugs withlow mammalian toxicity. Mer the discovery of thesetwo antibiotics, thousands of other antibiotics havebeen obtained but only a few hundred have foundmedical application .

. The antibacterial action of an antibiotic depends onits chemical structure. By altering its chemical structure, one can improve its antibacterial activity. Thisconcept is successfully exploited to produce more potent antibiotics. Such antibiotics are called semisynthetic antibiotics since chemical SYnthesis is only a partof the total process. For example, pencillin is a fermentatively produced antibiotic and ampicillin is a semisYnthetic penicillin derived from pencillin.

Fermented Products

(a)

55

(b)

(c)

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


But, antibiotics are only one part of the story. Steroids tell another tale of microbial versatility. Steroidsare complex chemical substance found in plants andanimals. In 1949 it was demonstrated that cortisone, asteroid, produces dramatic effects in treatment of rheumatoid arthritis. This discovery paved the way for extensive investigations ofvarious steroids as therapeuticagents. Today a large number of steroid hormones havebeen identified as valuable therapeutic agents in thetreatment of arthritis, rheumatism, leukemia,hemolytic anemias and many other diseases.

In the early 1950s it was discovered that certain fungican cause chemical changes in steroidal substanceobtained from plants or animals and thereby transformthem to therapeutically active steroids.

To transform steroids a microorganism known to becapable of producing the designed chemical change isgrown in suitable medium under optimum conditions.Mer adequate growth has occurred, the steroid isadded in the culture, and the designed chemical transformation occurs. The steroid so formed is then recovered for purification and may be processed for themarket.

Other health care products include antifungals, insecticides, antiparasitic agents, alkaloids, vaccines andinsect toxins. They are used widely in treating varioushealth related problem which are very frequently confronted by us.

Commercially useful enzymes are yet another usefulgift of microbial fermentation. Enzymes are powerfulcatalysts, they can accelerate the rate of reaction overa million times. Enzymes have become very handy in


Bronchitis

Ring worm infection

Candida infectionCandida infection

Typhoid

Tuberculosis,LeprosyUrinary tractinfection

Pneumonia,Meningitis, SyphilisTuberculosis

Cholera, Plague

Some antibiotics produced by fermentation

Disease controlled

Rifampicin

Gentamycin

Erythromycin

NystatinHamycin

Compound Producing organismAntibacterial antibioticsPenicillin G Penicillium

chrysogenum

Streptomyces griseus

Streptomycesaureofaciens

Chloramphenicol Streptomycesvenezuelae

Streptomyceserythraeus

Streptomycesmediterranei

Micromonosporapurpurea

Antifungal antibioticsGriseofulvin Penicillium

griseofulvin

Streptomyces nourseiStreptomycesp imp rina

StreptomycinTetracycline

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



teria are used for production of various enzymes. Inorder to improve the microbial strain, genetic engineering is employed to transfer a gene responsible forsynthesis of a desired enzyme, from one microorganism to another or from animals to microorganisms. Theaim of using such a technique is to obtain the desiredenzyme in greater quantities and at a lower cost.

Each enzyme catalyses a specific reaction. Thisproperty finds many applications, for example, amylases convert starch to glucose. This ability of amylaseshas enabled their use in production of glucose fromstarch. Bakeries use it for the hydrolysis ofgrain starchto enhance leavening, while the textile industry uses itfor the removal of starch during the desizing process.It is used in detergents to aid the removal of starchystains from clothes. It is used in animal feed, and as adigestive aid. Similarly, proteases hydrolyse proteins togive simpler smaller fragments (polypeptides) andeven amino acids. Proteases active under alkaline conditions are used widely in detergents for removal oforganic stains from clothes. In fact detergent enzymesconstitute about 40 per cent of the total market value ofenzymes.

Polysaccharide degrading enzymes such as amylases and cellulases are used in breweries and wineriesto accelerate fermentation. Candies are sweet, and thissweetness is courtesy fructose which is twice as sweetas glucose. Glucose obtained from starch can be converted to a syrup containing equal quantities offructoseand glucose by glucose isomerase. This syrup is usedto prepare candies and as a substitute for cane sugar.


Many other useful products such as cheese, ice-cream,coffee and fruit juice require processing with enzymesobtained by microbial fermentation.

Enzymes have clinical applications too. Enzymescatalyse various metabolic reactions in living cells.Therefore, it would not be surprising to know thatenzymes are used for treatment of various bodily disorders. Many digestive enzyme are secreted by the pancreas. Some children and elderly people suffer fromdeficiency of such enzymes which results in incompletedigestion of food. Fermentatively produced enzymessuch as amylase and lipase, which degrade starch andfats, respectively, are used as digestive aids. Otherexamples of fermentatively produced enzymes includestreptokinase used for dissolution of blood clots in thecirculatory system, L-asparginase as an anticanceragent and lysozyme as healing agent. Besides the useof enzymes as medicines, some fermentatively produced enzymes are used in industries for production ofdrugs or drug intermediates, e.g. Escherichia coli produces penicillin acylase which is used in preparation ofampicillin.

Another important application of enzymes is in diagnostic aids. Estimation of glucose in urine and in bloodwhich is particularly significant in the diagnosis ofdiabetes and its control is done very rapidly by usingthe enzyme glucose oxidase which in turn is usuallyproduced by fermentation.

Microorganisms play an important role in the life ofplants too. The story is again of friendship and enemity ..Plants suffer from various diseases caused by plantpathogenic microorganisms. Nevertheless, the story of

Fermented Products

Some useful fennentative enzymes

61

EnzymeAmylase

Protease

Cellulase

PectinaseRennin(Chymosin)LactaselipaseGlucoseIsomerase

Function

Hydrolysis of starch toglucose

Hydrolysis of proteinsto amino acidsHydrolysis of celluloseto glucoseHydrolysis of pectinlimited hydrolysis ofcasemHydrolysis of lactoseHydrolysis of fatsConversion of glucoseto fructose

ApPlication

Bakery, Detergent,Textile, Brewing,Glucose production.Detergent, Silk, Wine,Leather, CoffeeTextile, Brewing,DetergentFruit juice, WineCheese

Baby food, Ice-creamDetergentConfectionary

positive co-operation is equally fascinating. Compostprepared from cowdung and agricultural waste by anaerobic fermentation in a covered ditch is a familiarstory. Besides these, various agricultural aids are nowproduced by industrial fermentation. One of such products is gibberellic acid. It is a plant hormone and is usedin propagation of cash crop like grapes. Gibberellic acidis produced by the fungus Gibberella jujikuroi whengrown under nitrogen limited medium. Herbicides likeBialophos are obtained by fermentation of Streptomycesspecies. Bialophos does not have non-selective phytotoxic effects like antibiotics nor does it create environmental pollution like chemical herbicides. Yet anotheravenue of supporting agriculture is the use of microbialbiomass or fermented broth, (after removal of product)as a fertilizer. If the mycelia or broth is from antibioticfermentation then there is the added advantage of pro-

62 Friendly Fennentation

tection from certain diseases due to residual levels ofantibiotics.

The emphasis has, no doubt, been on several commercially valuable, fermentatively obtained products.In addition to these, the microbial biomass itself hasfound use as a bio-catalyst in various chemical reactions. This is another novel and innovative application.Microbial cells contain many enzymes that catalysechemical reactions. Thus, the cells can be used as a

---- -----~- ---------~------ --- --~----- - -.


single source for performing many chemical reactions.Such catalytic transformations are termed biotransformations. Use of microbial cells as a catalyst has anadded advantage. Factors necessary for enzymatic reactions and extended stability of some enzymes areinherently present in the bio-system. Biotransformation is exploited for production of useful compounds.For example, the production of vitamin C from glucoseby the strictly chemical route is a six step process.Strains of Erwinia herbicola can enable conversion ofglucose into vitamin C in two steps. An important advance in enzyme technology has enabled scientists toincrease the product yields manifold. This involvesimmobilization of the enzymes. Immobilization is aprocess in which microbial cells or the enzymes produced by them are encased within solid support materials. The advantage of this process is that the enzymesare not lost with every batch processed but may beretrieved for re-use. Immobilized cells are used both asbio-catalysts and in continuous fermentation.

Last but not the least is, alcoholic fermentation. Thisis fermentation of course, but the end-product here isalcohol. Alcoholic fermentation is the cornerstone of all'spirited' beverages.

Alcoholic drinks are intoxicating. Consumption ofalcoholic drinks has been a much debated issue overages. Though the adverse effects of too much alcoholon the human body are well-established, ,theproductionand consumption of alcoholic beverages have beenincreasing. In almost all civilisations, the history ofmicroorganisms 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-establishedlarge-scale and small-scale breweries and distilleries.Wine making or brewing at home is still practicedthough it is not too common.

The basic concept in alcoholic fermentation is theconversion of sugary and starchy raw materials intoalcohol and carbon dioxide by using yeast such asSaccharomyces cerevisiae. There are more than a thousand strains of yeast, each with different characteristics. The breweries maintain and protect theirspecial strains. Brewer's yeast is also available forhousehold fermentation. The variations in alcoholicdrinks depend on the fermentation process that the rawmaterial 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 alcoholicdrinks. Beer is produced from malt, and barley. Whitewine is produced from crushed grapes after removingthe skin an~ other insoluble parts ..Red wine is madefrom crushed purple grapes. The characteristic redcolour is due to the extraction of pigments from thegrape by the alcohol formed. Champagne is sparklingwine containing dissolved carbon dioxide. Fruit winesare made from a wide range offruits such as pomegranate, pit fruits, and berries. Brandy is the distillate ofwine, while whisky is the distillate from fermentedmash of grains and rum is the distillate from fermentedsugarcane molasses.

Fermented Products

Filter

•Filter

Wine making

65


The monitoring of fermentation conditions, incorporation of other nutrients, operational steps, distillationprotocol and ageing process varies from one type ofalcoholic drink to another. Microbial enzymes such a~amylase, cellulase and hemicellulase are used to accelerate fermentation and also to degrade the residualcarbohydrates after fermentation. The latter makes foreasier filtration.

1110

D

Brazilians opt for 'Gasohol' (gasoline + alcohol)


Alcohol as a beverage is well-known but the role ofalcohol as a chemical is often overlooked. Alcohol isused in many industries as a solvent. Distilled purealcohol (ethanol) is also used as a chemical feedstock.For this purpose alcohol is obtained by fermentation ofcheap raw materials like molasses. The alcohol obtained at the end of fermentation is then separated bydistillation. Alcohol has been used in Brazil as automobile fuel instead of petrol. This is possible as Brazil hassufficient land and water resources for extensive cultivation of sugarcane.

Besides alcohol, various chemicals such as aminoacids, organic acid, nucleotides, lipids, fatty acids, vitamins, glycerol and vaccines are also produced by fermentation.

.(po!dnu lofwnfd

'TIe fermentation industry has come a long wayfrom its modest and obscure beginning. It hasseen many phases from primitive origins when

man used microorganisms without any systematicknowledge to this date when we know a good deal aboutthem. The progress of the fermentation industry depends on three aspects: improvements in existing fermentation pro.cesses,. development of newerfermentation products and unrelenting exploration ofnewer more potent microorganisms. Increase in production at a lower cost is the moving force. Development of high performance microorganisms byselection, 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 aredifferent avenues for improvements in a fermentationprocess.

The techniques of genetic manipulation hold promise for making microorganisms compelled to producecompounds otherwise generated only by mammalianand plant cells. Genetic engineering has been applied

Looking Forward 69

to fermentation processes to enable bacteria to use awider variety of feedstocks, to biosynthesize new products to accumulate intermediate metabolites viablocked pathways, or to increase product yields byenhanced synthesis of special enzymes. In many commercial fermentation processes the cost of raw materi.:als is the most expensive component. Industriallyuseful bacteria can be modified to use cheaper feedstock by genetic manipulation. Research is being carried out to achieve better strains of microbes inlaboratories spread allover the world. Scientists haverelied heavily on recombinant DNA technology to obtain better breed (s) of microbes. This technology owesits origin to a key discovery - that of the wonderenzyme restriction endonuclease. This can be likenedto molecular scissors with which DNA molecules canbe 'cut' at certain sites. These enzymes are naturallyfound in many bacterial species. The original use wasto cut up any parasitic viral nucleic acid entering thebacterial cells. However, in the hands of scientists, therestriction enzymes became potent tools for tailoringgenetic material to suit our purposes. Another enzyme,DNA ligase helps in joining pieces of DNA Once thetool for genetic engineering became available, it became comparatively easier to get on with the job. Genesresponsible"for the synthesis of the desirable enzymeor antibiotics or those governing desirable traits wereidentified in the donor' species. Plans could now bedrawn up to transfer these genes to the recipient. Thusthe entire long-drawn sequential biochemical pathwayfor the chemical synthesis of a compound could easilybe shrunk to essentially an one-step process. For exam-

70

E.coll


Foreign DNA

Restriction enzymescut st specific point

'/,,' ...'

., •. ';"'i ~ 4-' ·;;l. '

t2 Cells divide \j- ', .. -

'<I,: ..i~'.',~-',,:, ..

Genetic manipulation has empowered scientiststo design microbes

pIe, the production of vitamin C initially involved atwo-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 generesponsible for the production of vitamin C from one ofthe two species and introduced it into the other species.

Looking Forward 71

The second species now had both the genes and thuswas an unique microbe. It could carry out single-handedly (so to say) the job that used to be a two-stepprocess involving two microbial species. Such is thepotential of genetic engineering.

It is providential that Nature has provided scientistsnot only with the 'molecular scissors' but also with themeans to 'ferry' genes out of the host cell and into therecipient. For this, scientists use plasmids. Plasmidsare circular extra chromosomal DNA molecules of smallsize (inthe average range of5-10kilobases compared withthe size ofan average chromosome which may be severalthousand kilobases). They are found in many bacteria,and sometimes there may be more than one plasmid in asingle bacteria. Several of the bacterial plasmid carrygenes for drug resistance. Plasmids which can best servethe role of a molecular taxi is one that is small in size, existin high copy number per cell and contain genes of drugresistance. Vrruses offer another 'ready- to-use' naturalroute to ferry DNA segments. In nature viruses readilyenter host cells and the viral genomes easily integrate,with the host's genetic material. Thus, the virus wasalmost perfect in its role as a molecular ferry whichscientists could manipulate to carry a genetic cargo oftheir choice. The only factor that scientists must guardagainst is the inherent pathogenicity of viruses.

Essentially, the plasmid or viral DNA is cleaved withrestriction endonucleases at a site where the cut willnot hinder normal functioning. The piece of foreignDNA, also cut by the same enzyme, is then inserted intothe viral (or plasmid) DNA. The ends are complementaty and they pair up easily.


I&'~ --' These are annealed

by the enzyme DNAli-gase. This results inthe

II . , ci~ U OU '->11 formatio~ of a vehicleG A A T T e DNA whIch now con-e T T A A G tains a piece of foreign

DNAas anintegral partRecognition sequence .\ I ofitself. Ithasnowayof

discriminating againstthe inserted foreignDNA since it recognises it as part of itself.When this DNAentersa host cell it reproduces just the way itwould have done normally. The foreignDNA, which by itself

II .~.. "II :~~"; I would not have beenResult of cleavage able to survive, letalone reproduce in the

Cleave as you please host cell thus gets afree ride and an assured chance of survival. Scientistsget a transgenic organism. Fermentative production of insulin used in treatment of diabetes and ofchymosin used in cheese production are recent successes. Transgenic microbes have helped the pharmaceutical industry tremendously. Just oneexample will help to highlight the point. Insulin, thehormone that keeps blood sugar under control innormal people, is used as a medicine by those suffering from diabetes mellitus. This is a condition charac-

LookIng Forward 73

terized by the presence of sugar in abnormal amountsin the blood and urine. In the early days of the pharmaceutical industry, insulin was sourced from the pancreas of freshly slaughtered cattle. The amountobtained was quite meagre and there was considerableunwillingness 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 hadgained ground.

Scientists chose a normal mouse and also a bacterial

species. These were to be the donor and the recipientrespectively. Mice produce insulin and thus, obviouslypossess the gene responsible for its sYnthesis. The nextstep was time-consuming. They had to identify the generesponsible for insulin sYnthesis from amongst thethousands of other genes in the genome. Then, theyhad to selectively remove the gene of choice and cloneit. The plasmid DNA had to be readied to accept a copyof the mouse gene. Once the mouse gene for insulinwas attached to the plasmid DNA the result was achimeric DNA The chimeric DNA was part mouse andpart plasmid in origin. The chimeric DNA was thenintroduced into the bacteria. This is when the fun began. Each time the bacteria divided, so did the chimericplasmid. Very soon the scientists had a colony ofgenetically engineered bacteria, each carrying a copy of themouse gene for insulin. Each time the foreign genesexpressed, they helped produce insulin. In effect, scientists had a factory producing insulin inside the test-


~0O)E. Coli

Foreign DNA

74

Restriction 1enzymes cut atspecific points

.I

Engineering insulin

Looking Forward 75

tube. Furthermore, this insulin was free of the stigmaof animal sacrifice. It also did not need to be tediouslypurified and was copiously available. A similar protocolusing the human gene for insulin was also successfullydemonstrated. The product available is marketed as'Humulin' (Le.Human Insulin). A similar success storyis that of chymosin production which previously usedto be obtained from calves. The use of recombinant

DNA technology has made the in vitro production ofenzyme easy.

Extremophiles are another group of microorganisms which have been exploited only to a limited extentin industrial fermentations. Fermentation, bioconversion and enzymatic catalysis constitute the answer tothe current pollution crisis in the world.

Municipal and industrial waste water treatment byemploying genetically engineered bacteria has beenproposed recently. Toxic and undesirable metals cannow be sequestrated from waste water by the bacteriaskilled in absorbing metals on the cell surface or byintracellular uptake. Metallothioneins are peptides thatare produced in response to increased metal concentration in body. Human metallothionein has been clonedinto E. coli and it has been proposed that these engineered bacteria be used as an immobilized cell systemfor removing metals from waste water.

Recombinant DNA technology has helped in engineering bacterial species which would flocculate betterthereby aiding in removal of more biomass for disposalor recycling purposes.


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

GlossaryCentrifugation - Separating molecules by size or density usingforces generated by a spinning motor.

Clone - Individuals having exactly the same genetic makeup.

Cure - A crucial step in preparation of fermentatively derivedfood during which the impregnated microbes are allowed towork on aiding the development of characteristic flavour andtaste of the food stuff.

Express - Refers to the manifestation of the characterscontrolled by the introduced genes following recombinationDNA technology in a living cell.

In vitro - Literally 'in glass'. It refers to any biologicalexperiment carried out outside the body.

Non-pathogenic - Inability to produce diseases. The term canbe applied to bacteria, fungi, viruses and other microbes.

Phytotoxic - Anything that is poisonous to plants. The termconsist of two parts. The prefix 'phyto' denotes plants and 'toxic'means pOIsonous.

Polysaccharides - Carbohydrates produced by combinationof many molecules of simple sugars.

Proteases - An enzyme that cleaves peptide bonds which linkamino acids in protein molecules.

Transgenic - A plant or animal in which foreign DNA (atransgene) is stably incorporated.

I FOUNDATIONS OF BIOTECHNOLOGY SERIES

Fermentation is a household word. Its origins are lost in the murkymist of time, but its future is as bright as the summer sun. Curdsand chocolates, alcohol and medicines, the list offermentation products is unending. We use all these all the timewithout any conscious thought about the underlying microbialactivity that makes fermentation possible.This lucidly written and profusely illustrated book is particularlymeant for school students of an impressionable age. It highlightsthe role our invisible allies, the microbes, play in making our livesmore comfortable and, in the process, introduces the readers tothe 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.~.ManagementAssociation, New Delhi. At p'resent he is Group Manager at R&D of HindustanAntibiotics Ltd (HAL), Pimpri.During his stay in the US, he has contributed to the deciphering of amino acidsequences of -several proteins of physiological importance. At HAL, he has playeda key role in the development of immobilized penicillin G acylase and 6-APAtechnology and has developed other important enzymes useful in theinterconversion of penicillins and cephalos- porins.Dr Shewale has over 60 publications and 11 patents to his credit. He is a fellow ofMaharashtra Academy of Sciences. He is also a recipient of MeritoriallnventionAward from National Research Development Corporation, New Delhi. BesidesFriendly Fermentation, he has authored another popular science book entitled'Enzymes Everywhere'.

J88172"361853

ISBN: 81-7236-185-8

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