Advances in Biotechnology 9789350435281, 9788183182676

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ADVANCES IN

BIOTECHNOLOGY s. N. JOGDAND Dept.o/Microbiology, Modem College,

Vashi, New Mumbai.

K6))I

GHimalaya Gpublishing GRouse MUMBAI· DELHI· NAGPUR· BANGALORE .HYDERABAD -CHENNAI. PUNE -LUCKNOW

© Author No part of this book shall be reproduced, reprinted or translated for any purpose whatsoev{ without prior permission of the Publisher in writing. .

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Branch Offices Delhi

ISBN

978-81-83182-67-6

Revised Edition

2007

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ICONTENTS I 1. FEATURES OF BIOTECHNOLOGY

2. BIOLOGY - BIOTECHNOLOGY - BIoncs, HISTORY OF BIOTECHNOLOGY 3. GROWTH OF BIOTECHNOLOGY AND ITS WORLDWIDE STATUS

1 - 2 3 - 11 12 - 25

Which nations will make the most out of bioindustrial revolution?Need of co-operation between developed and developing countries, Biotechnology Scenario 2000 onwards

4. BACKBONE OF BIOTECHNOLOGY

26 - 58

Introduction - Applications of Principles of Genetics to Biotechn9logy Introduction - Eugenics - 'Gene manipulation techniques useful in Biotechnology - Genetic Engineering - Protoplast fusion - Site directed mutagenesis Bioreactors - Major types of bioreactors - Continuous flow stirred tank reactor - Plug-flow column Reactor (PFR) - Fluidised bed reactors Ultrafiltration reactors - Electro-chemical reactors - Membrane bioreactors - Hollow fibre and rotating membrane - Airlift and bubble column fermentor - Deep jet fermentor - Tray reactors - Packed bed reactors - Immersing surface reactors Immobilisation - Definition - Why enzymes are suitable as biocatalysts? - Why cells are suitable as biocatalysts? - Advantages of immobilisation of biocatalyst Methods of immobilisation - Entrapment - Absorption - Covalent attachment - Encapsulation - Flocculation - Biospecific attachment Effectiveness factor - Downstream processing - Recent Developments

5. BIOPROSPECTING, BIOREFINERY, BIOENERGY

59 - 84

Introduction - Bioprospecting Biorefmery - Biomass.for energy production - Methane Algae as the Source of energy - Production - Biogas - Fuel alcohol production - Hydrogen gas production - Bioelectrochemical devices - Petrocorps

6. BIOSENSORS AND BIOCHIPS Biosensors - Biochip - A step towards biomolecular computer - Possible future applications

85 - 95

96 - 134

7. BIOTECHNOLOGY - MEDICAL APPLICATIONS Introduction - Vaccine production - New developments - Vaccines using genetically - Engineered organisms - Synthetic peptide vaccines - Minicells as vaccines - Disabling mutations for production of oral vaccines - Antiidiotypic vaccines - Genetic immunization Medical and Diagnostic Products - Diagnostic kits What are DNA probes? - Steps of use

DNA probes -

Monoclonal antibodies - Introduction - Production of hybridoma cells Monoclonal antibodies can reduce rejections - Monoclonal antibodies Facilitate better selection of donor - Monoclonal antibodies in cancer therapy - Monoclonal in immunoassays - Monoclonal antibodies in immunoassays - Monoclonal antibodies in immunohisto-chemistry - Monoclonal antibodies in diagnosis of malignancies - Monoclonal'antibodies in pregnancy tests Monoclonal antibodies in protein purification - Monoclonal antibodies in treatment of overdose - Monoclonal antibodies - Other application Interferon - Hormones - Medical uses of microbial enzymes - Liposomes as drug delivery system - Drugs production by transgenic animals - Gene therapy for human diseases - Gene therapy for haemophilia - Therapeutic Proteins - Blood Products 8. ANIMAL BIOTECHNOLOGY

I

135 - 147

Introduction - Types of cell lines - Maintenance of cell lines - Culture media - Stages of culturing continuous cell lines - Cell culturing in laboratory - Large scale culturing - Applications of animal cell culturing 9. BIOTECHNOLOGY AGRICULTURE

APPLICATIONS TO PLANTS AND

Introduction - Nutrient Film Technique (Nfl) - Exploring plant: Genome - Biotechnology and plant breeding conventional plant breeding - Nonconventional plant breeding - Non-recombinant approaches Somaclonal variation - Protoplast - Fusion Recombinant Approach - Plant Growth and Development - Cell culture - Tissue culture - Clonal propagation or micro propagation - Plant cell culture methods - Callus culture - Cell culture - Media and Material for Plant' Cell Culture - Embroy Rescue Embroy culture - Ovule culture -. Ovary culture - Auther and pollen culture - Endoesperm culture Applications of cell culture methods - Cell culture for higher productivity - Cell culture for stock plants chemical production by cell culture - Genetically engineered plants improved nutritional characteristics - Genetically engineered plants for chemicals ~ Artificials or synthetic seeds - Biotechnology aids seed testing - Somatic embryogenesis - Revolution in plant protection - Protection against diseases - Resistance to herbicides pesticides - Stress-resistant plants

148 - 185

10. BIOTECHNOLOGY - APPLICATIONS TO FOOD INDUSTRY

186 - 207

Introduction - Biotechnology for animal products - Single Cell Protein (SCP) - Sweetners - Applications in food processing - Debittering of fruits juices - Application in cheese industry - Degrading raw starch Bacterial silage additive - Application in brewing industry - Use of enzymes in food biotechnology - Bakery products - Dairy products fibre: for human consumption - Use of enzymes in food preservation - Flavouring compounds - Biogums

ll. BIOTECHNOLOGY - INDUSTRIAL APPLICATIONS

208 - 224

Introduction:- Antibiotics - Microbial Enhanced Oil Recovery (MEOR) - Mechanisms of ME OR - Microbes in Mining - Glycerol production from algae - Fats and oils - Plastics - Biodegradable plastics - Acrylamide production - Bacterial cellulose - Enzymes - Ligninase enzyme Proteases and other enzymes - Production of Vitamin C - New short-cut process - Biogums - Biopolysaccharides

12. BIOTECHNOLOGY - AP).>LICATIONS TO ENVIRONMENT PROTECTION

225 - 249

Introduction - Towards improvements in sewage treatment - Fermentation effluents - Degradation of Xenobiotic compounds - Microbial degradation of surfactants - Use of immobilised cells in waste water treatment Bioremediation - Biological odorization - Biosensors in environmental control.

13. BIOTECHNOLOGY IN INDIA

250 - 264

14. PRODUCT QUALITY AND PATENT PROTECTION

265 - 280

15. BIOTECHNOLOGY - HOW SAFE

281 - 290

Biological warfare - Safety efforts - Impacts of Biotechnology - Ethical Issues - Social- Legal- Environmental- Issues related to Biotechnology.

16. ENTREPRENEURSHIP IN BIOTECHNOLOGY Entrepreneurship What is needed for Bioentrepreneurship efforts in India.

entrepreneurship?-

291 - 3ll

"This page is Intentionally Left Blank"

FEATURES OF BIOTECHNOLOGY One of the most recently-quoted definitions of 'Biotechnology' is: ''The applications of scientific and engineering principles to the processing of material by biological agents to provide goods and services." The Spinks Report (1980) deflned 'Biotechnology' as the application of biological organisms, systems or processes to the manufacturing and service industries. Both these definitions are vague on the aspect of nature of organism or agents involved and, therefore, may raise a question as to: "Is agriculture itself a biotechnology?" But it is not so and most of the other definitions used employ this term only for the use of microorganisms and cultured cells. The whole world today is so much shaken up with the word 'Biotechnology' that it has become a strong belief that this is 'the field' which will give solution to our 'any' problem. Biotechnology, no surprise, is then variously described as "the last revolution of the current century", "the third wave in the evolution of human ambitions," etc. Biotechnology has the technical breadth and depth to change the industrial community of the 21st century because of its potentials (i) to give products which were never available before; (ii) to give products that are currently in short supply; (iii) to give new methods which will reduce costs substantially; (ill) to give safer, better quality products; and (v) to give products which will use cheap raw materials which are plentily available but not used. Today, the world is facing four major problems - malnutrition, diseases, energy scarcity and its high cost and environmental pollution. To overcome these problems has become the objective of biotechnology development.

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Biotechnology is applied today most vigorously in four major fields: (a) Medicine and pharmaceuticals. (b) Animal health, food industry. (c) Plant agriculture. (d) Chemical manufacturing. Considerable work and applications of biotechnology are also seen in the field of environmental protection, pollution control, energy production at less cost, etc. Recombinant DNA technology and genetic engineering have contributed a great deal to the development of biotechnology. However, biotechnology in its true sense is a multidisciplinary applied science, with its constituent areas - microbiology, chemical engineering, chemistry, biochemistry, genetics and immunology. Technological innovations are the result of convergence of several independent paths. The success of biotechnology still depends on the impact of modern biology on chemical engineers. Chemical engineers will have to become familiar with the language of biological scientists. Using rigorously-controlled conditions, high-purity chemicals, moderate temperatures, recovery of small amounts of products from dilute fermented liquors, suppressing unwanted metabolic pathways, keeping genetically-engineered cells viable and working over a long period are some of the new tasks for the chemical engineer to handle in the biological processes. Success in biotechnology means economic success as well as scientific success. It is essential that process engineers are involved at an early stage in the planning of a venture in biotechnology. Biological, biochemical.and chemical understanding of a particular systems may perhaps lead to a scientific success but a critical appraisal of the intended application on a large scale with considerations to man, material and money to be invested will give the judgement on economic success. And still it is true that development in biotechnology has spelt so much success that it has left no area of human activity untouched and has been in a stage to revolutionise human life styles. Progress in biotechnology has been so breathtaking that what chemical technology has taken four centuries to achieve, biotechnology has achieved it in just four decades. Teaching and learning in diverse fields of biotechnology is hampered by the inability of students and specialists in each of the constituent areas - microbiology, chemical engineering, chemistry, biochemistry - to understand each other's language. This is because each lacks the basic knowledge required to understand and interpret even the most fundamental of concepts under discussion. Due to the different language, jargons and definitions used, crossing the interdisciplinary boundaries often becomes difficult. Keeping this in mind, in the forthcoming chapters of this book, an effort has been made to introduce the reader to the multidisciplinary subject of biotechnology by exposing him to its vast applications (commercially successful or scientific developments). Stress is given on citing numerous examples in each field of application and indirecdy making the reader familiar with the mixed language. It will not be a surprise if the reader (from any discipline) is fascinated so much as to start thinking, speaking and applying his mind in biotechnological language. REFERENCES 1. Alan Wiseman, "Features ofBiotechnology and its Scientific Basis" in Principles ofBiotechnology, Surrey University Press, New York, pp. 1-4, (1983). . 2. Purohit Mathur, Fundamentals ofBiotechnology, Agro-Botanical Publishers (India), Bikaner, 1990.

3. Chemical Weekly, Vol. XXX, (1985). 4. Chemical Weeko/, Vol. XXXI, (1986).

BIOLOGY BIOTECHNOLOGY - BIOTICS Supremacy of man over the other members of the living kingdom lies in the evolutionary state reached by human beings over the millions of years. Man has made progress by observing nature, studying its basic principles, making applications of information obtained, correcting the mistakes and again trying to achieve better control. Naturally, while doing this, he developed knowledge about facts basic to his life. So he collected information through observations about the structure, organisation, function, heredity, growth, reproduction etc., which are common to the different life forms, although their nature and complexity vary. This information collectively can be referred to as the basic life sciences - Fundamental biosciences - which consider the physical, mechanical and chemical phenomena highly related to the existence of life but also account for the relationship between life and matter in the form of vitalism. Fundamental biosciences have contributed a lot to the progress of human beings. Knowledge in the fundamental biosciences today is almost 20 times of what it was in 190 3 billion, > 1 billion and> 1 billion dollars respectively. Japanese scientists have synthesized a peptide that, at very low concentration, lowers high blood pressure caused by defects in renin-angiotensin-aldosterone systems. Alpha-melanocyte stimulating hormone (MSH) which has a potential use in the treatment of melanomas (skin tumors) and other skin diseases has been synthesised. This may later have applications in cosmetics also.

7.3.5 Microbial Enzymes -

Medical Uses

Microbial enzymes have a lot of importance in medicine. According to one estimate, microbial enzymes will be 60% of the total S 1 billion diagnostic enzyme market. Microbial enzymes find their applications in therapy as well as in testing ki~s. Enzymes finding applications in therapy for variom disorders I~;'ly be either of microbial origin or produced in microorganisms by the introduction of appropriate gene from human beings for the production of respective enzymes. Certain types of cancer cells lack the ability to make their own aspargine (aminoacid) and hence depend for their requirements on supply through blood. Asparginase enzyme if injected in children suffering from blood cancer, acute lymphoblastic leukemia will show significant improvement. This is presumably due to the action of asparginase enzyme on aspargine in blood. Cancer cells though affected, normal cells do not have much disastrous effect. Biotechnology's role will be to produce cheaply the enzyme asparginase. Most of the medical supplies today are obtained from Erwinia.

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Methotrexate (MTX) is widely used as an anti-cancer drug damaging dihydrofolate reductase enzyme. This enzyme is required by cancer cells. MTX is not selective in action and also affects normal cells. Since different patients break down the drug at different rates, it is important to monitor the level of MTX in the body. This can be done by in-vitro testing by effect on dihydrofolate reductase enzyme of MTX from the blood sample. Here dihydrofolate reductase enzyme obtained from Lactobacillus casei is used. Gaucher's disease is one of the ten known hereditary lipid storage diseases doing damage to the nervous system. Children suffering from this disease lack enzyme which normally breaks down lipids in the body, glucocerebroside. This material then accumulates in the liver, spleen, bones producing swellings, damage to nerves and death in children before reaching the age of two. If the right enzyme is injected to cells enforged with this lipid, the child could be relieved of the symptoms of Gaucher's dis 15% of pepsin is considered unsuitable for typical Dutch cheese manufacturing. Proteases from different microorganisms are used for development of flavour in cheese.As there is shortage of calf rennet alternate proteolytic enzymes are being investigated.

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None of them are as suitable as chymosine. Proteasc:;s from Mucor miehei and Mucor pusillus show nearly the same specificity as chymosine. Minor differences in flavour development is observed in long maturation requiring cheeses in rennet and microbial enzymes. Thermostability of microbial enzymes is problem in application. Now by mild oxidation thermostability of microbial enzymes is reduced without affecting original activity and the problem of residual active enzyme and its interference in formulation of dairy products and baby foods from whey is solved. Recombinant DNA technology was used to transfer calf rennet gene to yeast. This is effective and reduces the need of suckling calves. Lipases: Lipases are used along with proteses for preparation of EMC (Enzyme modified cheese). Lipases are also used in certain Italian cheeses to give them their characteristic flavour. In this case enzymes are used to create natural flavour and not for development of flavour concentrate. The enzyme is added to the milk before the addition of rennin and is activ~ under normal ripening conditions.An example is the use of Picantase, a lipase produced by the fungus Mucor michei for the production of Remano and Provolone. Brittlefat can also be modified to give a unique flavour. The modified fats are used in confectionary (toffe.es) as coffee whitner and in bakery products. Enzyme Modified Cheese (EMC): Proteases and lipases from different microorganisms are used for the production of cheese flavours. The technology consists of the incubation of the natural cheese slury with specific proteases and lipases, usually at 37°C for a period of 2 days to two weeks, followed by inactivation of the enzyme and formulation of the end product into paste. EMC is availablle in variety of strength and with different strength with regard to the flavour concentrate (e.g., 5 to 20 times the intensity of normal cheese). EMC are used as cheese substitutes in number of products such as snacks, pizzas and cheese dips. Cheese Industry: Starter culture used has Streptococci to ferment milk. If streptococci are attacked by phages to which they are very much susceptible, milk will not ferment properly and thousands of dollars of milk will be wasted. To avoid this, by genetic engineering technique, Streptococci which are resistant to phage attack but efficient milk fermentors, are being produced. Rennin enzyme (obtained from the stomach of calves) is required during cheese-making. To reduce the costs of rennin, gene of calf rennin enzyme is beiRg cloned in bacteria and yeasts. Geneticallyengineered fungi-bearing gene of calf cells for rennin enzyme are now used in the UK to obtain rennate enzyme preparation. Chymosin enzyme will also be present in this rennate preparation as it is there in calf rennet.

10.7.4 Degrading Raw Starch A Japanese researcher has discovered a microorganism that produces amylase which is easily absorbed by raw starch and which could be used to control starch ageing in food products. The enzyme will decompose raw starch to glucose when it is just mixed with water. Therefore, in the food production process, sugar addition for ageing control will not be necessary. Common amylases cannot decompose raw starch but this enzyme can. It can find its applications in noodle and confectionery making.

10.7.5 Bacterial Silage Additive A concentrate of biological silage additive is unveiled by the ICI Agricultural Division. The product known as 'Ecosyl' consists of a selected strain of L. planatarum. At the recommended application rate, it adds 1 billion live Lactobacilli to every tonne of grab. By dominating fermentation, it ensures correct biological action to make nutritious and stable silage. The dry product is supplied in a novel plastic mixing bottle sufficient to treat 33 tonnes silage. It is applied to grass for silagemaking through the conventional liquid application equipment.

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10.7.6 Brewing Industry In beer-making the use of bacterial enzymes to reduce the costs on malting and also the use of combination of various proteolytic enzymes to remove haze in beer are already in practice. Beers with low concentrations of carbohydrates, i.e. light beers, are popular in the US, the UK and many other countries. Light beers are available in the market for many years. Dextrin makes up 1/4th of carbohydrates in the original fermentation liquid. Brewers' usual yeast cannot ferment dextrins. Saccharomyces diasticus which is not used in the industry can turn dextrins into alcohol, thus reducing the carbohydrates of beer. The genes responsible for this have been introduced into the normal brewing yeast. At present, the work is at research stage but very soon, beer with no carbohydrates and better taste will be made using this gen~tically-engineered brewers' yeast. Scientists working at a Danish brewery are collaborating with workers at the Lo Trobe University, Melbourne who have isolated a barley gene which encodes for the enzyme (barley beta glucanase). The enzyme degrades betaglucan molecules which can clog filters in the brewing process and form haze. The gene responsible for this enzyme production has been cloned in brewing yeast that eliminates haze formation and improves beer quality. Immobilised yeast cells are likely to be used in the future and with a high cell volume, primary fermentation will occur in just 16 hours instead of 7 days and maturation which today takes several weeks will occur in a few hours. If the two processes are combined successfully, the overall process time will be about 24 hours, without affecting quality. In Finland it has already progressed on a semiindustrial scale. Research in Beer Brewing Technology: Brewing Research Foundation International (BRFI), has received approval for sale of the world's first beer made with gene technology. This beer is called as 'Nutifield Lyte lager.' It is produced with the help of a genetically engineered strain of yeast. The beer contains about 1% by volume more alcohol than beer made with unmodified yeast, because the genetically engineered yeast can convert more sugar into alcohol. The new yeast carries an extra gene that helps to break down dextrins which the natural yeast cannot degrade. Dextrins account for 25% of the sugars that might be converted into alcohol during fermentation. The gene called STA2 responsible for dextrin breakdown comes from a variety of baker's east (S.cerevisiae, var, diasticus). This strain in used by industrial distillers, but cannot be used for brewing because it produces off flavours. The gene makes are enzyme called glucoamylase which breaks down dextrins to alcohol. Otherwise brewers add extract of this enzyme to the fermentation vat. These extracts taken from fungus may be impure and can destroy peptides responsible for the foamy head on beer. In contrast, there is no such problem when genetically engineered yeast produces this enzyme. Nutifield Lyte lager beer has been cleared by the UK govenment. Further an additional gene from Aspergillus niger for glucoamylase is being inserted in brewing yeast for more conversion. Japanese and German researchers are working to eliminate a fermentation by-product called diacetyl, which produces a buttery off-flavour in beer. During fermentation, some yeasts leak a substance called alpha-acetolactate and this gets converted to diacetyl. Ultimately diacetyl breaks down to tasteless compound called acetoin. A gene for alpha-acetolactate decarboxylase enzyme that suppresses diacetyl formation is cloned by brewery at Yokahama, Japan. In Germany, Department of Microbiology and Genetics at Berlin University of Technology employs yeasts that make an alpha-acetolactate decarboxylase enzyme that converts alpha-acetolactate directly into acetoin. So there is no diacetyl formation at all. The use of genetically engineered yeast cells in future will display tolerance to very high ethanol concentrations, for production of high gravity beers.

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Use of enzymes in the brewing process Enzyme Used

1. Thermostable bacterial-amylase

2. Fungal amyloglycosidase

3. Bacterial or fungal l3-glucanase 4. Plant proteases (e.g. Papain) 5. Acetolactate decarboxylase

Application Liguifies cereal adjuncts flavour extraction fruit and vegetables prior to addition to mashing vessel. Hydrolyses the limit dextrin in brewer's wort prior to yeast fermentation stage. (This results in lowering of carbohydrates but slight increase in alcohol, thus result is low carbohyd rate beer for diabetics.) Lowers viscosity of wort by hydrolysis of barely glucans, (This improves extraction and filtration of mash). Chill haze prevention in the beer. Converts alpha-acetolactate to acetoin and avoids off flavours otherwise due to diacetyl formation, allows fast maturation of the beer.

10.7.7 Wine Industry Vinoflow is a new enzyme preparation developed by Novo Nordisk Ferment in Switzerland which helps to overcome clarification and filtration difficulties which are a major challenges to winemakers. Vinoflow is a blend of pectinases and glucanases and is used immediately after alcoholic fermentation. The high temperature of the young wine at this stage (more than 15°C) is necessary for the enzymes to work more efficiently. The wine treated with Vinoflow became much clearer and more stable. The colour and the body of wine also improved.

10.7.8 Fibre for Human Consumption Genetically-engineered yeast has been used to obtain a range of fibres for human consumption. It is called 'fibrecell' and is glucan of yeast cell wall and is related to glucans that form fibre components of oat etc. It has desirable properties such as controllable bulking, controllable degradation by bacteria in colon and cholesterol-reducing properties. It is tasteless, odourless and nongelling. The product contains 90% fibre as compared to 18-20% in the oat bran. Very soon the product will be commercialised as a food additive.

10.8 FOOD PRESERVATION - USE OF ENZYMES In sensitive food preparations, the use of glucose oxidase/catalases has found applications in packaging as an oxygen scavenger for milk powder, egg products, coffee. Catalase has been found alone as agent for sterilisation in milk production and cheese-making. Oxidoreductases such as lipoxygenase has been used in flour treatment and as a flavouring component. It also contributes to flavour development in fruits and vegetables. Catalase has been shown to improve creamy flavour in dairy products. Some oxidoreductases such as sulfahydril oxide may have application to remove off flavour in heat-sensitive products like long life milk. In eggs produce before drying yeast or bacteria were used to ferment glucose in egg white. This was to prevent nonenzymatic browning reaction during storage. Browning (glucose protein reaction) results in discoloration, loss in protein solubility and off flavours. Therefore, by removing glucose from egg products this can be prevented. Yeast, if added for this purpose, causes yeasty flavour, also chances of contamination are there. Use of glucose oxidase/catalase enzyme preparation is beneficial now. Glucose oxidase converts glucose into gluconic acid and hydrogen peroxide while catalase breaks down peroxide into water and oxygen.

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Now 'Novamyl' is the new antistaling enzyme for bread developed by Novo Nordisk Bioindustries Inc. (US). It has passed the tests of the baking institute. It extends shelf life of bread by 3 days compared to other similar enzymes in the market, without causing gumminess that can result from enzYme overdose.

10.9 A NEW YEAST DEVELOPED TO COLOUR THE FARM CULTIVATED SALMON IN USA Salmon consumption is growing in USA by 12% a year and future consumption rates expected to continue at double digit rates. The worldwide market for salmon is rated at 100 million per annum. Universal bioengineering has recently developed in response to growing development of farm cultivated salmon a 'Red Star' brand of 'Phaffia' yeast as new source of astaxanthine - the natural pigment in the pink fresh of wild salmon. Synthetic material has been traditionally used cor this purpose by fresh farmers, who must compensate for the lack of astaxanthine in farm settings.

10.10 FLAVOURING COMPOUNDS AND FLAVOUR ENHANCERS Flavour is an important component of consumer's appreciation of food contributing to its smell, taste and interacting with mouth-fill and colour of final product. Many food flavours have been generated by trial and error while increasing stability of the food. For example in vinegar, cheese, yoghurt, beer, or wine - preservative effects of microbially generated molecules (such as acetic acid, methylketones, propionic acid and ethanol) were accompanied by the desired flavour. The presence of complex mixture of acids, alcohols, esters etc. gives the individual character and identity to the food and beverage. Some flavours are derived from plant sources (from India, Indonesia and Africa). With advent of sophisticated analytical techniques complex flavour mixtures have been extensively analyzed and compounds are classified into classes like green, sweet, fruity, roasted etc. Although various flavour compounds can be synthesized chemically and are identical to natural ones, there is growing preference for 'natural' products. Therefore there is interest in biotechnological production of flavour.

Flavouring complexes (1) Cheese flavour - Use of lactic acid bacteria and fungi is made. (2) Enzyme modified cheeses (EMC) - Use of short chain fatty acid-specific lipases (3) A blue cheese-type of flavour is generated by Penicillium species e.g. Penicillium roqueforti. The free fatty acids that are generated from cheese by lipolysis are relatively toxic to P. roqueforti and as a detoxifYing mechanism the fungus converts them into methylketones. Fatty acids - Beta oxidation - Beta keto acids - decarboxylation - Methylketones RCH2 CH2COOH

~

RCOCH 2COOH

~

RCOCH 3 + CO 2

As both short chain and long chain fatty acids are partly metabolized a variety of methylketones arise. The main ones produced when milk fat is used as substrate are (1) 2-pentanone (2) 2-heptanone (3) 2-nonanone. Exploiting bioconversions such as oxidation, reduction or synthesis by either microbial fermentation or by using specific enzyme system also produces flavour compounds. Cheaper guar gum can be converted to more valuable locust bean like gum by the action of alpha galactosidase from guar seeds cloned in Saccharomyces cerevisiae. Enzyme used has peculiar property of being able to remove galactosyl residues that are alpha - 1,4 linked.

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Flavour Enhancers: Food additives, which enhance sensory response are produced either by microbial fermentation or by enzymatic reactions or in combination with chemical methods. Inosine 5' - monophosphate (5' IMP) and Guanosine 5' - monophosphate (5' GMP) can be produced by degradation of RNA by 5' - phosphodiestarase. Microbial or plant RNA is used. 5' IMP arises through conversion of adenosine 5' monophosphate (5' AMP) by adenosine deaminase. The production of 5' IMP and 5' GMP from the yeast Candida utilis is shown in the flowsheet. RNA used is by-product of single cell protein (SCP) production for food from this organism. Candida utilis

1Moll",~

Biomass

l.

Drymg

!

. Hot alkal'1 Extraction

1

Ce!trifugation, Filteration ---+ Discarded biomass (SCP) Extracted RNA

! !

Precipitation,

Centrifugatio~

Discard centrifugate

Recovered RNA

!

Dissolve in Buffer pH5 Hydrolysis with Exonuclease from Penicillium citrinum

! ! Adsorption on Activated Charcoal

AMP/GMP/CMP/UMP Mixture

~

CMPJUMP

!

Elution (with Methanol/Ammonia)

l ! Deamination of AMP (by adenylic acid deaminase from Aspergillus oryzae) AMP/GMP Mixture

l

,

Fractionation (Ion Exchange Column)

/ 5'TMP

5'GMP

Fig. 10.1 IMP and GMP Production by Candida utilis

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Monosodium Salt of L-glutemic Acid (MSG): Hundreds of thousands of tons of MSG is produced annually worldwide. MSG is produced by fermentation of sugars with Corynebacterium glutemicum. With strain selection, classical genetics, and optimization of fermentation has resulted in product yields of 30-50grams per litre. Further improvements are possible by manipulation of glutemic acid metabolic pathway by genetic engineering. Flavour precursors

,

Diacetyl: Diacetyl gives typical buttery flavour. Citrate

Citrate permease

.~

citrate ~ citratase Acetic acid + Oxaloacetic acid

1

Oxaloacetate decarboxylase

, - - - - - - - - - Pyruvate + CO 2

/1

,Acetic acetalfehyde

Acetoacetic acid

..--{ I

'It

~

~

Acetoin

I

Acetiyl CoA

t

* a acetoacetate decarboxylase enzyme

Diacetyl

CO 2

Fig. 2 (Diacetyl Production)

It is proposed to increase the level of diacetyl precursor by reducing the amount of alphaacetoacetate decarboxylase by genetic manipulations. Acetaldehyde: Acetaldehyde is a significant flavouring component of yoghurt and fruits. Procedure have been described for the conversion of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) and alcohol oxidase (AOX). Oxidoreductases require cofactors, which are relatively complex to produce and to regenerate. Systems involving hydrolases (such as proteases, lipases and pectinases), which do not require cofactors, will be better. ADH Ehanol + NAD+ CH 2CHO

t

CH 3 CHPH

ADH

•

(ADH) NAD

light

Acetaldehyde + NADH

(MN) 0, (,0) FMNH 2

Fig. 10.3 Acetaldehyde Production

°2

Catalase \....H 2 O

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A process with conversion of 10-20% and a concentration of 2.5gram per litre after a period of 9 hours has been reported. Modern biotechnological methods will greatly improve the conversion rates. Lactones: Lactones have pleasant coconut- and peach-like fruity flavour and can be isolated from various classes of foods (fruits, vegetables, nuts, meat, milk products). Fermentative production has been described with yeast (Candida species) using ricinoleic acid (12-hydroxy-octadecyl-9-enoic acid) as starting material. Beta-oxidation also plays important role here. Sporobolomyces odorus, Trichoderma virideac, and Polyporus porus are other fungi, which will carry out the reaction. 12-hydroxy-Octadec-9-enoic acid CH 3 - (CH 2 )s - yH - CH 2

-

CH = CH -

(CH2 h

-

COOH

-

CH = CH -

(CH 2 )s -

COOH

OH 1O-hydroxy-hexadec-7-enoic acid CH 3 - (CH 2 )s - CH - CH 2

I

+

OH

~ ~

4 hydroxy decanoic acid

CH3

--

(CH 2)s -

.

CH -

I

CH 2

-

CH 2

-

CH 2

-

COOH

OH

i

V-decalactone

CH3

-

(CH 2)s -

CH -

0" I

CH 2

I

/CH C

2

II

o Fig. 10.4 Lactones Production

Esters: Esters are responsible for fruity flavoured aromas. Ethyl acetate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate are produced by various species of Pseudomonas, Lactococcus, Lactobacillus and yeasts (Hansenula anomala) and (Candida utilis). Uses of lipases are thought as other strategy. Pyrazines: Pyrazines are heterocyclic, nitrogen containing compounds, which are often associated with roasted and nutty flavours. Methoxy alkylpyrazines have been found in variety of vegetables including bell papers (specifically 2-methoxy-3-isobutyl pyrazine), potatoes and green beans. Fermentative production of 2-methoxy-3-isobutyl pyrazine has been described by Lactobacillus lactis, Pseudomonas perotens and P. taetrolens and tetramethyl pyrazine by Bacillus subtilis, Corynebacterium glutemicum. Green Components: Green components like C-9 compound nonenal and nonadienal and C-6 compounds like hexanal and hexenal play an important role in overall flavour of cucumber and apple/tomato type of aromas. They arise through degradation of unsaturated fatty acids via lipoxygenasecatlaysed formation of hydroperoxides followed by cleavage by hydroperoxide lyase. Lipoxygenase from different sources vary in their pH optima and substrate specificities. Soyabean lipoxygenase produces mainly C-13 hydroxyperoxides, whereas that from tomato produces C-9 hydroxyperoxides.

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Enzymatic systems for the preparative scale production of such green flavour compounds via oxidation of fatty acids have been described. Modern biotechnological methods can be useful here to overcome the lack of availability of lyase enzyme. Pungent taste: Pungent taste like that of mustard, cress and horseradish are due to formation of isothiocyanates from odourless precursors known as glucosinolates by the action of enzyme myrosinase.Immobilised myrosinase enzyme can be used for continuous production of horseradish aroma. Table 10.2

No.

Flavouring Compound

Flavour

1

Diacetyl

2

Acetaldehyde

3

Lactones

Pleasant, coconut, peach like

4

Esters (ethyl acetate. Ethyl butyrate, ethy isovalerate, ethyl hexonate)

Fruity

5

Pyrazines

Roasted, nutty

6

Noneal, Nonadienal, hexanal

7

Isothiocyanate

Pungent

8

Methylketones

Blue cheese type

9

IMP, GMP

10

Monosodium, (MSG)

Food

Buttery Yoghurt, fruit

Glutamate

Cucumber, tomato, apple Mustard, Cress, Horseradish

,

10.11 BIOGUMS Although biogums are not m1Jch known in India, it is a big business in the US and Europe. Biogums are used as suspending, thickening and gelling agents mainly in foods and pharmaceuticals. Worldwide sales of microbial polysaccharides were US $ 300 million as per 1992 reports. These biopolymers are valued primarily for their ability to act as thickeners, and as hydrophilic colloids to emulsify, suspend, and stabilise mixtures in water-based systems. They often exhibit compatibility and synergy with one another, under wide variety of pH, temperature and salt conditions. The Kelco Division of Merck & Co. is the world's largest leading manufacturer of biogums. The company has recently announced plans to undertake $ 29 million expansion of its biogum production at San Diego and at Okumulgas (Oklahoma). Biogums are produced by fermentation at both the plants. Many microbial exopolysaccharides (EPS) have been described in some structural detail and might have 'useful industrial properties', but polymers from pathogenic microbial species are unlikely to be acceptable. Only those microbial polysaccharides which show good yield and have properties superior to xanthan, curdlan, or gellan or traditional plant and algal gums are likely to develop. Several microbial polysaccharides are now widely accepted commercial products. Major technological

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development is required in the recovery of microbial gums from fermentation broth and its drying. Effective use of ligno-cellulosic wastes or whey as carbon source for producing microbial gums would be advantageous. Although many microbial polysaccharide structures have now been elucidated, industrial usage of polysaccharides (gums) still relies extensively on material obtained from plants or marine algae. Such traditional commercial polysaccharides include starch, dextran, alginate, gelatin, carrageenan, gum Arabic, gum tragacanth (used in toothpaste), china grass (used in jams and jellies) and the plant glycomannans - locust bean gum, gum guar and konjac mannan which are widely employed in the food and pharmaceutical industries. All the gums from plant or algal sources do not have desired rheological properties. Microbial polysaccharides provide replacements for gums in current use, or novel materials with unique or improved rheological characteristics, which may find new applications. Although plants produce a wide range of polysaccharides, their diversity is considerably less than those produced by microorganisms. This more diversity in microbial polysaccharides is due to some 200 different sugars found in them compared to only about 25 sugars associated with polysaccharides of plant origin. Advantages of Microbial Gums (1) The collection of gums from plants is laborious process and requires skilled labour. (2) Seasonal variations affect both quality and quantity of gums that can be obtained from plants, which is not a problem with microbial gums. (3) With traditional supplies of gums from plant or marine algae sources problems can arise due to crop failure, drought, war, climatic conditions or to marine pollution, famine and disease which is not the case with industrial production of microbial gums. High technology equipment, well-trained staff and adequate power and water supplies, however is required for production of microbial gums. (4) Microbial gums have unique rheological and phsico-chemical properties. The rising commercial importance of microbial gums is due to their ability to alter the flow properties or rheological characteristics of aqueous solutions. (5) The viscosity of microbial gums is not significantly affected by acids, alkalies, salts, surfactants (soaps and detergent chemicals) and organic solvents even after a prolonged contact qf 3 months. Better resistance of solutions of microbial polysaccharides to degradation by acids, alkali, free radicals, hydrolytic enzymes, shear forces is due to their ordered conformation (single/double/triple helix structure of regular repeating uni.ts of monosaccharides). (6) They often exhibit compatibility and even synergy with one another and under wide variety of temperature, pH and salt conditions. (7) Microbial polysac'charides show strong pseudoplastic behaviour (reduction of viscosity with shear and recovery of viscosity when shear rate is decreased) which distinguishes solutions of microbial polysaccharides from other thickeners. Important Microbial Gums (1) Xanthan - This is made up of beta-D-glucose units, which are linked in a way identical to the linkages between the glucose units of cellulose. Each alternate glucose in the chain is attached with a trisaccharide side chain consisting of two mannose sugar molecules and a glucuronic acid molecule. More than 20,000 tonnes of xanthan is produced worldwide per annum. The viscosity or thickness of xanthan solution decreases when it is subjected to shear pressure. As soon as the pressure is released the gum returns to its original viscosity. Tllls property of instantaneous viscosity reversal

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is known as non-Newtonian behaviour. In other words, xanthan gum show pseudoplasticity. Xanthan gum is best suited for enhanced oil recovery operations due to its special rheological properties. The commercial production requires a very high level of engineering and technical expertise as well as marketing skills. Physical properties of xanthan make it useful as a stabilizing, emulsifying, thickening and suspending agent. Xanthomonas campestris produces this biopolysaccharide as a by-product of its metabolism. x.campestris has been genetically engineered to use whey as source of carbon for production ofxanthan. Genetically engineered X. campestris produces 4241mcg/ml ofxanthan using whey while its wild type produced only 224 mcg/ml of xanthan with whey as a substrate. It is used as a gelling and stabilising agent in salad dressings, ice creams, toothpastes, cosmetics, water-based paints, etc., and also as a drilling lu.bricant in oil wells. The gum itself is colourless. Xanthan is possibly the 'benchmark' product, gaining approval for food use many years ago. It is a relatively inexpensive product because of the very high potential conversion of substrate to polymer. Xanthan gum is the natural mammade polysaccharide of microbial origin. It was the first biotech gum developed in USA. Xanthan gum is white to cream coloured free flowing powder soluble both

in cold and hot water but insoluble in most organic solvents. Its industrial importance is on its ability to control the rheology of water based systems. Even at low concentrations xanthan gum solution shows high degree of viscosity in comparison with other polysaccharide solution. This property makes it very effective thickner and stabilizer. Xanthan gum is compatible with all commercia thickners and stabilizers. Applications and usage level include Salad dressings

(0.1-0.5%)

Baking products

(0.1-0.4%)

Soups and sausage

(0.5-0.5%)

Meat products

(0.2-0.5%)

Confectionary

(0.1-0.4%)

Dietic products

(0.3-0.5%)

(2) Pullulan - This is produced by Aureobasidium pullulans (Black yeast). This is a glucan gum composed of maltotriose units, and small number of maltetraoses units (1,4 ex linked), which are coupled through 1,6 ex bonds to give linear molecule. Pullulan forms gels on heating. Films of pullulans have extremely low permeability to oxygen as compared to cellophane or polypropylene film. Pullulans are biodegradable while cellophane and polypropylene are not. Therefore fruits, vegetables or other food material can be preserved with coating of pullulan solution. Transparent sheets of pullan are manufactured commercially for this purpose. It is also used in fibre, film, packaging industry. It is used as flocculating agem in clay suspension in mining. Pullulan is not degraded by many amylases. Pullulan is fully water-soluble. Approximately 10 tonnes of pullulans was produced in Japan in 1990. (3) Curdlan - This is produced by Alcaligenes faecal is var. myxogenes strain 10C3 or Agrobacterium. It is a polymer of beta-D-glucose. Curdlan gum becomes a gel on heating. It is used as gelling agent in cooked food. It is also, used as a molecular sieve for filtering antibodies and proteins. It is also used as a support for the immobilization of enzymes and as binding agent. Curdlan is also supposed to have anti-tumor activity. Curdlan is manufactured and used as a gelling agent in various food preparations in Japan, but is not an accepted food ingredient in either the U.S. or the E.U. (4) Scleroglucan - Scleroglucan is a homopolymer of glucose and is produced by Sclerotium glucanicum. Scleroglucan has chemical composition showing backbone consisting of~-D-glucopyranosyl

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monomers linked (1-3) and every third sugar is substituted with a further glucose monomer linked f3 (1.6). Due to this conformation it is more resistant to degradation at elevated temperatures. The gum is highly viscous and is pseudoplastic, and is useful in enhanced oil recovery with oil-well drilling operations. Scleroglucan is non-ionic, unlike the other microbial polysaccharide viscosifiers, and thus is compatible with cationic surfactants Scleroglucan is also used in ceramic glazes, latex paints and printing inks. (5) Dextran - This is produced by Leuconostoc mesenteroides. It is composed of D-glucose. Dextran sucrase is the enzyme secreted by bacteria which is involved in synthesis of Dextran. Dextran is used as blood expander or as plasma substitute during transfusions. Dextran can be crosslinked to form a gel or anion or cation exchangers such as diethylaminoethyl, (-DEAE) or carboxymethyl (CM) can be attached to sugar molecules. These then can be used for purification of proteins. (6) Alginate - Current annual production of alginates is more than 25,000 tonnes. Brown algae are the major source of alginates. Pseudomonas spp. and Azotobacter spp. produce alginates. It is produced by Azotobacter vinelandi. It is used as thickening and gelling agent in dairy products, to control ice crystal formation in icecream. Alginates are also used for viscosity control in dairy products. Small amounts of alginates are also added to some other food products to prevent drying-out and to stop the product from becoming tough or rubbery. Propylene glycol alginate is incorporated as suspending agent into fruit-flavoured drinks and is also used as stabiliser in beer. Alginate gels give products of uniform size and shape when used for fruit pieces, onion rings etc. Alginates are also used to control the rheology of fibre-reactive dyes in printing and paper coating. Microbial alginates have to face some regulatory issues for use in foods. (7) Gellan - It is produced by Pseudomonas elodea, ATCC 31461. It can also be produced by Sphingomonas paucimobilis. Gclrite is the gellan which can be used in microbiological media to replace agar and carrageenan. Improved clarity and low use-levels are the advantages. It is thermostable gel useful in plant cell culture media. Gellan is also used as gelling agent in dental and personal care toiletries. Gellan is also used in deodorant gel products (to replace carrageenan-locust bean gum mixture). Gellan when mixed with starch can be used as size in paper manufacturing. Initial FDA approval for use of gellan in foods as additive in low-solid jams, jellies, frostings, and icings is granted. (8) Zooglan Gum - Wisconsin Alumni Research Foundation have developed woglan gum U.S. Patent No. 5,118,803. This woglan gum WARP: P90046US is formed from the fermentative action of a Zoogloea ramigera cultured on whey, whey permeate, or other sugar containing media. Its properties are enhanced over other zooglan gums because of its high degree of modification by succinate groups by maintaining some succinate on the gum, not removing it all as is usual practice. It is excellent in removing metals such as cadmium, cobalt, nickel, and iron, from wastewater (e.g., sewage, factory, other) by binding them. It is claimed to have superior properties as a thickening, suspending, stabilizing, lubricating, and binding agent in food and non-food commercial application. Suggested applications are waste water treatment, food products, paper products, building materials, well drilling materials, cleaning products, cosmetics, pharmaceuticals. adhesives, pastes, building materials, cleaners, polishes, seed coatings, binders, wet-end additives and coatings for paper products, petroleum and water well drilling muds, cosmetics, pharmaceutical suspensions and emulsions. (9) Zanflow industry.

It is produced by Envinia fahitia. It is used in carpet printing and in paint

(10) Polymer S-130 and S-194 - It is produced by Alcaligenes spp. It is used as suspending agent for pesticides and as lubricant in oil industry. (11) Hyaluronic Acid - Hyaluronic acid is produced by Streptococcus as extracellular polysaccharide. Hyaluronic acid is used in cosmetics and pharmaceutical industry as hydrating agent.

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Hyaluronic acid is also used in eye and joint surgery. A characteristic property of use of hyaluronic acid which is important is the high viscosity in aqueous solutions at relatively low solute concentrations. These solutions also exhibit elastic properties. Other new gelling polysaccharides have been isolated from various bacterial species. Each tends to have specific properties of interest. Enterobacter XM6 gels in association with monovalent or divalent cations. The gels melt and set close to 30°C and show absence of syneresis. Two other polysaccharides which gel in the presence of cations are 207 beijeran from Azotobacter beijerinckia and a novel polymer from a mutant of Rhizobium meliloti - an acetylated homopolymer of 1,4D-glucuronic acid. Following chemical de acylation, both these polysaccharides gel in the presence of suitable cations. The IGI Biotechnology (USA) has unveiled production of polysaccharide substitute to Gum Arabic. The product is produced by fermentation of raw cane or beet sugar. The fermentation process produces a white sprat dried powder, which is composed of repeating alpha-linked fructose monomer unit. It can be used as a substitute for Gum Arabic, which is often erratic in supply due to seasonal or political instability. This development should be of great importance to sugar-producing countries like India. REFERENCES 1. D. L. Collins Thompson, J. D. Cunningham and J. T. Trevors, "Food Microbiology and Biotechnology - An Update," in Biotechnology Applications and Research, (Editors), Paul M. Cherimisinoff, Robert P. Quellette, Technomic Publi. Co., USA, pp. 188-195, (1985). 2. Steve Prentis, "Biotechnology from Farm to Supermarket" in Biotechnology: A New Industrial Revolution (New Revised Edition), Orbis, London, pp. 145-155, (1985). 3. ET update. News Magazine ofScience and Technology, "Project on Embryo Transfer," Vol. I, 1991. 4. G. A. Beech, M. A. Melvin and J. Taggart, "Food, Drink and Biotechnology" in Biotechnology: Principles and Applications, (Editors), 1. J. Higgins, D. J. Best, J. Jones, Blackwell Scientific Publications, London, pp. 73-113, (1985). 5. Keshav Trehan, "Microbial Production of Food", in Biotechnology, Wiley Eastern Limited, New Delhi, pp. 79-88, (1990). 6. Chemical Weekly, 1991, Vol. XXXVI, Vol. XXXVII - 1992. 7. J. c. Senez, "Single Cell Protein, Past and Present Developments", in Microbial Technology in the Developing World, (Editors), E. J. Dasilva, Y. R. Dommergues, E. J. Nyns, and C. Ratledge, Oxford Science Publications, Oxford University Press, New York, pp. 238-239, (1987). 8. John H. Litchfield, "Production of Single Cell Protein for Use in Food or Feed," in Microbial Technology, Vol. I, (Editors), H. J. Peppler, D. Perlman. Academic Press, New York, pp. 93-146, (1979). 9. Chemical Weekry, Oct., 20, 1992.

BIOTECHNOLOGY - INDUSTRIAL APPLICATIONS 11.1 INTRODUCTION Every scientific research that has economic implications and applications for the benefit of human beings ultimately emerges out in the form of industry to commercialise the scientific principles. No doubt, then, many developments in the field of biotechnology have resulted in successful essential industrial processes. That way producing more sophisticated products like monoclonal antibodies, interferons, hormones, biosensors or others like food, agricultural plantlets, seeds are all biotechnology-based industrial processes. But their uses and conventional thinking treat them more as a part of other respective branches of biotechnology. And then discussing industrial applications of biotechnology means discussing the extension work of old (relatively) fermentation processes for products like vitamins, antibiotics, steroids, enzymes, alcohols, glycerol, organic acid and some new chemicals like plastics, polyacrylamide etc. Thus it talks more about producing chemicals through biotechnology. Applications of microorganisms to oil recovery and metal recovery though suggested and under development for long many years, there have been ups and downs in the importance given to these applications. These fields obviously suit in the discussion in this chapter than anywhere else. Without elaborating much on the earlier state of achievements, an attempt is made here to put before the readers further advances. It won't be untoward to mention here the success of biotechnology which can be exemplified by the fact that the cost of steroid (cortisone) by chemical process was $ 200 per gram in the 1930s, which came down to $ 6 per gram by 1980s with microbiological conversions (that started their application since 1950s) and has further reduced to $ 0.46 per gram now due to further improvements in biotechnological processes and less dependence on high temperature, pressure and expensive chemicals required otherwise for chemical methods.

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Industrial Applications

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11.2 ANTIBIOTICS There are about 100 major antibiotics in the market and around 5,000 have been identified and isolated. Four major classes of antibiotics are Penicillins, Tetracyclines, Cephalosporins, Erythromycins and the sale of these four antibiotics alone as bulk drugs is over 4 billion dollars per year in the US. Antibiotics mostly are not proteins but arise out of a series of reactions as secondary metabolites. They are produced by a restricted group of organisms. Genetic engineering cannot be applied as easily to antibiotic improvements as in the case of other protein products because a number of genes controlling the number of reactions are involved in the control. Biosynthetic pathways are not fully understood for most of the antibiotic formation. After the sensational discovery of penicillins and their subsequent effective applications, mutations and other gene manipulation techniques have enabled us to get thousands of times more yields than its natural producer's capacity. Biotechnology is not only gene manipulation and the proof of this in this field is semisynthetic penicillins which are obtained from penicillin G which is a fermentation product. Enzymatic and chemical conversions coupled with fermentation can produce dramatic results as in the case of semisynthetic penicillins. Still newer antibiotics are constantly being searched for with any of the following targets in mind: (I) broad spectrum action; (2) versatility of application; (3) antineoplastic agents; (4) high potency; (5) high yields in process; (6) reduced toxicity; (7) improved production techniques. Eli Lilly and Company and Merck, Sharp and Dohme (MSD) have reported new betalactum antibiotics in fermentation broth of streptomycetes. These compounds are cephalomycins and have a structure similar to cephalosporin with methoxyl group. Cephalosporin C is a semi-synthetic antibiotic, active on both gram positive and gram negative organisms but is destroyed by betalactamase enzyme. MSD has discovered a new class of betalactum antibiotic, Thienamycins, from Streptomyces cattleya. They are capable of inhibiting betalactamase action. Three thinamycins are: Cefoxitin, Clavulanic acid and Thinamycin. Clavulanic acid is now marketed in combination with betalactum drug amoxicillin. Pharmaceutical hybrid known as Augmentin is also resistant to betalactamase action. The world's first-ever antibiotic produced by genetic engineering was developed jointly by John Innes Lab (UK) and Kittasato Institute (Japan) by r-DNA technique. Genetic material from two Streptomyces spp. producing actinorhodin and medermycin were combined and resulting hybrid compound named Mederhodin A was successfully produced. Both original antibiotics are too toxic for human use. Recently, scientists at the Institute of Microbial Technology (IMTECH), Chandigarh, in India have developed enzymatic conversion of Rifamycin B, an antituberculosis drug, to Rifamycin S which is more effective and faster in action. Rifamycin B is produced by Nocardia medditerrainie while its conversion to Rifamycin S is achieved by an enzyme from Curvularia lunata. Patent application is filed and commercial production will be commenced at the Indian Drugs and Pharmaceuticals Ltd. (IDPL). There are various methods of obtaining new antibiotics. Some of them are: 1. Mutabiosynthesis - The fact that many enzymes do not have substrate specificity but can act on a variety of substances resembling the original substrate (substance) is used in the new system developed which is called Mutasynthesis (Fig. 11.1). In a multi pathway convergent biosynthetic system, the organism is mutated so that endogenous synthesis of one of the antibiotic precursors is blocked. The missing component is then replaced by analogues of it and added in culture. This analogue then udergoes conversion. Similarly, instead of mutation, inhibitors may be added to block precursor synthesis. An example of the second type is

.

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antibiotic cerulenin that is used to block the formation of polyketides which form the backbone of many antibiotics. Aminoglycosides are a group of antibiotics from which new or hybrid antibiotics can be obtained in this way. By mutasynthesis, one-step fermentation is possible by incorporating just a gene for synthesis of a chosen precursor in (production) organism. Precursor used may be a natural or chemically synthesized analogue. Examples of new antibiotics obtained in this way are: hybrimycins, mutamycins, hydrm:ygentamycins. The cell's ability to take up the analogue and its acceptance by enzymes is the limitation in the technique. .

Glucose

---L

2 Deoxystreptamine ~--~Neamine

~--- Neomycin B

Neosamine Ribose

. Exogenously fed 2-Deoxystreptamine analogue

Neamine analogoue

--~6-Deoxyneomycin

B

Fig. 11.1 Production of new antibiotic by mutabiosynthesis

2. Biosynthetic pathways of antibiotic production are not clearly understood. But amplification of some gene on the pathway may help in increasing yields, to some extent. Overexpression of all genes by cloning them on multicopy plasmids might provide maximal production. 3. Specific chemical modification of aminoglycosides currently produced are expensive and difficult but microbial conversions if done will be beneficial. Ex. Amikacin obtained from Kanamycin by chemical modification is expensive. Bacillus Circulans can do this conversion by the addition of hydrm:yaminobutyric acid. If the gene responsible is added to Kanamycin producing organism, it will be still economically beneficial. 4. Successful isolation of antibiotic producing organisms will be possible if it is remembered that overproducing strain is the one which has the ability to tolerate high concentrations of antibiotics (Ex. Neomycin, Kanamycin producers). There is also some work going on to improve the production methods. Amongst this application of immobilised cells, immobilised enzymes are significant and the commercial successes and research successes on this front are mentioned in the topic on immobilised biocatalysts. Potential advantages of antibiotic production by immobilised system are continuous operation, reduction of non-productive growth phase, high yields, fast reaction. Problems faced here are contamination, availability of cofactors, stability of biocatalyst,. O 2 transfer rate, diffusion efficiencies. Monoenzymes systems are more successful than multistep reactions. Also Mexican researchers have recently developed a process for producing penicillin by solid fermentation which being a nonsterile process is economically advantageous. This process is claimed to produce 17 times more penicillin than that by the conventional methods. There is also lot of work going on regarding regeneration of protoplasts to observe if antibiotic production varies and also protoplast fusion as a method of strain improvement particularly in streptomycetes. There are no definite reports regarding the improvement of yields at the commercial level.

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By genetic engineering, strains of Streptomyces can be developed which would make extra proline or prevent proline breakdown, thus making it possible for the organisms to produce more of proline containing antibiotic. David Hopwood of Great Britain has expressed the possibility of activating silent genes to produce new drugs or of transferring drug producing genes into organisms that are easy to grow. Mutabiosynthesis -

Examples

Producing Strain

Block in Biosynthesis of

Analog Added

Multabiosynthesis Product

•

Streptomyces fradiae (Neomycin)

2-Deoxystreptamine

Streptamine 2-epistreptamine

Hybrimycin AI, A2 Hybrimyein BI, B2

•

Bacillus circulous (Budyrosin)

2-Deoxystreptamine Neamine

2- 5dideoxystreptamine streptamine 6-N -Methyleneamine

5 deoxybutirosin 2hydroxybutyrosin 6-N Methylbutirosin A, B

•

Micromnospora inyensis (Siromycin)

2-Deoxystreptaminc

Streptamine 2 epistreptamine 2,5 dideoxystreptamic

Mutamycin I Mutamycin 4 Mutamycin 2

•

Streptomyces griseus (Streptomycin)

Streptidine

2 deoxystreptidine

Streptomutin A

11.3 MICROBIAL ENHANCED OIL RECOVERY (ME OR) The increasing prices of petroleum and decreasing stocks (reserves) of petroleum compel us to recover oil from wells in the most profitable manner and to the fullest capacity. Oil recovery is carried on in three steps: drilling of potential wells and oil comes to the surface by inside pressure also assisted by pumps. (2) Secondary (40-60%) - by injecting water inside to push oil out. (1) Primary -

to dislodge oil that is sticking to the porous rock and is trapped in rock and to mobilise it upwards in the well. For tertiary or enhanced oil recovery (EOR), chemical, physical, biological strategies can be assessed. Microbial enchanced oil recovery (MEOR) has cost-effectiveness, simplicity and improved efficiency. MEOR should also have reliability and effectiveness of a high degree. Most of the oil in wells is found as a coating on the grains of the rock. The oil sticks tenaciously to these grains and has to be removed and mobilised out of the well. MEOR can be carried out in three ways: (3) Tertiary -

(1) Stimulation of indigenous microbial population of reservoir by injecting nutrients to enhance

their microbial activity. (2) Use of microbial products -

biopolymers, biosurfactants, acids, .1lcohols, ketones.

(3) Injection of specific microorganisms instead of depending on indigenous population in the first case or external microbial products in the second case. Here injected microorganisms will carry on useful activity, giving products that cause mobility of oil.

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The first method is comparatively unpredictable and useful microbial population of indigenous type may not be sufficient to achieve EOR. The second method uses (Fig. 1l.2) biopolymers and biosurfactants which are to be pumped into the reservoir. Many factories have come up to produce such products for MEOR. Since ordinary water is too thin to push out the oil, thickening agents, viscous materials are often employed as lubricants for the heavy drill which penetrates the rock as well as to displace oil. Various types of muds containing water, clay and polysaccharides do this task. Xanthan gum produced by bacterium Xanthomonas campestris is being used for this purpose for many years. Xanthan gum is an efficient thickening agent, allowing the water/gum mixture to act as a piston pushing oil towards the well. Surfactant such as emulsion which reduces oil water interfacial tension and enhances oil-flow is also used. Polyacrylamide is also used. Polyacrylamide is resistant to temperature, salinity and shear. Xanthan gum is also resistant but expensive. Scleroglucan which is produced by aerobic fermentation of selected strain of Selerotium spp is twice as viscous as Xanthan gums (causing better oil displacement) and will be useful in MEOR. Scleroglucan gum production is easier because it occurs in an acidic condition (fungi as production organism) and hence biological contamination of it prior to use does not occur. The third method of injecting specific microorganism and nutrients into the reservoir and then exploiting microbial activity for oil recovery is more attractive. Genetics may be used to combine desirable characteristics in one or a few strains. Desirable characteristics are: (i) Small size to permit efficient penetration of rock.

(ii) Tolerance of reservoir conditions like high pressure, high temperature, more salinity, lack of oxygen. (iii) Nonfastidious and capable of using hydrocarbons. (iv) High yields of products that permit mobility of oil. (v) Tolerance of biocides, corrosion inhibitors used.

(vi) Organism of choice should be dominant in mixed growth. Bacillus and Clostridium spp are found to be more useful than Xanthomonas which gives Xanthan gums. Cyclic recovery is being tried in Kansas. Microorganisms + nutrients + biocatalyst are injected into the recovery well. Shut-in period for days to weeks is allowed depending on the downhole conditions and the production phase ranges from weeks to months. The cycle can be repeated when production falls back to pretreatment period level. Microbial product called Wel-Pres-5 from Ram Biochemicals of Raleigh North, Carolina, produced 10-18% increase in 3 out of 6 wells. Acids produced by microorganisms help to dissolve rock while surfactants produced decrease oilwater interfacial tension and polysaccharides proliuced help displacement of oil. Scientists in Britain have identified one organism to be the best in their 11 isolates which can be used for MEOR. This organism grows at soac, tolerates a pressure of 200 atmospheres and produces organic acid in substantial quantity which can dissolve calcium carbonate in rocks to open up new channels for oil mobility. This method will be far better than using hydrochloric acid which is consumed before it reaches the bottom and is costly and corrosive.

11.3.1 Mechanisms of MEOR Research on MEOR began with the research paper by Zobell in 1940 and subsequent US patent in 1946 making use of sulfate reducing bacteria (Desulfovibrio halohydrocarbonoclasticus) for the release of oil.

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Zobell in 1940s has given an excellent discussion of the mechanisms of MEOR, which is still valid today: (1) Bacteria may produce acids which promote the dissolution of carbonate rocks, thus releasing droplets of entrapped oil. (2) Sulfate reducing bacteria can reduce the sulfate in gypsum, anhydrite and similar sulfate minerals, solubilising the minerals and releasing oil. (3) Bacteria may produce gases including carbon dioxide, hydrogen and methane which can increase the reservoir pressure and expel oil from the micro traps. (4) Bacteria will grow firmly attached to the surface, so will dislodge oil stuck to the surface. (5) Bacteria growing in aqueous culture often produce surface active agents, detergents or wetting agents which reduce surface tension and interfacial tension between oil and water liberating oil from sand particles. (6) Bacteria may break large hydrocarbon molecules to smaller ones, thus reducing the viscosity of oil. Dissolution of methane, CO 2 and other gases in oil should also reduce its viscosity.

WATER 1HICKENEDWI1H GUM

J

l

1

1

~~~~ Fig. 11.2 Oil recovery.

Sugar fermenting bacteria (Clostridium acetobutylicum) did enhance the rate of recovery and volume of oil recovered from rocks (petroleum-bearing) in the period of 1940-1973. Further, it was observed that before attempting MEOR, a study of the reservoir is necessary to decide which MEOR method will be suitable. Study of rock porocity, temperature, pressure, salt content etc. will decide the suitability of organism and process.

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SPRAY

+ ROCK

euso. converted to copper metal Fig. 11.3 Dump or Heap leaching for recovery of metal from ore.

If the situation underground is properly researched and microbial system appropriately designed with the specific reservoir or well problem in mind, MEOR will be more successful. But for this a dialogue, an understanding has to be there between the biotechnologist and those in oil field operations.

11.4 MICROBES IN MINING Mining is perhaps one of the oldest industrial activity. Conventional mining for metallic minerals involves (1) crushing of vast quantities of rock from the potential sites and its grinding; (2) then its treatment with chemicals to recover metals. If rocks are having high-grade ores, then this process is economically worthwhile. When high-grade rocks are in short supply but a large amount of metal is still in the ground, processing low grade ore by this conventional process is not economical. Microbes can be used to extract and concentrate them in a cheaper and more efficient way.

11.4.1 Biohydrometallurgy A new dicipline known as Bihydrometallurgy, which is combination of biotechnology and metallurgy is developing fast. The method was first used commercially in late 1970s to extract uranium left in old mines in Canada. At roughly the same time it was applied to two gold mines in South Mrica. Biohydrometallurgy has so far recorded the greatest impact on copper mining. Smelting copper by traditional methods had cost between $ 60-90 per lb. The introduction of biohydrometallurgy cut the cost to less than $ 30 per lb. Smelting 1 ton of copper results typically in 2 tons of S02 being pumped into atmosphere. Biological extraction avoids this. The copper released during bioleaching is extracted as sheets through an intervening process in which electricity is passed through copper solution. The metal collects on negative terminals. This part of the process is still costly and research is underway to develop bioadsorption filters such as algae which could be used to make the process entirely biological.

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Biohydrometallurgy in future may provide a method for underground mining without the environmental damage associated with conventional methods. There is a mine in San Manuel i\riwna consisting of 5 holes drilled into an ore deposit which are fractured by detonating an explosive charge underground. Instead of standard mining practice a mixture of acidic water and T. Ferroxidans is pumped down the central hole where bacteria do their work. The resulting solution rich in valuable copper is pumped from four other holes, processed and recycled. Despite the potential of this technology and envrionmental advantages, mining industry is reluctant to use it. So far it is used only as a last resort to recover low grade metals from sites where traditional methods are not profitable. Problem with biological process is that it is slow and hence rate of return on capital is slow. Conventional methods can recover most metals from an ore in a matter of months or years depending on size of deposit and level of resource applied to production, whereas biological recovery may take decades. This causes delay in cash flow. Researchers are working to speed up the process. The researchers are also working on heterotrophic leaching for extraction of manganese - a process carried out under air starved conditions similar to anaerobic fermentation stage. By adding sugars to the solution containing ore, the researchers speeded up significantly the extraction of manganese. In this process mineral itself becomes a substitute for oxygen, acting as electron acceptor at the end of oxidation of sugar. In future genetically engineered organisms may make the process faster. Recovery of metals with the help of microbes can be done at two places: (1) applying microbial leaching for low-grade ores; (2) concentration from waste waters (scavenging) or from dilute metal solutions. Recovery by the first way is secondary recovery (or primary) in mining while in the second case, it is a recycling process and a means of avoiding heavy metal pollution disasters.

An organism like Thiobacillus ferroxidans derives energy from iron sulfide in the presence of CO 2 and N 2 • In the process it manufactures H 2S04 and FeS04 • H 2S04 and FeS0 4 attack the surrounding rock and leach (dissolve) many metallic minerals. Thus, it will convert insoluble copper sulfide into copper sulfate. As water is added in the site, it percolates and CuS04 (copper sulfate) which is scattered is collected in the pool. A solution of copper sulfate in this pool can be passed over iron where the copper deposits and can be scraped off. Uranium also can be leached in a similar way and is converted from U4+ to U6+ in the Canadian mines. 14% of copper produced in the US is by biotechnological process of leaching. Microbial leaching can be done in three ways: (1) Vat leaching. (2) In situ leaching. (3) Heap leaching (Fig. 11.3). Vat leaching is generally applied for the extraction of uranium, gold, silver and !:opper from their ores. It is applied for ores with high metal concentrations. Under controlled conditions, it can be carried out using suitable selective bacteria. Economic factors are to be considered before making the actual application.

In situ leaching is done by injecting a leach solution (chemiCal oxidants like H 20 2 oxidise and solubilise uranium) into unfractured uranium ore body via injection well and collecting the leached metal from the production well. In situ microbial leaching is possible for sulfide minerals but this method is at research level. Heap leaching (Fig. 11.3) or dump leaching is the one which is mostly used today as far as microbial leaching is concerned. At present, microbial leaching is mainly employed with wash material

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for conventional mining and extraction from discarded residues. Dumps up to 1200 ft. high and 4 billion ton in weight are constructed from waste residues after primary mining. Water is sprayed. Thiobacillus organisms are ubiquitous and so are present in dumps and no inoculation is required. Area on which heaps are built is usually covered with clay or asphalt so that metal-rich liquids are collected in pools. This is called secondary recovery in mining. But stopping conventional mining early and allowing microbial leaching to occur in place just by spraying water may assist natural leaching. Microbial leaching has been successful in copper, uranium, cobalt, lead, nickel extraction and is being tried on cadmium, mercury. Microorganisms active in leaching are Thiobacillus fen-oxidans, Thiobacillus thioxidans, Sulfolobus spp. Leptospirillum ferroxidans, thermophilic and acidophilic bacteria.

T. thioxid(;,ns and T. fen-oxidans both attack sulfur in metal sulfides and convert it to thiosulfate and tetrathionate respectively and then to H 2S04 , Both together are more effective. Leptospirillum jerroxidans and T. organoparpus can degrade pyrite (FeS 2 ) and chalcopyrite (CuFeS 2 ), neither of which can be extracted from anyone of the bacteria alone. Thermophilic and acidophilic bacteria can act on pyrites, chalcopyrites, covellite (CuS), pentlandite (FeNi9Sg).

Sulfolobus spp. oxidise S and Fe for energy and use CO2 or simple organic substance as carbon source, oxygen is the ultimate acceptor of electrons in chemical odixation. Molybdenum (MO+6) and ferric iron (Fe+++) can act as electron acceptors in the absence of oxygen. Since Sulfolobus are resistant to high concentration of molybdenum, they are under immense study. Mechanism of microbial leaching is not clearly understood. It occurs mainly in two ways: (1) Direct leaching and (2) Indirect leaching. Direct leaching involves enzymatic attack by bacteria on the components of minerals that are susceptible to oxidation. Inorganic ions do not enter the cell. Electrons released by oxidation reaction are transported through membrane proteins tq oxygen. H 2 0 is produced. Transferred electrons give up energy which is coupled to ATP formation in cell. In indirect leaching, the organisms do not attack the atomic structure of the mineral but convert Fe+ 2 to Fe+ 3 . Fe+ 3 then reacts with other metals because it is a powerful oxidising agent. Acidity is important here to keep ferric iron and other metals in solution. ' Advantages of microbial leaching: (i) Useful for low grade ores. (ii) Degradation of a variety of mineral forms. (iii) Selective leaching possible, one metal solubilised while the other remains insoluble. Flotation method then is used for separation. (iv) It avoids surface disruption, smelting etc. (v) Application most useful is desulfurification of coal so that coal for burning is free of sulfur and further pollution (acid rain) problems avoided. (vi) Suitable for less developed countries as it eliminates the need for some costly imported heavy mining equipment. Limitations of microbial leaching are: (1) process is slow; (2) in situ applications still under development; (3) control difficult; (4) more possible for acid-producing minerals and not acid-consuming minerals. . Microbial leaching is looked to as not to replace the existing conventional metal extraction processes but to support it if suitable.

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Metal extraction from waste water (recycling) is discussed in the chapter on environmental biotechnology. An Update on Bacterial Leaching and Biomining: Bacterial leaching in recent years has been used successfully in many countries to recover metals from wide variety of ores. The principal metals recovered are Cu, Ni, Zn, Pb, Co, Au and Uranium. In August 1985, copper worth $ 350 million and uranium worth $ 20 million were recovered by microbial means in USA alone. Microbial metal industry by 2000 A.D. is expected to be $ 90 billion. The most important bioleaching bacteria are Thiobacillus ferroxidans, Lipospirillum fen'oxidans and number of spp. belonging to Sulfolobus. T. ferroxidans is best studied among them. Considerable progress in development of a system for the genetic manupulation of T .ferroxidans has been made over past few years. Plasmids have been isolated for carrying new genes into cells. TF gene also has been shown to be expressed in E.coli. A means of introducing vector DNA into T .ferroxidans is still lacking. The application of r-DNA technology will result in better understanding of molecular biology of these oganisms. The generation of new mutants will permit investigations into genetics and regulation of sulfur and iron oxydation. An advantage of r-DNA technology will be for introducing multiple copies of gene into cells. Such amplification may permit identification of enzymes that act as bottlenecks. This knowledge will help to improve growth rate and leaching performance of the bacteria. Tolerance of TF can also be increased to certain metals and other ions encountered during leaching. Locally isolated TF should be used for gene manipulation so that it can compete with indigenous microbes. Although it has been proved technically feasible to bioleach copper, zinc, cobalt, molybdenum and variety of other metals form their sulphide ores by using Thiobacillus ferroxidans, commercial operations are limited due to 16-20 hours generation time required by this bacterium. Under normal growth conditions it takes upto 30-40 hours to obtain 100 million cells per m!. During bioleaching when microoganisms interact with mineral substrates T ferroxidans produces ferric sulphate which dissolves a wide variety of sulphide minerals. Researchers at Indian Institute of Sciences (lISe), Bangalore, have suggested that applying negative direct current potential in the range of minus 500 millivolts (MV) to minus 1000 MV to a bacterial culture containing ferric ions converts ferric to ferrous ions, and promotes bacterial activity and growth. The growth period of bacteria is reduced 10 fold.

11.5 GLYCEROL PRODUCTION FROM ALGAE The Dead Sea has been colonised by a few unusual microorgnisms. Concentration of salt is 8 times that of the oceans. This kills most of the microorganisms. They die due to water contents of cell being extracted due to high osmotic pressure outside. Dunaliella bardawil algae has evolved a way of preventing dehydration. It produces high concentration of glycerol so that the cell contains a high concentration of dissolved material. This prevents drawing out of water by high osmotic pressure outside. In Israel near the Red Sea coast, this algae is grown in specially constructed ponds covering 2 hectares. Organism is photosynthetic, so it uses energy from the sun and requires only a few nutrients. On harvesting and drying 40% of dry wt. of algae is glycerol and 8% is betacarotene. After extraction of glycerol and betacarotene, the remaining biomass is protein rich feed. Algae thrive in brackish-water, so they can be grown in semi-arid regions. This will come up as low technology, less capital-intensive application of biotechnology. Less attention required, less contamination chances are the plus points.

Dunaliella salina can be cultured to accumulate 50% of dry wt. glycerol.

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11.6 FATS AND OILS Researchers at the Hindustan Lever Research Centre have succeeded in manipulating genes of two edible varieties of yeast to produce a hybrid cell capable of producing and accumulating high amounts of fats of the order of 50% of the biomass and growing at high rates also. Protoplast fusion technique has been used for getting the new yeast. Pilot plant operations are successful and hopes are high for fat production from molasses at the cost of Rs. 9,000 to Rs. 10,000 a tonne. India's perennial deficit in vegetable oils had motivated Hindustan Lever to explore these new biotechnological means. In Japan the National Chemical Laboratory for Industry (Tsukaba) has developed a method for the production of oil and fats by the use of microorganisms. In this process, Morbicella fungi are producing 50% w/w fats plus oils. Fat and oil can be obtained by mere centrifugation in aqueous suspension. This can later be evaporated for recovery. Crushing these fungi in a ball mill to rupture cells gives oil containing 7-12% of gamma-linolenic acid. This technique has been transferred to various firms for commercialisation. In New Zealand also single cell oil-producing yeast has been developed and tested on a pilot plant scale. From the concentrated cream of yeast or from dried yeast, oil recovery will be done. Continuous whey fermentation with cell recycle or a process based on a series of batch fermentors is tried.

11.7 PLASTICS The plastic industry in the US is over $ 50 billions per year and is obviously a tempting market for biotechnological enterprises. A synthesis of alkene oxides which are used in the manufacture of plastics and polyurethane foam is being produced since the seventies. Alkenes can be polymerised, e.g. polypropylene (used to make containers), and polyethylene (polythene). Before polymerisation, alkenes are converted into alkene oxides. This is done at present by a chemical process. Alkenes are derived from oil or similar materials. A Californian firm Cetus has employed three enzymes out of which two are fungal and one bacterial to perform the same function as the current chemical methods do. Puranose 2 oxidase Glucose

+

02 - - - - - - -..... ~

H 20

2

+ Glucosone

Chloroperoxidase Alkene + H 20 2 + Chloride ion ----.;'------... alkene chlorohydrin Epoxidase Alkene chlorohydrin - - - - - - - - . . . Alkene oxide

In 1981, one more process was patented in which the organism Mcthylococcus capsulatus inserts oxygen atom in propylene or ethylene gases that are supplied to produce propylene oxide and ethylene oxide in one step. The organism grows at 45°C at which alkene oxides are gaseous. Therefore, recovery becomes easier. Advantages of enzymatic or bacterial process are: (1) The chemical process requires chlorine gas while for the enzymatic process, sodium chloride will do and bacterial process even does not require that. (2) Both biotechnological processes are working at low temperature; therefore energy-saving occurs. (3) Flexibility offers the same installation to be used for the different types of alkene oxides. (4) No pollution as compared to the chemical process.

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11. 7.1 Biodegradable Plastics Environmentally, friendly products are the need of the hour and considerable research and development have taken place in obtaining biodegradable plastics. Plastics that are manufactured today like polyethylene, polyvinyl chloride and polystyrene are not biodegradable and persist in soil environment. acids.

Bioplastics that are thought of are poly-[3 hydroxybutyrate (PHB) and more recently polylactic '

Poly-[3 hydroxybutyrate (PHB) is a thermoplastic polyester consisting of repeat units of formula CH(CH3) - CH2 - CO - O. PHB has been recognised as an energy reserve material that accumulates in many microorganisms like Alcaligenes, Azotobacter, Bacillus, Nocardia, Pseudomonas, Rhizobium spp. Alcaligenes eutrophus and Azotobacter beijerinckii can accumulate up to 70% of their dry weight of this material. These organisms produce the polymer particularly in an environment of nitrogen and phosphorus limitation. If the organisms produce a minimum of 40-50% of their dry weight of this polymer, then commercialisation of the process is possible. Extraction of PHB is done using solvents like halogenated hydrocarbons and purification is achieved. Moulding or extrusion of dried cells directly is possible when the PHB contents are high. A lot of work has been done on engineering polymeric properties of PHB. However, PHB is considered to be a useful biodegradable plastic with applications in certain specialised areas like biomedical uses and speciality coating. A process that converts starchy food wastes and byproducts into lactic acid-based plastic will be soon available for commercialisation. There are two key steps in this process: one that converts glucose found in starchy food wastes into lactic acid and the other that converts lactic acid into polylactic acid. The Argonne National Laboratory in the United States has licensed these key steps in its Biolac process to Kyowa Hakko USA Inc., a subsidiary ofJapan's Kyowa Hakko which deals in fermentation products. Biolac plastic has commercial applications in products such as compost bags, coatings for paper, seeds, pesticides and fertilizers. The US produces 5 million tons of food-wastes in the manufacture of fried potatoes and 1-2 million tons waste in the cheese industry. So Kyowa Hakko USA Inc. will use the Biolac process to produce polylactic acid from these food waste sources.

11.8 ACRYLAMIDE PRODUCTION Acrylamide is a petrochemical product used as a paper reinforcing agent, water treatment agent, flocculant in enhanced oil recovery (EOR). At present, acrylamide is produced by a chemical process, using acrylonitrile and Cu catalyst. Earlier a sulfuric acid catalyst was used. Now a biotech process has been developed which uses acrylonitrile as a raw material and uses biotech reactor of a special design under water. After enzymatic reaction, the acrylamide water solution is sent for purification and condensation reaction to obtain the final pure product. The Nitto Chemical Industries Company Ltd. is constructing a 4000 toupes per year acrylamide plant for the biocatalyst process. Advantages of the biotech process are: (1) normal temperature required; (2) easy recovery; (3) simple equipment; (4) low capital investment. The Nitto Chemical Industries Co. of Japan was using Rhodococcus rhodocrons strain SP 774 for its first 4,000 tonnes per year plant of acrylamide production. Now improved yields are obtained by Pseudomonas chlororaphis strain B 23 and the new Rhodococcus rhodochron~ strain 11 gives 10 times higher yields -than the old one. Now the 6,000 tonnesjyear is the capacity of production. Less impurities, direct concentrated acrylamide solution are the advantages of the new strain.

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It may be produced by addition of water to acrylonitrile CH2

= CHCN +

H 20

~

CH2

= CHCONH2

When reduced copper is used as catalyst the yield is poor and unwanted polymerisation or conversion to acrylic acid (CH 2 = CHCOOH) may occur at the relatively high temperatures (80-140°C) and the catalyst is difficult to generate. These problems may be overcome by the use of immobilised nitril hydratase (nitrilase). The immobilised nitrilase is used by Nitto Chemical Industry Co. Ltd. Immobilised enzyme is prepared by entrapping the intact cells of Rhodococcus in cross-linked 10% w/v polyacrylamide/dimethylaminoethyl methacrylate gel and granulating the product. It is used at lQoC and pH 8.0 to 8.S in sekmibatchwise process. Acry lonitrile (substrate) concentration is kept less than 3% (w/v). The process takes a day. Product concentration achieved is 20% (w/v). A New Lactic Acid Bacterium for Lactic Acid Production: France's Orstom Research Institute has recently isolated a bacterium from palm tree wine.The wine is a traditional Mrican drink made from palm tree sap. It is called Bacillus thennoamy-Iovoron. It produces around 100 g/lit of lactic acid in less than 48 hours. A temperature range of 47-S8°C is required for limiting contamination. Some vitamins and nitrogenous compounds are required. It can be used in future for cheaper and more efficient production of lactic acid from starch based agro-industrial wastes. Enzymes Provide a New Route to Phenolic Resins: Enzymol International Inc.(Columbus, Ohio, US) has developed the process from the technology bought from Mead Corporation (Dayton, Ohio). Conventional phenolic resins are made by polymerising phenol and formaldehyde using an acid catalyst. However, reproducibility is the problem. The resins have too broad a molecular weight distribution and they tend to oxidise and turn yellow. Enzymol process uses a peroxidase enzyme obtained from soyabean to polymerise phenols in aquous solution of organic solvents at SO-60°C. H 20 is added to activate the enzyme. Molecular weight of product is controlled by choice of solvents and ratio of solvent to water.

11.9 BACTERIAL CELLULOSE Acetobacter xylinum which produces cellulose ribbons may be used to obtain cellulose. Harvesting and recovery are easy. After sterilisation and washing, cellulose can be used for parchment paper and other products made from wood pulp otherwise. Bacterial cellulose is much cheaper and energy-efficient than cellulose from wood pulp. This cellulose can also be used to coat the surface of synthetic fibres which permits the fabric to breathe more like cotton.

11..10 ENZYMES 11.10.1 Ligninase Enzyme Ligninase enzyme has been discovered by an American research group in white rot fungus Phanerochaete chrysosporium as an extracellular enzyme. Ligninase can be useful in (i) breaking down the lignin barrier of plant material in the extraction of cellulose; (ii) it also opens up lignin with its rich store of oxygenated aryl rings for exploitation as a source of aromatic chemicals; (iii) it can also oxidise phenols which are common pollutants in water and to reclaim waste water; (iv) ability of ligninase to split c-c bonds gives it intriguing possibilities as a low energy tool for cracking down petroleum. Bulk production of ligninase today is difficult because the enzyme is produced only in certain phases of fungus development. Consequently, optimisation of ligninase yields is difficult. Also the current method of fungus production makes the scale up difficult.

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Ligninase is a powerful peroxidase and is most stable. Lignin and cellulose (lignocellulose) make up as much as 95% of the earth's land produced biomass. 25% of this is lignin. If ligninase production becomes possible, then this lignin can be attacked and will serve as a potential source. The chemical route of separating cellulose from lignin in agricultural biomass is expensive and cellulose has to be purified before use. In France, ligninase enzyme from improved patented strain of Phanerochaete chrysosporium is obtained and is claimed to produce 30 times more enzyme than the US results. Also the enzyme is 100 times faster in action than that of the US. INRA's (Institute National de la Researcherche Agronomique) results showed degradation of 246 mcg/hr of lignin per 360 units of ligninase as compared to US report of 2 mCg/hr. Cloning of gene from P. chrysosporium in other organisms is under search since P. chrysogenum growth for ligninase production is difficult.

11.10.2 Proteases and Other Enzymes Genentech and Genencor (San Francisco, US) have produced enzyme subtilisin (protease) from 80 different variants. Site specific mutagenesis was used as a genetic engineering technique. By using casette mutagenesis, researchers have replaced a number of aminoacids to make a series of 19 new subtilisins which are not normally present in nature. Stronger patent protection is given to this ingenious product. New protease enzyme active at low temperature for detergent use has been developed by Showa Denko K. K., Japan. It is obtained by the cultivation of alcalophilic bacterium. Production is 1,000 tonnes per year. Proteases are used extensively in detergents along with amylases and lipases. Novo Nordisk has introduced 'Durazyme', a detergent protease which is long-lasting, resistant to bleach via protein engineering. Candida rugosa lipase can separate products by hydrolysis of tallow - the fatty acids and glycerine - into two layers within 20 min. at 40°C which is very much faster than the existing processes. An enzyme has been discovered in bacterium Acidothermus Cellubolyticus (from hot springs) which can be used for clarification of fruit juices even at 75°e. A pectinase is obtained from Aspergillus niger which can be used to get increased juice yields, reductions in viscosity, increased colour extraction and enhanced clarification.

Bacillus sterothermophilus has a lipase that can help break down fats into smaller molecules for use in confectionery. This enzyme works at 65°C, so other organisms are killed and product sterilised simultaneously; also fats liquify at this temperature, making solvents unnecessary. This saves money and avoids hazards from solvent residues. Other enzymes that are being or are likely to be produced on a commercial scale by microorganisms are amylase, streptokinase, lipase from Aspergillus which can be used as a detergent enzyme. Also protease from Thermomonospora fusca has detergent action at 80°e.

11.11 VITAMINS 11.11.1 Vitamin C -

New Short-cut Process (Fig. 11.4)

A two-step Vitamin C process based on biotechnology is on the horiwn. Agreement between Genentech, Lubrizol Corp. and Pfizer has been reached.

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This process is simpler and more energy-saving than the conventional Reichstein Grussner process (6-step). Commercialisation of the new process will make the first-ever use of genetic engineering to produce chemicals in industrial quantities. Current production of Vitamin C is 40 million lb/year with a turnover of 400 million dollars/ year. Gene is isolated from Corynebacterium spp that codes 2, 5 diketo' D-gluconate reductase. This enzyme catalyses conversion of 2, 5 diketogluconate to 2 keto I gluconate which is an important intermediate in ascorbic acid production. This gene is then inserted into Erwinia herbiccala bacterium with the help of a plasmid. Unmodified Erwinia has the ability to ferment D-glucose to 2, 5 diketo D-gluconate. Inserting the new gene allows this transformed bacterium to ferment D-glucose to 2 keto L-gluconate in one step. The second step in the production of ascorbic acid is acid or base catalyzed cyclisation of 2 keto L-gluconate to ascorbic acid. Older process (6 steps)

,/;,'/;' !

(

I

New process modified

I I I

I I

herbiccala

I I ./ )

Ascorbic acid

D-sorbitol

1

fermentation by Acetobacter

suboxidans

L-sorbose

L2 ~:cid

1

Diacetone I sorbase (bis acetonide) oxidation KMnOJ Sod. hypochlorite

1

,/

kcro 1 gluconic acid

Hydrogenation

I

Erwinia

directly or Via methyl easter

D-glucose

"ulyud

Diacetone 2 keto 1 gluconic acid

cleavage

Fig. 11.4 Short-cut Process for Vitamin C production.

11.12 BIOGUMS, BIOPOLYSACCHARIDES (TABLE 11.1) Although biogums are hardly known in India, it is a big business in the US and Europe. The Kelco Division of Merck & Co. is the world's leading manufacturer of biogums. The company has recently announced plans to undertake $ 29 million expansion of its biogum production at San Diego and at Okumulgas (Oklahoma). Biogums are produced by fermentation at both the plants. Biogums are used as suspending, thickening and gelling agents in foods and pharmaceutical formulations. The expansion will be in research engineering and production technology, and also to increase production capacities of Xanthan gum, gellan gum, rhamisan and welan gums. The IGI Biotechnology (USA) has unveiled production of polysaccharide substitute to Gum Arabic. The product is produced by the fermentation of raw cane or beet sugar. The fermentation process produces a white spray dried powder, which is composed of repeating alpha-linked fructose monomer unit. It can be a good substitute for gum arabic which is often erratic in supply due to

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seasonal or political instability. This development should be of great interest to sugar-producing countries like India. Table 11.1

Biopolysaccharides

Producing Organism

Applications

1.

Xanthan

Xanthomonas Campestris

Stabilizer and agent for suspension, gelling and viscosity control. For EOR (enhanced oil recovery) As food additive (for improved mouth feel).

2.

Dextran

Leuconostoc mesenteroides

Plasma substitute (blood expanders) as hydrophilic layer for burns to absorb fluid exudates. For separation and purification of biomolecules, as polyelectrolytes.

3.

Alginate

Azotobacter vinelandi

Thickening and gelling agent in dairy products, to control ice crystal formation in icecream.

4.

Gellan

Pseudomonas elodea

In microbiological media to replace agar and carrageenan.

ATCC 31461

5.

Zanflo

In carpet printing and in paint industry.

Erwinia

fahitia 6.

Scleroglucan

Sclerotium glutanicum

7.

Polymer S-130

Alcaligenes spp

As pseudo plastic. In EOR. In Ceramic glazes, latex paints, Printing inks. As suspending agent for pesticides in oil industry as lubricant.

Alcaligenes fecalis var.

As gelling agent in cooked foods, for immobilisation of enzymes.

S-194 8.

Curdlan

myxogenes Strain 10C3

9.

10.

Pullulan

Aureobasidium pullulans

PHB (Poly Hydroxy butyrate)

Azotobacter Spp Alcaligenes Spp

In Fibre, Film, Packaging industry. Flocculating agent in clay suspension in mining. Biodegradable plastic.

REFERENCES 1. Steve Prentis, "Biotransformation - The Way Ahead ofIndustry," inBiotechnology -ANew Industrial Revolution (New Revised Edition), Orbis, London, pp. 170-187, (1985). 2. David M. Updegraff, "Early Research on Microbial Enhanced Oil Recovery," in Developments in Industrial Microbiology, Vol. 31, (Editor), G. E. Pierce, Society for Industrial Microbiology, pp. 135-142, (1990).

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3. Vivian Moses, "Downhole Icrobially-enhanced Oil Recovery Systems," in DevelopmentsinlndustrialMicrobiology, Vol. 31, (Editor), G. E. Pierce, Society for Industrial Microbiology, (1990). 4. Jonathan Katz, Dr. David B. Sattele, Biotechnology for all Consultants, (Editor), Dr. W. T. Mason, Hobsons Scientific, Cambridge U.K., Microbiological Resources Centre (MIRCEN), UNESCO, (1991). 5. C. L. Brierley, D. P. Kelly, K. J. Seal and D. J. Best, "Materials and Biotechnology," in Biotechnology - Principles and Applications, (Editors), 1. J. Higgins, D. J. Best, J. Jones, Blackwell Scientific Publications, London, pp. 163-212, (1985). 6. Dr. S. Ramachandran, Secretary, Dept. of Biotechnology, "Industtial Opportunities in Biotechnology," in Chemical Week~, 5th May 1992, Vol. XXXVII, No. 35. 7. Chandana Chakrabarti and PushpaM. Bhargava, "Chemicals through Biotechnology: Facts, Hopes, Dreams and Doubts," in Chemical Week~, 21st April 1992, Vol. XXXVII, No. 33. 8. KeshavTrehan, "Hybrid Antibiotics," in Biotechnology, Wiley Eastern Limited, New Delhi, pp. 219-223, (1990). 9. Keshav Trehan, "Mining and Metal Biotechnology" in Biotechnology, Wiley Eastern Limited, New Delhi, pp. 89-103, (1990).

10. Chemical Week~, Vol. XXX, 1985. 11. Chemical Weekly, Vol. XXXI, 1986.

12. Chemical Week~, Vol. XXXVI, 1991. 13. Chemical Week~, Vol. XXXVII, 1992.

BIOTECHNOLOGY APPLICATIONS TO ENVIRONMENT PROTECTION 12.1 INTRODUCTION Increasing industrial, agricultural and domestic activities have brought about many untoward changes in the environment. The obnoxious gases, toxic chemicals, heaps of solid wastes have become a point of concern for our living beings. Nature's built-in mechanisms have been thrown out of gear due to the quantity and complexity of wastes generated by modern society. No sooner did man become aware of these consequences, he started efforts for the repair and control of things that have gone wrong due to pollution. Also future activities are planned with environmentally friendly processes and products in mind. It started with the physical and chemical processes to curb the pollution but very soon, the help of microorganisms was hailed, for Biotechnology makes its contribution to environment protection in various ways. The new approaches are being practised in reactor design and sewage treatment facilities. This chapter also includes information on effluents from the fermentation industries. The immobilised cells and enzymes have a significant role to play in the removal of pollutants, so an account of them is taken too. Bioremediation and biological deodorisation have been discussed to explain the new methods of pollution control. And lastly, for effective environment monitoring, biosensors will be very much useful due to their specific and rapid response, so biosensors are also included here.

12.2 ENVIRONMENTAL PROTECTION AND BIOTECHNOLOGY Environment can be defmed as man along with its surroundings (which consists of biotic and abiotic components). Its a set of relationships between man and nature.

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Pollution can be defmed as, undesirable change in physical, chemical and biological characteristics of air, water,land that can harm human life, lives of desirable species, our industrial processes, living conditions, cultural assets and waste our material sources. Environment protection means limiting impairment of environment and conservation of resources. Industrial growth, Urbanisation and Wrong cultural practices are mainly responsible for different kinds of pollutions and negative effects on environment. In economic term we consider only energy costs, material costs but costs incurred due to damage to the environment are often neglected. Environment protection will pay us back in terms of money, economy, productivity, social justice, cleaner surroundings and health. Environmental Biotechnology Encompasses (a) Environmental monitoring. (b) Restoration of environment quality. (c) Resources recovery from wastes.

(d) Application of r-DNA technology for degradation of toxic pollutants. (e) Control of global changes.

(f) Maintenance of biological diversity. Thus, Industrial pollution management is only one of the many issues that are tackled by Environmental biotechnology. Biotechnology works in environment protection area with following different possible options(a) In-process treatment. (b) End of pipe treatment. (c) Remediation of pollution sites.

(d) Modification of existing processes. (e) Introduction of new processes and products.

First three of these options are short-term remedies and bring about correction or clean-up of environment, while the last two options are long-term strategies and are meant for prevention of damage to environment. We are normally more concerned about industrial pollution, which is more obvious and of local consequences. Biotechnology works for treatment of industrial effluents, solid wastes, and waste gases. Biotechnology is applied for degradation of toxic xenobiotic compounds. Biotechnology can be applied for decontamination of polluted sites, land, ground water. Biotechnology can be significant in pollution abatement because it does not merely convert wastes from one form to other but removes or eliminates them in total. Biotechnology is different in the approach because in many biotechnology treatments it either generates energy or produces food (SCP Mushroom, Biomass) along with the pollutant degradation. In the strategy of prevention of pollution, biotechnology applications in paper industry, tanning industry are well proved and accepted. Biopulping as an option to chemical pulping and biobleaching as optioin to chemical bleaching can prevent use of hazardous chemicals which later enter into waste streams. Similarly, use of enzymes for unhairing and degreasing stages also considerably reduces use of toxic chemcials which otherwise after treatment enter effluent steam. In new products to replace environmentally unfriendly plastics are the bioplastics which are finding more and more applications in food, pharmaceuticals cosmetic, agriculture, in packings, coatings and containers.

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12.3 TOWARDS IMPROVEMENTS IN SEWAGE TREATMENT At present, sewage is treated by aerobic processes like activated sludge process and trickling ftlter process and aerobic methods of sludge digestion. These processes together bring about the digestion of organic compounds by microbial activity. The present methods have certain disadvantages: (1) Aerobic systems are open constructions, therefore organisms and less control.

uncontroll~d

growth of population of

(2) Environmental pollution due to odour emission and fog formation. (3) Large space/area occupied by the treatment plants. (4) Low efficiency of oxygen transfer in present aerobic systems. (5) Optimisation of physiological capabilities of microorganisms involved is not seen. (6) Little attention paid to making methane production a highly economical productive process. Four new approaches which have the potential to overcome the above-mentioned disadvantages and increase the efficiency of sewage treatment are: (1) Use of starter cultures for treatment process. (2) Tubular loop reactors/Air-lift fermentors. (3) Aeration with pure oxygen. (4) Methane production optimisation. Use of Starter Cultures for Treatment Processes: Conventional treatment processes involve the use of microorganisms which develop as a natural population and no attempt is made to optimise the organisms involved. Starter cultures having specific abilities to degrade certain wastes will be useful for new heterogeneous sewage as well as for industrial wastes and accidental spills. Industrial eftluents cannot be channelised in sewage plants as these eftluents are toxic and lack biodegradability and thus they may cause harm to the nonadapted organisms of the usual sewage. Also organisms from the usual sewage cannot be a suitable seed for treatment plants of industrial eftluents. Starter culture may also be useful, as it will shorten the start up time that is generally required after shutdown of the process for one or other reason. A week's time is required otherwise to reestablish the culture of process organisms which can be avoided with the starter culture use. Starter cultures containing genetically engineered organisms will fmd their application to degrade alkanes, aromatic compounds, DDT, polychlorinated diphenols, phenols etc. which are tough pollutants present in the eftluents. Starter cultures with high protease, lipase, cellulase activity are available in the market. Also organisms active at different pH in the range from less than 5 to more than 9 are also available. Starter cultures will find special applications in tank cleaning, pipeline cleaning etc. Starter culture containing genetically engineered Pseudomonas putida organism is already patented for breakdown of octane, xylene, metaxylene, camphor. Starter cultures can also be used to deodorise animal excrements. In the US starter culture industry grossed 2-4 million dollars in 1978. There are 20 manufacturers who produce starter cultures and the potential of starter culture industry is expected to be near 200 million dollars.

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Tubular Loop Reactors/Air-Lift Fermentors: Disadvantage of activated sludge process is the large amount of space required for the installation of the treatment plant. The British Chemical Company (ICI) uses tubular reactor which is embedded 100 metres into the ground (SO-ISO metres deep) and is O.S to 10 metres in diameter. Airflow giving liquid circulation of 1-2 m/sec. is used. Oxygen transfer rate is 10 Kg 0v'm 3jHr. Bubble contact time is 3-S min. instead of IS seconds in the conventional process. BOD reduction of 92% is achieved with raw effiuent of 110-3S0 mg. dm-3 . Average residence time is 80 min. Very less sludge is produced. Two German companies, Bayer (Biotower) and UhdejHoechst (Biohigh reactor) have constructed bioreactors 30 metres in height. In UhdejHoechst reactor the circular settling basins are located around the top rim of the bioreactor. The dimensions of this type of activated sludge fermentor allow a considerably be~er oxygen transfer. Hoechest system has baffies to facilitate mixing. In the Bayer biotower process, two component nozzles have been developed as injectors by means of which kinetic energy of liquid stream is used to disperse air in small gas bubbles in a manner similar to a water spray pump. These injectors are installed in groups of four (injector dusters) above the bottom of the tower reactor (1 injector/I-2 m 2 bottom surface). Aeration with Pure Oxygen: This has been tried both in the US and Germany. The objective is to improve the efficiency of oxygen transfer, since the loading capacity of conventional plant and size of microbial population are limited by the low solubility of the dissolved oxygen. Pure oxygen has 4.8 times partial pressure of oxygen from air, so aeration with pure oxygen markedly increases the oxygen content of activated sludge system. DO (dissolved oxygen) in such system is 90-9S% utilized and less off odours (1% of that from conventional aeration) that are produced can sufficiently be treated chemically or thermally to prevent unpleasant odours. High loading capacity in the oxygen process allows smaller aeration tanks and smaller settling basins. Since the amount of sludge produced is also small, the costs of facilities for further treatment are also reduced. . On the negative side, higher capital investments, complicated installations, the need for careful control of process parameters and highly trained personnel are the matters of concern. There are several firms that currently market treatment facilities which use pure oxygen. Methane Production: Methanogenesis is a widely applied process in the disposal removal and methane is used as a fuel. In most waste treatment plants, however, concentration is dont: on stabilisation of sludge (for safe disposal in the environment) and not on methane production optimisation. By controlling parameters of methanogenesis, it can be made more efficient and gas can be sold in the market. Methanogenic organisms are able to use only the restricted group of substrates for the production of methane, induding acetate, methanol, formate and H2 + CO 2. In waste disposal systems, about 7S% of methane is derived from acetate and the rest from H2 + CO2. The starting materials of aerobic decomposition process are complex polymers like rellulose, starch, fats, proteins, none of which methanogenic bacteria is able to use. Methanogenic bacteria are thus dependent on other anaerobes for the initial breakdown of organic matter to the level of acetate and H2 + CO2. These two component processes can be handled separately to make it a multi-stage and an efficient complete process. Also control on parameters suitable for a multi-stage process - temperature, organisms used, rate of feeding of waste substrate, pretreatment, physical separation of groups of organisms with a separate stage for separate activity, improvements in mixing as a whole - will result in the optimisation of methane production.

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High Performance Compact Reactor: 'Otto', a company in Germany, has developed a new kind of effluent treatment plant that needs less surface area than the conventional plant. 40 Kg. load per cubic metre volume can be treated while 0.75 - 1.0 Kglm 3 load is achieved in the conventional activated sludge process. High performance compact reactor process can handle some contaminated waste water that is not normally biologically degradable. This is because waste-water stream is added to the large recirculated water circuit to dilute it first. Central to the process is a loop reactor aerated from above using a 2-phase jet. This operates in conjunction with sedimentation facility. The inflows to the reactor circuit - sewage feed and sludge return - are cleaned and then flow by gravity to sedimentation basin where the sludge and purified water are separated. The compact and enclosed reactor eliminate the emission and odour problem. The problems of surplus are also minimised by 40% than the conventional systems. The process has been established in Europe and at least 11 plants are already in operation and several more are due to come on stream. The Coca-Cola group has used an anaerobic-aerobic hybrid reactor to tackle their High COD effluents from the syrup plants. COD is 10 times higher than that of any normal food processing unit. A conventional suspended slurry on the fixed film anaerobic reactor cannot handle such high COD effluent. In the hybrid reactor developed waste is pumped into the suspended slurry at the bottom of the reactor that removes most of COD. This then will flow up through a stack of cubes that support the fixed ftlm media. The system will produce 600-900 Btu methane. The treatment plant handles 100,000 gallon of wastes per day. The product of many sewage plants is a sludge that consists of about 95% of water. The water content can be reduced to 80% by costly centrifugation. Israeli researchers have reported a method that eliminates these expenditures of centrifugation with a proprietary "biological method" that quickly separates the sludge into liquid and solid phases. The liquid is said to be almost free from odour and is suitable for irrigation. The solid contains organic matter that can be successfully processed to make animal fodder. The other method which is considered useful is the addition of ferrous sulfate and polyacrylamide to the biological sludge. It compacts sludge 5% to 10% more than the floc produced by aluminium sulfate and polyacrylamide. The increase results in 5% to 10% rise in floc's ability to release water. This is important because it reduces costs of transporting the dewatered cake of sludge to land fills. Both sulfates cost the same.

12.4 FERMENTATION EFFLUENTS Large-scale fermentation processes generate effluents with high levels of particulate organic matter and soluble organic matter. As a result, BODs of many of the fermentation effluents are much higher than that of domestic sewage and ip some cases, are parallel to or higher than the paper mill and dairy effluent BODs (Table 12.1). The initial medium is generally rich in organic matter while biomass and primary/secondary metabolites produced after fermentation still represent a small amount of organic contents. Hence, the spent wastes account for 90% of original organic matter i~ antibiotic fermentation plant. Thus, fermentation effluents pose a potential pollution problem and are expensive to dispose of. A different strategy can be worked out to tackle such a high BOD generation problem and its ultimate treatment. The following approaches will help in isolation or in combination: 1. A careful selection of raw material will have a significant effect on types and quantities of effluent produced. The use of pure sugars and other refmed material instead of complex organic material will reduce wastes with a high BOD residue. Consideration of product yield, recovery cost,

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Table 12.1 BOD Strengths of Efiluents

BOD

Effiuent 1.

350 20,000 - 45,000

Domestic sewage

2. Sulphite liquor from paper mills 3.

4.

5. 6. 7.

8. 9.

10. 11. 12.

Beer(a) spent grains press (b) hop press liquor (c) mash fllter cloth wash (d) yeast wash water (e) spoilt beer (f) bottle washing Maltings(a) suspended solids (b) wastes (c) grain washings Industrial alcohol stillage (molasses) Distillery stillage Yeast production Antibiotic wastes Penicillin(a) wet mycellium from fllter ( b) flltrate (c) wash water Streptomycin spent liquor Aureomycin spent liquor Solvents

15,000 7,430 4,930 7,400 Up to 550 1,240 20 1,500 10,000 10,000 3,000 5,000 40,000 2,1500 210 2,450 4,000

100,000

- 204 -

25,000 25,000 14,000 30,000

-

70,000 10,000 13,800 5,900 7,000 Up to 2,000,000

effluent disposal problem and byproduct recovery when done together may permit the use of pure inputs (apparendy cosdy) than cheaper raw materials. 2. Biomass generated will always have high BODs. Fungal mucelia have BOD values as high as 40,000 to 70,000 mgdm· 3 . Hence, any biomass should be normally kept separate from remaining effluent. Wherever possible such biomass may be sold as a by-product in the market. But in all cases remove them and do not allow them in the effluent treatment plant. 3. Concentrations of liquid fractions of spent waste and byproduct recovery wherever possible will be a good solution to reduce high BOD wastes. Using the existing market potential and creating new markets will be necessary (Table 12.2). Table 12.2 Recovery of Byproducts from I.

Brewery(a) spent grain (b) spent grain (c) spent grain (d) spent grain (e) yeast (f) yeast (g) yeast

wet or dry - animal food for silage preparation as low grade fuel as fertilizer mixed with spent grain - protein-rich feed as source of vitamins debittered - as baker's yeast

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Distillery (a) light grains - wet or dried screenings - animal feed (b) dark grains - light grains + stillage solubles as Animal feed (c) distiller's dried solubles - as animal feed supplement as media adjuncts in antibiotic production (d) evaporated spent wash - as fuel for boilers (e) recovery of K salts from sugar beet stillage (f) stillage - SCP production using Geot'richum candidum, Candida

uti/is, Candida tropicalis After different byproduct recovery, only 1% wastes are produced. III.

Aminoacid fermentation - lysine & glutemic acid production (a) spent liquor - rich in aminoacids - animal feed supplement (b) salts - crystallization - as fertilizer

12.5 DEGRADATION OF XENOBIOTIC COMPOUNDS Before discussion of applications of biotechnology to the area of hazardous wastes it will be appropriate to understand the status of various chemicals we are talking about. In this respect Anthropogenic is the specific term for man-made compounds. Xenobiotic means stranger to life. Chemicals from agrochemical industry, petrochemical industry, mining, metal processing, aromatic industry are xenobiotic in nature. Recalcitrant compounds are those compounds that persist in nature and ar not degraded following their release in nature, even when conditions are adequate for microbial growth. Recalcitrance can be due to - (1) unusual substitution by chlorine, bromine etc., (2) unusual bonds or bond sequences (tertiary or queternary C atoms, (3) excessive molecular size, (4) failure to induce degradative enzymes, (5) difficulty to transport into cell, (6) insolubility (7) excessive toxicity of parent compound or intermediate metabolites, (8) adsorption on clay. Recalcitrant compounds accumulate in nature and cause environmental and health hazard. Solvents, wood preservative chemicals, coaltar wastes, plasticizers, refrigerants, biocides, polychlorinated biphenyls, plastic, PVC, polystyrene, polyethylene, detergents, oils etc. are some of the examples of a long list of compounds which belong to the category of recalcitrant compounds. Natural biodegradation is a slow process, but it is the ultimate fate of most of the compounds that enter in nature. Biodegradation is better by consortia (mixed cultures) than by pure cultures. Cometabolism is important phenomenon in biodegradation of xenobiotics. Here growth of organisms occur at cost of one substrate while the other is cometabolised. To combat pollution, industrial effluent treatment, solid waste treatment or waste gases treatment is necessary. In such circumstances, organisms that are capable of carrying out degradation should be used. Such organisms can be obtained by enrichment selection or by adaptation, mutation programmes or by gene manipulation work. Use of such organisms or their enzymes in appropriate reactor systems is the way of biotechnological approach to treat various pollutants. Mechanisms of biodegradation and organisms active in biodegradation differ with the compounds and it will further depend on other complexities occurring in a particular environmental situation. Temperature, pH, availability of oxygen, redox potential, competition by easily degradable substrates, concentration of pollutant, toxicity etc. are the various factors which affect the outcome of biotreatment.

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Manipulation of these factors in favour of desired degradation is the essence of successful biotechnological process. Chlorinated Hydrocarbons: Cl and C2 hydrocarbons are used extensively as solvents. Microbial degradation is less advanced. Dichloromethane is degraded but pathway is not elucidated. Dichloromethane undergoes primary dehalogenation by halidohydrolase enzymes to give chloromethanol. This spontaneously decomposes to formaldehyde. Dioxygenases remove aromaticity of halogenated aromatics. Then they undergo dehalogenation. C-halogen bond is labilised by introduction of oxygen atom on halogen carrying carbon. This results in formation of halocatechols. Relaxed specificities of enzymes is importnat. Halocatehols are critical metabolites in degradation of aryl halides. Ortho cleavage pathway is used. Meta cleavage is not possible due to toxicity of metabolites of cleavage. Ordinary pyrocatechases are inefficient in cleavage of halocatechols. Thus, ring cleavage is important. Subsequent metabolic pathway involving cycloisomerization eliminates remaining ring substituetns. In conventional treatment accumulation of toxic phenols and their black oxidation products result. Other Substituted Simple Aromatic Compounds: In sulfonated aromatics carbon constituent bond is highly polar and must be labilised at earliest possible opportunity. Sulofonated naphthalenes which are used as emulsifiers (wetting agents) in manufacture of azo dyes are inert in treatment plants but in continuous culture degradation occurs. (Dioxygenation, substituent elimination, re-aromatization). Nitrotoluenes (from amunition factory) are not completely mineralised but aromatic amines are produced. These condense with carboxylic group present in cellular macromolecules to produce polyamides. These are further resistant to microbial attack. PCB: Polychlorinated biphenyls are extremely recalcitrant. It absorbs on biological material and sediment. It is practically immobile in soil. PCB degradacion is similar to that for other aromatic hydrocarbons. As degree of chlorination increases rate of metabolism decreases. Degradation of PCB to uncharacterised low molecular weight compounds is reported. Most studies are done on pure PCB isomers. Benzopyrene: This is polyaromatic and recalcitrant compound. Its transformation gives rise to carcinogenic hydroxy and epoxy derivatives. It is not mineralised in activated sludge process. Many cultures are reported to degrade it with hydroxylases to give complex mixture of conjugated derivatives. Polystyrene: It is extremely resisant to degradation. According to one report pulverised car tyres, containing styrene-butadyene rubber were partially degraded biologically following addition of surfactant. End product could be a soil conditioner. Growth on styrene by microbial community resulted in removal of polymerization inhibitor. Pesticides: Along with the better productivity in agricultural sector, the demand and consumption of pesticides has continuously increased. Chemical pesticides though useful in protecting the crops, have several adverse effects on envrionemnt. Pesticides may enter environment as point dicharges or non-point discharges. Pesticides and their residues may come from manufacturers wastes or from users' wastes or after application it may get added excessively to the environment. Residual pesticides may enter in food chain and can get concentrated as it passes higher in the food chain and finally may come back to human. This phenomenon is referred to as biomagnification. Degradation of chemical pesticides is one of the problems facing us in our pollution abatement efforts. Pesticides are hydrocarbon skeletons with substituents. There is a definite pattern that is exhibited by pesticides during their biodegradation. Stages are (1) All carbon chains are degraded by f3 oxidation. (2) Resulting C2 fragments are further metabolised by TCA cycle. (3) Substituents on aromatic rings are removed partially or completely.

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(4) Aromatic ring structures are degraded by dihydroxylation and ring cleavage mechanism. (5) Substituents may be halogens, nitro, amino groups which cause intereference in oxygenation and cause reacalcitrance. Methyl, methroxy, carbonyl or carboxy groups may be present and can be removed. (6) If dihydroxylation occurs on substituted aromatic compound then previously described pathways will be followed with minor modifications. (7) Saturated ring structures are more refractory than aromatic analogs. They do not support growth of microorganisms. However, they may undergo cometabolism. (8) If the pesticide compound contains aliphatic then degradation is still difficult.

+ aromatic + alicyclic +

hetrocyclic portions

(9) If moieties are connected by ester, amide or ether bond in molecule then cleavage of such bonds by microbial enzymes is the first step of attack and then subsequent degradation occurs. (10) If such attack cannot occur then degradation will be commonly initiated at aliphatic end of the molecule. (11) If extensive branching is present at aliphatic end then degradation may start from aromatic end. Microbial Degradation of Surfactants: Surfactants find use for cleaning purpose in domestic or industrial applications. Surfactants may be hard or soft according to their susceptibility to biodegradation. Surfactants my be classified as cationic, anionic and nonionic. Non-ionic surfactants fmd use in agriculture sprays and in cosmetics. Hard detergents have branched alkyl side chains. Alkyl benzene sulfonate caught public attention as pollt;tant as early as 1950s. Linear alkyl benzene sulfonates (LAS) are unbranched and are therefore biodegradable. LAS degradation occurs by terminal methyl group oxidation (requires oxygen and process is aerobic) followed by ~ oxidation of linear side chain. Ring degradation does not occur until side chain is eliminated. Branching does not necessarily confer recalcitrance. But the process of P oxidation is inhibited. Mechanism of branched chain degradation is not clear. C-S bond is stable and increases biological resistance of detergent molecule. Desulfonation reactions are not completely elucidated but could be affected by hydroxylases and monoxygenase mechanims. Ultimate fate of sulfonate is conversion to sulfate via intermediate sulfite. In aryl sulfonates carbon consituent bond is highly polar and must be labilised at earliest opportunity. There are evidences of plasmid coded desulfonation and meta cleavage of aromatic ring. Sulfatase enzyme is involved in initial attack on linear sulfates. Corresponding alcohols are produced which are then further metabolised. Sulfatase enzymes attack C - 0 bond of C - 0 - S linkage. In non-ionic detergents their wetting and emulsifying properties are used in agricultural sprays and cosmetics. Linear primary alcohol ethoxylates are rapidly and completely mineralised. Their high molecular weight analgogs are increasingly recalcitrant. Degradation occurs by terminal methyl group oxidation followed by ~ oxidation to produce short chain alkonate ethyoxylate with no surface active properties. In secondary alcohol ethoxylates hydrophobic alkyl chain is degraded from both ends by oxidation and (0 oxidation. Ether linkage found in these compounds contributes to recalcitrance. Possible mode of cleavage of this linkage is - (a) monooxygenative, (b) hydrolysis, (c) carbon oxygen lyase, (d) oxidation of carbon atom alpha to ether linkage followed by ester hydrolysis. ~

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Table 12.3 Mechanims Used in Biodegradation of Xenobiotic Compounds

No. Category of Xenobioitc Cmnpounds

Mechanism Used by Organisms

1. Aromatic hydrocarbons

Ring cleavage meta pathway produces catechol, substituted catechols. Attacked by dioxygenases.

2. Substituted benzene, toluene, xylene, a1kylbenzene

Microbial oxidation.

3. Halogenated organics Dehalogenation, ring cleavage, halide elimination afterring cleavage. 4. Aliphatic and halogenated aliphatic hydrocarbons of crude oils and petrochemical products.

Degradation via mono-dioxygenases.

5. n-alkanes 6. Chlorinated methanes, chlorinated ethanes, chlorinated ethylene, chloroform, tetrachloromethene, tetrachloroethane.

Terminal and sub terminal oxidation. Reductive dehalogenation

7. Polychlorinated biphenyls 8. Aliphatic hydrocarbons (Hexadecane)

Reductive dechlorination. Anaerobic degradation.

Dye Waste Treatment by Bacteria: The treatment of dyestuffs in waste is now a costly process, involving absorbants, concentrators and incinerators. Now the bacteria can do the job economically by decomposing dyestuffs into colourless compounds. The new bacteria found by a group from Tokyo Instinlte of Technology, can decompose around 20 different commonly used dyes, includng blue, red, yellow and purple pigments. This filamentous bacteria can do it both in liquid culture and on agar medium. Thus, it can be useful for liquid as well as solid wastes. The bacteria are easy to culture and thrive in pH range of 4-7 and temperature 20-30°C. Engineered Bacteria Developed to Metabolise Polyhalogenated Compounds: A strain of bacteria has been engineered by researchers at the University of Minnesota to express two enzymes that enable it to decompose highly halogenated compounds. Bacteria are known to metabolise many organic compounds, but attempts to culture bacteria that metabolise polyhalogenated pollutants have been largely unsuccessful. Polyhalogenated compounds can be broken down by bacteria but only in a two step process ~ anaerobic bacteria must first metabolise them by reductive dehalogenation, and then aerobic bacteria must degrade the compounds by using oxygenases. However, seven genes encoding two multi-component oxygenase enzymes have now been engineered into Pseudomonas putidaJ enabling it to metabolise polyhalogenated compounds by sequential reductive and oxdative steps. One of the enzymes, cytochromine p450 monoxygenase, reduces the polyhalogenated compounds under low oxygen conditions, and the other, toluene dioxygenase, oxydyzes the products to non-toxic compounds. Biotech Process to Destroy Halo-alcohols: A bioprocess developed in Wales to remove unwanted contaminants in the manufacture of Kynene ULX used in the paper making process has been commercialised by Hercules in two plants, one in France and the other in Sweden. Carbury Herne

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(UK) and Hercules (USA) are collaborating on the use of biocatalysts to detoxify halo-alcohols, a byproduct in the reaction between epichlorohydrin and aqueous solution of polyaminoamide. The reaction produces polymers which are then used to give strength to paper. Halo-alchohols produced as byproduct however, have no value and are carcinogenic. So far two different strains of bacteria have been developed to degrade the halo-alcohols. These work through hydrolysis reaction which effectively restructures the molecule to form an epoxide. Of the two principle form of halo-alchols. 1, 3 dichloropropan 2-01 (DCP) and 3-chloropropanediol (CPD). DCP is first converted to epichlorohydrin and then into CPD. The CPD is then converted to glycidol then glycerol, which is finally broken down into carbon dioxide, water and chloride ions. Hercules claims the process reduces the level of DCP and CPD by a factor of 10 and to level of 1 ppm of DCP and 5 ppm of CPD. Recycling of Rubber: Sulfer-loving microbes such as Rhodococus, Sulfolobus and ThiobacillusJ may provide in future an economical way to recycle the mountains of used tyres. Battele Pacific Northwest Laboratory is developing a process in which bacteria attack the sulfur that binds together a tyre's basic rubber polymers. The bacteria leave carbon-rubber backbone intact for reuse. From 2.5 to 3 billion used tyres are stored in USA and 200 to 300 million are added each year. Some are burnt causing pollution, while the recovery of hydrocarbons by pyrolysis is uneconomical. In Battelles process, tyres are ground to about 75 ~m then mixed with bacteria in aqueous solution along with nutrients for the microbes. After several days, the rubber is recovered from the solution. Three different bacteria produce rubber of different surface chemistry. The process has been tested at 41 scale and scale-up at 20-401 is planned. Clean-up of TNT: Naturally occuring Pseudomonas bacteria that have liking for 2, 4, 6 trinitrotoluene (TNT)are used to clean-up TNT contaminated soil in a process developed by Argonne National Laboratory. Addition of mollasses increases bacterial growth and increases TNT removal tenfold. The bacteria break down TNT to non-hazardous materials such as CO2, fatty acids and butanediol. Contaminated soil is slurried with 85% water and treated in four 300 gallons reactors. Mollasses is added twice a week, and 10-20% of the volume was replaced with fresh slurry once a week. The estimated cost was $ 250-300/yd3 as compared to $ 400/yd3 for incineration.

12.6 USE OF IMMOBILISED MICROBIAL CELLS IN WASTE WATER 'FREATMENT Pollutants whether natural compounds like lignifieq wood or artificial ones like organophosphates, chlorobenzenes are not as recalcitrant as what they are thought to be. For most of the chemicals over the course of millions of generations, microorganisms have evolved metabolic activities either to use these chemicals as nutrient sources or to detoxify their surrounding. And still some other chemicals are degraded by cometabolism activity. Inspite of this, for the pollutants wh.ch are tough, microorganisms can be developed to degrade them by mutations and genetic transfers. Although it is ultimately the enzymes which carry on tP~ ~egradation activities and also it is true that using enzymes in pure form as a catalyst has certain advz..r:~ages, still :lSing complete cells rather than purified enzymes is a more convincing proposal. Advantages that whole cells have over isolated eazymes in applications are: (1) Enzymes when within the cells are better protected from denaturation by the intact cell wall and membrane. (2) Degradation activity may be composed of a series of reactions working in concert and it will be difficult to develop different systems for different enzymes working together. (3) Isolating and purifying the component enzymes is quite expensive. Also chances of denaturation are more.

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(4) Sometimes, induced enzyme production occurs in cells which is required for degradation activity. Advantage of this fact cannot be taken and such induced enzymes will not be available if purified enzyme systems are used. (5) Degradation rates and resistance to toxic pollutants will he great when the mixed culture cell population is used and not so with only enzyme system is used. As using whole cells rather than isolated enzymes has the above-mentioned advantages, further using immobilised cells instead of free suspended cells has additional advantages as follows: (1) Overall capability of cells becomes more enhanced because of the reuse of cells that are made. (2) Cells do not contaminate the outgoing reaction mixture. (3) Cells are more evenly dispersed by immobilisation, so diffusional restrictions are minimised. (4) Immobilised cells are used with more ease to exploit the kinetic features of continuously stirred and packed bed type of reactors. (5) Product inhibition will be minimised by using immobilised cells in packed bed type of reactors. (6) Immobilised cells work better in continuously-operated systems. Uses of Immobilised Cells and Enzymes for Waste Water Treatment: Applications fall into 4 categories: (a) BOD/COD reduction. (b) Specific pollutant detoxification. (c) As biosensors. (d) To get the product from the waste. (1) Cells trapped on the slime layer of trickling filter beds are conventional immobilised cells in (2)

(3) (4)

(5) (6)

(7)

use. In activated sludge method also, flocculation of cells is the method of immobilisation which permits economic recycling. Natural flocculation can be enhanced by the use of synthetic polyelectrolytes which improve the normal partial cell immobilisation. Several organisations have immobilised cells with a view to removing the particular substances from waste water. At the East Hyde Sewage Works, waste water is forced through a sand bed on which species such as Hypomicrobium are deliberately grown with added methanol to cause nitrate reduction. Another plant at Rye Meads, processing 81000 m 3 of water per day, uses a similar technique but without methanol addition. Nitrate reductions between 50-90% are obtained. Mohan and Li examined the use of Micrococcus denitriftcans cells encapsulated in liquid membranes for the reduction of nitrate to nitrite. Capsules containing 500-600 cells are formed by emulsification using cells, surfactant, oil membrane strengthening additives. These capsules retained 78% of their activity for 120 hours compared with free cells whose activity reached zero in just 16 hours. The immobilised cells also tolerated lO-4M HgC12 while free cells were sensitive. Immobilised cells have recently been used as biosensors to continuously evaluate the Biological Oxygen Demand (BOD) of waste water. These analytical devices usually work by measuring the loss of enzyme activity caused by exposure to the pollutant, the degree of inhibition of

Biotechnology - Applications to Environment Protection

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16) (17)

(18)

237

the enzyme being proportional to the concentration of the pollutant present in the sample under test. Use ofimmobilised a-amylase enzyme has been proposed for treatment of waste waters from the wheat starch industry and also to clarify colloidal starch-clay suspensions of 'white waters' from the paper mill industry. The brown, partially chlorinated lignin derivatives commonly known as 'kraft lignins' are present in the pulp mill effiuents. These are normally treated in aerated lagoons or activated sludge systems and BOD/COD is reduced. But it fails to reduce the colour. Kraft lignins can be decolourised by using white-rot fungus Coriolus versicolor entrapped in calcium alginate beads and supplemented with various carbon and energy sources. Also the lignolytic fungus Phanerochaete chrysosporium may be used. Immobilised cells removed 80% of the colour after incubation for 3 days in the presence of sucrose. Biological method eliminates carcinogenic chlorinated lignins. Nitrosomonas europaea cells immobilised in alginate have been used to oxidise ammonia in waste waters to N02 and N0 3 so as to reduce BOD and prevent algal growth. Further degradation of soluble nitrates has been achieved by Pseudomonas denitriftcans immobilised in alginate which have been used to denitrify drinking waters. Cells have been provided with an exogenous carbon source and reduce nitrates and nitrites completely to gaseous products. When operated in continuous reactors, half-life of 30 days is shown by cells. With the adding of additional nutrients, fresh cell growth occurs and the rate of reaction increases. Degradation of phenols remaining in the waste waters of hospitals, laboratories and coal processing to coke can be achieved either by using the fungus Aureobasidium pullulans adsorbed by fibrous asbestos or by using cells of Pseudomonas spp either adsorbed to anthacite coal or entrapped on alginate gel. Treatment of phenol containing wash waters is advantageous since it avoids damage to the conventional biological treatment systems. Formation of CH4 continuously for over 90 days from the waste waters using a population of methanogenic cells entrapped in agar, collagen or polyacrylamide membranes has been achieved. Microbial population isolated from the sewage sludge digester has been used to degrade waste waters from the alcohol fermentation factory to CH4 • Cyanide present in aqueous wastes can be effectively detoxified using immobilised mycelia of Stemphylium wti in which cyanide hydralase has been induced. Cyanide is converted into formamide. Flocculation by polyelectrolytes was the preferred method of immobilisation. Immobilisation stabilised the enzymes and allowed them to be used continuously. The immobilised cells were, however, inhibited by nickel and so they cannot be used for treating wastes from electroplating. Production of glucose from waste cellulose can be regarded as a means of disposing of waste material and also producing valuable products. Unfortunately, despite the undoubted potential, no practical process using immobilisation techniques has yet emerged. Immobilised enzymes can be used to hydrolyse lactose in whey which has high BOD and is in large volume in the wastes of the cheese-making industry. Zoogwea ramigera can be used to accumulate copper continuously. Thus microorganisms have a potential to remove toxic metals in wastes, even uranium included. Erwinia rhapontici can be entrapped in calcium alginate gels in 20 litre columns and several tonnes of isomaltulose can be produced from sucrose. Cells used are structurally intact, nongrowing, not viable and with operational half life of 1 year. Using immobilised yeast cells, molasses in wastes can be converted into alcohol.

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(19) Pseudomonas putida has dehalogenases which degrade mono -

and dichloroacetates and propionates from herbicides and pesticides. Dalpon degradation activity is stable for 20,000 hrs. in chemostat. (20) An enzyme extract obtained from mixed microbial population adapted to grow on Parathion was found to hydrolyse a number of organophosphate insecticides. Enzyme could be immobilised with operational half-life of 280 days. (21) Nitrate pollution of ground water is a health hazard. The current methods for removing nitrate are ineffective and impracticable. Processes that use live microorganisms to denitrify water are slow, frequently incomplete, difficult to set up and maintain. In a new technique developed by Mobite GmbH, Germany, and the University of Michigan, US, a series of oxidoreductase enzymes are coimmobilised in polymer matrix along with the electron donor species. The redox process which is driven by electric current results in complete conversion of nitrate into gaseous nitrogen. Such electrobioreactors could be developed for the removal of other water contaminants such as pesticides if appropriate enzymes and cofactors can be identified.

12.7 BIOREMEDIATION Products, chemicals arising from modern technologies, are posing a great danger to the natural breakdown processes and built-in mechanisms of nature to maintain ecological balance. New pollutants, complex as they are in nature, are accumulating in nature systems. Liquid, gaseous and solid wastes created by modern technology were so far treated with physicochemical processes (burying, burning etc.). Bioremediation is the microbial clean-up approach. Microbes can acclimatize themselves to toxic wastes and new resistant strains develop naturally. But if microbial physiologists, biochemists, geneticists and ecologists join hands with chemical engineers, efficacy, efficiency, economy and environmental safety can be generated in the use of microorganisms for environmental protection. Bioremediation engineers from the BioTrol Company in the USA used Flavobacterium to remove pentachlorophenol from contaminated soil. Aeration of this soil helped the process. At the Gulf of Alaska, oil spilled from ships was cleaned by adding nutrients to promote growth of oil-eating microbes. The microbe referred to as GS-1S has been found by an American microbiologist which can eat up uranium from wastes (water) of a nuclear weapon manufacturing plant. GS-1S microorganisms in bioreactor convert uranium in water into particles that precipitate and settle to the bottom. Collection and disposal then become easier. GS-1S bacterium metabolises uranium directly deriving twice as much energy as it normally would do in the presence of iron. The organism grows overnight. The organism can be useful for waste water treatment of uranium mining. Yet another novel and interesting method has been developed by scientists in the Southampton University in England to extract metals from waste. Metal ion concentration reduces and metal accumulates in coats. Metal if it is of magnetic nature can be easily removed using a big magnet. When organisms grow with added nutrients in the presence of metal containing waste water, they accumulate metals in their coats and so reduce the concentration of metal ions in the solution. Then magnetic fields of up to 8 Tesla (80,000 Gauss) compared to 1 Gauss of earth's natural magnetic field are produced by the superconduction coils. When a field of this strength is made on a SS filter, it produces a force factor on particle which is 64,000 times that of ordinary bar magnet, so it pulls away coated microorganisms from the liquid passing through the filter. Uranium, rhodium, platinum, gold, nickel, mercury, cadmium concentrations can be reduced from 10 ppm to < 1 ppb in ionic concentrati(>n after treatment.

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Approximately, two million Trabants, Germany's unwanted cars, are now accumulated in the junkyard and posing a waste disposal problem. While engines break down easily, their plastic shells are indestructible. This is duroplastic and not thermoplastic. Therefore, it does not melt out. Burning them would add toxic gases. Microbial help is now being sought. Organisms are now being searched which will digest resin in the duroplastic. Penicillium degrading cellulose, the other constituent in duroplastic, has been already patented. . At present, more than 100 companies with the help of 1,000 different spp. of bacteria and fungi are continuing to fight against some of the most notorious pollutants such as vinyl chloride, PCB's, benzenes, oil spills etc. Bioremediation employs biological agents to render hazardous wastes to nonhazardous or less hazardous wastes. Demonstrations and help is given by EPA (Environment Protection Agency) of the US to interested industries in developing new bioremediation techniques. For example, a system called Geolock/Biodrain involves high density polyethylene plastic walls that can be driven into the ground to cordon off polluted soil from good soil. In-situ bioremediation then can be carried out with little disturbance to the good soil. Some fungi, even as dead biomass, trap metallic cations in aqueous solutions. This is due to their special wall composition. Many fermentation industries produce fungal biomass as an unwanted byproduct which can be used for this purpose. The biomass of fungi Rhizopus arrhizus can absorb 30130 mg of cadmium/gm of dry biomass. Fungus has anions in its cellwalllike amines, carboxyl and hydroxyl groups. 1.5 kg of myceliuin powder could be used to recover metals from 1 tonne of water loaded with 5 gms of cadmium. Algasorb is a product patented by Biorecovery Systems Company which absorbs heavy metal ions from waste water or ground water in the above-said manner. Algasorb is produced by trapping algae (dead) in silica gel polymeric material. The trapping protects algal cells from being destroyed by other microorganisms. Algasorb functions very much like commercial ion exchange resin and on saturation, heavy metals can be stripped out of it. Controlling pollution at source or containing it is the approach which is more useful. Heavy metals like Hg, Cd, Pb are often present as pollutants in the waste water of modem industry. The consequences of Hg as a pollutant have been experienced in Minamata disease and similar instances. Some algae and bacteria can accumulate great quantities of metals in the surrounding. Pseudomonas aerugenosa can accumulate uranium, Thiobacillus can accumulate silver. Acid rain which is due to the sulfur in emission from polluting factories is acrually due to S in coal which is used for burning. By removing S in coal before burning, this problem could be solved. The chemical method is cosdy. Organisms from hot springs are able to use sulfur for deriving energy. A small-scale plant at Ohio in Japan has shown that this microbial process to remove S from coal works and it will be very soon a commercial operation. Several major companies in the US sell mixtures of microbes and enzymes to clean up the chemical wastes, including oil, detergents, paper mill wastes, dioxin, pesticides. In the super bug of Dr. Anand Chakravarti, genes able to degrade all four hydrocarbon components of oil were engineered together and so it could degrade Xylene, naphthalenes, Octanes and camphors. Plasmids (extrachromosomal DNA) bearing necessary genes are presen~ in the oil-eating microbes. Capacity of microbes to eat up oil is further proved in oil spills incidences at many places. Biological transformation of organic pesticides and herbicides which are not natural, does take place in the soil. Persistence varies and depends on such transformation actions in natural states. Half

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lives, i.e. time for 50% degradation, for some are - 2, 4, D (2,4 dichloro phenoxy acetate) - 0.5 months, Dalapon (2,2 dichloropropionate) - 0.5-1.5 months, Dichlorobenil (2, 6 dichlorobenzonitrile) 1.5 - 6 months and DDT (4,4 dichlorodiphenyltrichloroethane) - 6 months. Several microbial genera like Bacillus, Arthrobacter, Nocardia, Pseudomonas attack them as part of the mixed population. Chlorobenzoates which are used as herbicides and some which arise out of polychlorinated bisphenyls are attacked by Pseudomonas. Researchers in the Michigan State University have reported that the white rot fimgus Phanerochaete chrysosporium can degrade DDT and Dioxin to CO2 when placed in N deficient medium. 90% degradation in 48 days occurred. Also this fimgus has been shown to degrade halogenated aromatic rings and dechlorinate alkyl chlorides to produce CO 2 , In the US, a novel method using naturally occurring bacteria to clean up contaminated soil is being tried. The idea is to pump water, methanol, nutrients into the polluted soils to encourage certain bacteria in the ground to grow. Methylotrophs metabolise methane and methanol and in the process also degrade many common pollutants. T~e organisms are ubiquitous in soil but are few in number; therefore, this method is used to boost up their activities. Bacteria destroying Trichloroethylene and other toxic chemicals are to be cultured in polluted soil for pilot plant stu,dy. Methane oxidising Metahnotrophs are responsible.

12.8 BIOLOGICAL DEODORISATION Deodorisation technology has assumed greater importance as environmental problems have attracted the attention of many more people. Offensive odour is complained about by people before they receive any damage therefrom and is often linked to air pollution. As offensive odour affects human sense, it is put under administrative regulations. Also since studies on the actual state of offensive odour done are very limited, an assessment depends on the human sense of smell. Deodorisation processes are roughly classified into physical, chemical and biological ones. Waterwashing and absorbing methods are dominant types of physical deodorisation processes, while chemicalapplied washing and incineration methods are major chemical processes. Biological processes featuring low running costs and easy operation/maintenance have recently come to the fore. In a biological deodorisation process, bad smell ingredients are decomposed by exploiting the metabolism of the microorganisms. The processes already in use are: (1) Soil process using microorganism living in soil; (2) Packed-column process based on porous carriers, allowing microorganisms to propagate therein; and (3) Active sludge process in which offensive odour gas comes into contact with the active sludge and offensive odour ingredients are decomposed by microorganism acting as a component of active sludge. Packed column process has many advantages: (a) high deodorisation capacity;

(b) simple/compact design; (c) easy operation; (d) low running costs;

(e) no by-products.

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The carriers designed to propagate microorganisms are mainly produced from peat, resulting from the deposition of reed. Studies are being made on how to use cardboard for the same purpose. Carriers with a wide surface area and suitable for propagation of microorganisms are to be sought. Elucidation of the deodorisation mechanism of microorganisms is still unsolved. An exhaust gas treatment system for H 2S and S02 based on bacteria is already mooted in Japan. The bacteria employed for this by Dowa Mining Company is Chiobacillus ferroxidans which obtains energy by oxidising Fe+ 2 to Fe+ 3 . These organisms live in natural environment in the neighbourhood of mines. These bacteria have maximum capacity to digest abundant iron. In the case of H 2S treatment system, solid sulfur is recovered from H 2S merely by circulating bacteria in intimate contact with ferrosulfate and ferric sulfide.

The new system of Dowa mining consists of H 2S reaction tank, bacteria tank, a sulfur recovery unit and a simple closed circuit. Compared with conventional process using caustic soda for neutralisation, the new system reduces the cost to about one-third. It makes possible greater energy conservation, compact construction, room temperature treatment and easy operation control. The new system has a potential application in petroleum plants, gas production plants and chemical processing plants. Bioscrubbing is the microbiological method used for scrubbing of waste effluents. It can be used to remove, to detoxifY certain substances present in wastes. Detoxification of cyanide can be done using enzyme Rhodanese of Bacillus steraeothermophilus (Cyanide ~ thiocyanate). Immobilised enzymes converting cyanide into formamide and other examples are mentioned in the portion of immobilized enzymes in this chapter only. Noxious, toxic, odorous gases, reduced sulfur compounds being treated by Thiobacilli aerobically or anaerobically is also an example of Bioscrubbing. Hypomicrobium spp can be used to oxidise dimethyl sulfide (malodorous component of animal wastes is an example of bioscrubbing for deodorisation).

Aerobic process H 2S + 202 ~ H 2S04 (CH3hS + S02 ~ 2C02 + H 2S04 + 2H20

Anaerobic process 5H2S + 8NaN03 ~ 4Na2S04 + H 2S04 + 4Hp+ 4N2 (CH3hS + 4NaN03 ~ 2C02 + NaS04 + 2NaOH + 2Hp + 2N2

12.8.1 Processes for Biological Purification of Waste Air These have been used in the last few years. They are based essentially on the principle of absorbing the harmful and odorous substances in solid or aqueous phases and degrading them further by means of microorganisms. The advantage of biological purification resides in the fact that in contrast with absorption alone, which works only at relatively high capacities of absorbant for the odourous substances, the microorganisms keep their concentrations in the medium low through their metabolism.

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Table 12.4 Biological Degradation of Malodorous Substances

Source

Main Constituents

Microorganisms

Systems

1. Pig faeces

Lower fatty acids

Streptomyces griseus S. antibioticus

Surface culture

2. Culture of

Geosmin

BacillUs Cereus B. subtilis, Sarcina spp, Mimaspp

Submerged culture

Carbon monoxide, ethylene, acetylene

Aerobic soil bacteria

Earth columns

Bacillus cereus

Submerged culture

Anabena Circinalis 3. Atmosphere

4. Actinomycetes in water reservorrs 5. Industrial waste

Phenol

Bacillus cereus

Sand ftlter

6. Foundry waste air

Phenols, amines, formaldehyde ketones aldehydes

Adapted activated sludge

Biowasher

7. Meat processing

Lower fatty acids

Nocardia spp Microthrix Pervicella

Activated sludge process

8. Pulp industry oil refilling photographic industry, animal

Reduced sulfur compounds, thiosulfate, H 2S, methyl mercaptans, dimethyl sulphide

Thiobacilli

wastes

243

Biotechtwlogy - ApplicatWns to Environment Protection Table 12.5 Microbial Degradation of Substances with Intense Odours

I.

2.

Substrate

Degradation products

Degradation pathway

Methanol formaldehyde

CO 2, Water

Assimilation via serine, transhydroxy methylase

Pseudmtwnas AM I

Lower alcohols

Acetyl CoA

13 oxidation

Many bacteria and fungi

Subterminal attack by monoxygenases

Pseudmtwnas multivorans

& fatty acids

Microorganisms

3.

Methyl Ketones

4.

Dimethylamines

Methylamine and formaldehyde

Hydroxylase

PseutUmwnas aminovorans

5.

n-propylamine

Propionate

Amine dehydrogenase

Mycobacterium conpolutum

6.

Phenol

Acetaldehyde and pyruvate

Meta-cleavage

7.

Phenol

Acetyl CoA and succinate

Ortho cleavage

8.

p-cresol

Protocatechuic acid

Ortho cleavage

9.

M-cresol

Fumerate and pyruvate

Gentisic acid pathway

Pseudmnonas putida Trichosporon cutaneum Psudomonas fluorescence Pseudmwmas

10.

Benzaldehyde

Benzyl alcohol and benzoic acid

Dismutation

II.

Aniline

2-hydroxyacetanilide 4-hydroxyaniline

12.

Aniline

Pyrocatechol

13.

Pyridine 4 methylpyridine

14.

Indole

Pyrocatechol

Chromobacterium Piolaceum

15.

Indole

Tryptophan

Neurospora cmssa

16.

Camphor

Lactonic acid

Pseudmtwnas .putida

17.

Dimethyl sulphide

spp

Acetobactor ascendms Aspergillus ochraceous

dioxygenation

Nocardia spp Pseudmtwnas spp

Hypomicrobium spp

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.Advances in Biotechnology

Biofilters, beds of packing bodies through which liquid is trickling and biowashers, i.e. spray waters or bubble columns, are used. The waste air (Table 12.3) from garbage composting plants, from carcass processing facilities, from agricultural enterprises, of sewage plants and even from industrial plants such as foundries have also now been purified successfully by biological processes. Biological processes are also used for fermentation plants. In the near future, purification of sulfur containing power plant waste gases must be considered and also binding of malodorous substances from the waste air of chemical plants.

12.8.2 An Efficient and Economical Bioscrubber System to Remove Styrene from Waste Gases An efficient and economical bioscrubber system to remove styrene and Volatile Organic Compounds (VOC) from industrial waste gases has been developed. The process uses water to strip styrene and VOCs from industrial waste gases in a packed column scrubber. The styrene-laden waste water is pumped to fermenter, where selected 5trains of naturally occurring bacteria decompose styrene to CO 2 and water. Clean wash water is then recycled to the scrubber. Since 1994, the first commercial unit has run continuously on 20,000 mafhour waste gas stream at German automotive parts manufacturer, cutting the styrene concentration from 400 ppm to 5 ppm. The operating costs are reported to be only about 20% of those of comparable biofilters, while the capital costs is at least 40% less.

rno was one of the first organisations to appreciate the true potential of biological treatment. They have developed a special low-cost biofUter containing compost and wood bark which treats VOCs. They have also developed a fast-acting biofUtration system which removes toluene, xylene, propene and styrene. Fungi are used in this system well dispersed on creamic carrier. rno has also developed two stage system in which a photoreactor with UV radiation improves the biodegradability of off-gases containing hydrophobic pollutants which are then biotreated. Styrenes, Nox and alkanes are being treated this way. The last compound is tried by improved bioscrubber. 12.8.3 Biotech Processes for Producing Elemental Sulfur Biotechnology can clean up sulfur from gas streams and produce elemental sulfur. A flue gas desulfurisation process called Biostar has been developed by Paques BV (Netherlands) and Hoogovens Technical Services Energy and Environment BV. First sulfur dioxide is absorbed and converted to sulfite by reaction with sodium hydroxide, then sulfate reducing bacteria convert it to H 2S, which in turn is converted to elemental sulfur by Thiobacilli. Another Paque bioprocess, for H 2S has been installed commercially in a Dutch paper mills and is producing 0.2 m.t/day of sulfur from a gas stream, reducing the H 2S content from 12000 ppm to 40 ppm. NKK Japan uses Thiobacillus Jerroxidans bacteria in its Bio-SR process. A ferric sulfate solution absorbs H 2S from a gas stream, producing elemental sulfur and ferrous sulfate solution. After sulfur is filtered, the solution is regenerated to ferric form by the T.Fen-oxidans. Sulfate reducing bacteria (anaerobic) may also offer a way to deal with the mountains of gypsum accumulated from wet-scrubbing of S02 from stack gases. In a process developed by Idaho National Engineering Laboratory, bacteria and slurried gypsum are mixed in stirred tank along with a nutrient of starch from potato wastes. The bacteria produce H 2S which can be converted to elemental sulfur. INEL also is developing a way to treat stack gas directly by sparging it into a tank containing water, bacteria and starch. The S02 dissolves to form sulfurous acid, from which the bacteria produce H 2S. The process is yet to be scaled up. At present the technology to remove sulfur from process streams and effluents are fast developing and will provide sulfur in a big way. Recovered sulfur may one day exceed the total demand and there will be no native sulfur production from mining.

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12.8.4 Bioreactor Promises NO Reduction in Flue Gas Idaho National Engineering Laboratory USA is reported to be experimenting with a bioreactor for removing nitrogen oxides from flue gas. Tests using a gas stream containing 250 ppm. nitric oxide showed that bacteria can remove upto 99% of the NO, leaving a residula conc. of only 2.5 ppm. Pue gas from coal typically contains 100-400 ppm. of NO. The flue gas is passed through a column of 100 mm. diameter and 1 meter long. Compost inside the column immobilises the Pseudomonas denitrificans bacteria and also serves as source of nutrient. A sugar solution dropped over the bed every few days provides a food supplement. With flow rates of 1-2 lit./min. the residence time is of the order of 1 min. Researchers attribute the bacterias impressive performance to the fact that in gas phase the mass transfer is better than that in similar liquid system. The bacteria grow best at 30-45°C so the system has to be in the coolest part of the flue gas duct.

12.9 PHYTOREMEDIATION Plants can treat municipal and industrial wastes. Plants are remvoing heavy metals and organics from ground water and soil for a fraction of cost of traditional treatment. Phototech Inc., Phytokinetics Inc. and researchers at universities are convicing about its effectivity. Plants are regarded as solar driven pumps and filters with exploratory liquid phase extractors. Phytoremediation involves a number of different clean-up schemes as well as number of different types of plants. Key of success is selecting right plant. Some specialisation can be identified. Poplars have been found to remove trichloroethane effectively, and to block sewage seepage of polycyclic aromatic hydrocarbons (PAHs), while grasses are suitable for spilled hydrocarbons at petroleum sites and sunflowers are proving useful with heavy metals. Some systems use plants alone. In rhiwfltration, plant roots filter contaminant from waste water or ground water. In phytoextraction, plants are used to take up contaminant from soil or ground water and are then harvested for disposal or often recycled to recover metals. Phytostabilisation uses plants to keep lead and other site contaminants from leaching out to neighbouring areas. Poplars are used for containment at site in North Salt Lake City, Utah, where they separate a refinery from neighbouring wet lands system and keep polycyclic aromatic hydrocarbons (PAHs) from entering the ground water as reported by Phytokinetics Inc. at Utah. Phytokinetics uses 1800 poplars, willows and cypresses to prevent nitrate from Missouri fertiliser factory from entering the drainage water and streams. In future genetic engineering of species of plants, shrubs and flowering plants to optimise removal or containment is expected. Plants often have excess reducing power from photosynthesis that can be tapped to reduce toxic metal ions, as per the researchers at University of Georgia. They have demonstrated this possibility by genetically engineering a plant not only to absorb and cocentrate Hg2+ ions from soil but also to reduce them to less toxic elemental mercury. Mer A gene from soil bacteria having this capacity is inserted into Arabidopsis thaiiana plants. While control plants died in n:::dium containing 25 mm HgC12' the transgenic plants grew well at 50-100 MM HgCl2 producing elemental mercury. The gene may be further modified according to researchers to reduce and lower the toxicity of other heavy metals like Pb2+ and Cu2+. Mining industry and dumping of wastes is generally responsible for contamination of soil by heavy metals. Plants capable of accumulating high concentrations of metals such as zinc, nickel, cobalt, lead, copper, cadmium could provide an effective and practical method of cleaning of heavily polluted soils. The Alpine Peunycress (thiaspecaerulescens) has been found to be hyperaccumulator of zinc and cadmium. Research is on to identify the fastest growing and strong accumulating genotypes and to improve metal uptake by genetic engineering if possible. Also harvesting of crops and disposal of metal enriched harvested material are the other problems to be tackled.

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12.10 ENVIRONMENTAL MONITORING Biosensors employing various enzymes, cells, antibodies, or oranelles in immobilised· form are widely used in environmental monitoring. Sensitivity, specificity, accuracy, stability are the important features of such biosensors. Biosensors used for environmental monitoring in addition should be portable and convenient for continuous monitoring in most of the cases. Many biosensors work on the fact that activity of immobilised enzyme is inhibited in presence of toxic chemical or heavy metal ion. Table 12.6 Some Examples of Biosensors Used for Environmental Monitoring

Pollutant

Environment Rivers, Lakes, Waste water

Bwsensur Algae-toximeter

Blocking of electron transport is measured as fluoreecence emitted. as low as 5 ppm. of herbicide is detected continuously

• Phosphates

Immobilised alkaline phosphatase electrode

inhibited by phosphate

• Methanol

Use of Alcohol oxidase electrode

low reactivity with ethanol

• Benzopyrene

Cytochrome P-450 metabolism as substrate or antibody binding

• Mercury, lead cadminum

Yeast Alcohol dehydrogenase enzyme

• Fluoride

Urease enzyme or Acetylcholineesterase sensor

• Herbicides

• Pesticides

Butyrylcholineessterase

• 3-chlorobenzoate

Pseudonwnas spp.

• Phenol

Rhodotorula spp. phenol oxidase

• Derivatised aromatic compound

Pseudonwnas cepacia

• Lead

Fuel combustion

Urea or Acetylcholine is used as substrate, 2 ppm of fluride

is detected.

3 ppb of ethyl paraoxon, 4 ppb. of malathion is detected

or

Oxygen type electrode linear relationship upto 9 ppm

Enzyme inhibition

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Field ofbiosensors is multidisciplinary, spanning fundamental and applied aspects of biochemistry, electrochemistry and electronics. The field of biosensors is perhaps the one from which rapid and substantial returns for research investments are expected. Biosensors using enzymes, antibodies and even microorganisms are currently in use in clinical, immunological, genetic and other research and investigation fields. Also there is large scope for such probes (biosensors) in the field of environmental analysis and control.

Advantages of biosensors: (1) (2) (3) (4) (5) (6)

Rapid analysis. Specificity. Sensitivity. Accuracy. Large number of samples processed with less labour. Reproducibility.

The following biosensors are developed so far: (1) (2) (3) (4)

BOD sensor. Ammonia sensor. Nitrite sensor. Sulfite ion sensor.

1. BOD Sensor: The biochemical oxygen demand (BOD) is one of the most widely used and important tests in the measurement of organic pollution. A conventional BOD test requires a five-day incubation period and results still depend on the skill of the operator. A rapid test with biosensor uses yeast. Trichosporon cutaneum organism is sandwiched between an oxygen permeable Teflon membrane and a porous membrane. Then the membrane was directly fixed on the surface of platinum cathode of an oxygen probe. A continuous flow system using a new microbial sensor was developed for automatic estimation of 5-day BOD. When the sample solution containing glucose and glutemic acid was injected into the system, organic compounds permeated through the porous membrane and were assimilated by the immobilised microorganisms. Consumption of oxygen by immobilised microorganisms began and caused a decrease in the dissolved oxygen around the membranes. As a result the current of electrode decreased markedly with time until a steady state was reached within 18 min. The steady state current depended on BOD of the sample solution. Then the current of microbial electrode sensor finally returned to the initial level. A linear relationship was observed between the current decrease and 5-day BOD of standard solution below 60 mg I-I. The current was reproducible with + 6% of the relative error when BOD 40 mg I-I of the standard solution was employed. The standard deviation was BOD 1.2 mg Pin 10 experiments. Results of BOD sensor and that of JIS (Japanese Industrial Standard) method were very much comparable when estimated on untreated waste water of a fermentation factory. BOD of various other effluents were estimated with this sensor. BOD values depended on the compounds in waste waters. Stable responses to standard solution (BOD 20 mg p) were observed for more than 17 days (400 tests). 2. Ammonia Sensor: A nitrifying bacterium Nitros01fUJ1UlS spp utilises ammonia as a sole source of energy and oxygen is consumed by respiration. 2NH3 + 302 -+ 2HN02 + 3H 20

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Therefore, ammonia may be determined by a microbial sensor using immobilised Nitrosonwnas spp and oxygen probe. NitrifYing bacteria were immobilised on a porous acety1cellulose membrane. Ammonia in waste water can be determined. The porous membrane retaining the nitrifYing bacteria isolated from activated sludges was carefully attached to the teflon membrane of the oxygen probe so that microorganisms were trapped between two membranes and covered with nylon net and fastened with rubber rings. When a sample solution containing ammonia was injected into the system for 12 min., it permeated through the porous acety1cellulose membrane and was assimilated by the immobilised nitrifYing bacteria. A steady state current is obtained within 8 min. The time required for determination of ammonia was long by the steady state method. Therefore, the pulse method was employed for determination. The current reached is 80% of that of the steady state method. The assay can be done within 4 min. and electrode recovery time is 8 min. by the pulse method. The total time required for the assay of ammonia was 30 min. by the steady state method and 12 min. by the pulse method. A linear relationship was observed between the current difference and concentration of ammonia below 1.3 mg P. Minimum concentration for determination was 0.05 mg NH3 1-1. A good agreement was obtained between ammonia determination by the conventional method and that by the microbial sensor. The microbial sensor could be used for more than two weeks and for 1400 assays. 3. Nitrite Sensor: The principal gaseous oxides of nitrogen of interest in air pollution sampling and analysis are nitric oxide (NO), and nitrogen dioxide (N0 2). These gaseous oxides of nitrogen are the primary absorber of sunlight and produce the photochemical smog. Nitrobacter spp. utilise nitrite as the sole source of energy and oxygen is consumed by respiration. 2N02_

+

02

~

2N03_

Oxygen uptake by bacteria can be directly determined by the oxygen electrode attached to the immobilised bacteria. Therefore, N0 2 generated in the buffer (pH 2.0) can be determined by a microbial sensor, using immobilised Nitrobacter spp and oxygen electrode. A nitrogen dioxide electrode using a gas permeable membrane, immobilised Nitrobacter spp, and oxygen electrode is described. The porous membrane retaining the immobilised bacteria was cut into a circle and soaked in the buffer. This immobilised bacterial layer was carefully attached ~o the surface of a teflon membrane of the oxygen electrode, and covered with gas permeable teflon membrane and fastened with rubber rmgs. The oxygen contents in the sample solutions are checked using a differential sensor system (a sensor without an immobilised bacterial layer and with an immobilised bacterial layer) to prevent an influence of oxygen in the sample solution. The differences between the initial and the steady state currents were directly proportional to the concentration of sodium nitrite. A minimum concentration for the determination of sodium nitrite was 0.01 mm. Linear relationship was observed between the current decrease (difference between initial and steady state) for sodium nitrite concentration below 0.59 mM. (Current decrease 0.63 J,LA observed). The microbial sensor was stable for more than 21 days and 400 assays. 4. Sulfite Ion Sensor: The determination of sulfite and sulfur dioxide in waste waters and atmosphere is important in any environmental analysis. There is an ever-increasing demand for new, simple and inexpensive methods for the measurement and control of sulfite and sulfur dioxide pollution.

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Hepatic microsome is a subcellular organelle which contains many different oxidases and enzymatically oxidizes sulfite to sulfate with the consumption of molecular oxygen. Sulfite in sample when injected, S02 permeates and is oxidised by sulfite oxidase. Oxygen is consumed. Current decreases till steady state is reached. This steady state current depends on concentration of sulfite ions. REFERENCES 1. Venetia A. Saunders and Jon R. Saunders, "Environmental Biotechnology," in Microbial Genetics Applied to Biotechnowgy - Principles and Techniques ofGene Transfer andMutation, Croom Helm, London, pp. 384-411, (1987). 2. Steve Prentis, "Biotransformation - The Way Ahead for Industry," inBiotechno/qgy -A New IndustrialRevolution, New Revised edition, Orbis, London, pp. 170-187, (1985). 3. P .S.J. Cheetham and C. Bucke, "Immobilization ofMicrobial Cells and Their Use in Waste Water Treatment," in MicrobialMethodsfor Environmental Biotechnowgy, Society for Applied Bacteriology, Technical Series, (Edited), by J. M. Grainger and J. M. Lynch, Academic Press, pp. 219-231, (1984). 4. Isao Karube, "Biosensors in Fermentation and Environmental Control," in Biotechnowgy - Applications and Research, (Editors), Paul N. Cherimisinoff and Robert P. Quellette, Technomic Publi. Co. USA, pp. 135-155, (1985). 5. "Extensive Application of Biotechnology," in "Environment Protection," Chemical Weeko/, Sept. 10, 1991, Vol. XXXVI. 6. Atlas, Ronald and Bartha Richard, "Microbiology and Some Novel Pollution Problems," in Microbial Ecowgy : Fundamentals and Applications, Addison Wesley Pub. Co. (1981). 7. New Approaches to Sewage Treatment, pp. 277-282. 8. Patricia H. Clarke, F.R.S., Microbiology and Pollution, The Biodegradation of Natural and Synthetic Organic Compounds. Phil. Trans. R 80C Lon. B 290, pp. 355-367 (1980), in New Horizons in IndustrialMicrobiowgy (Editor) S. Brenner, B.S. Hartkey F.R.S.P., J. Rodgers, London Royal Society 1980. 9. Chemical Weeko/, Vol. xxx, XXXI, XXXVI, XXXVII ofl985, 1986, 1991, 1992. 10. P. F. Stanbury and Allan Whitaker, "EffiuentTreatment," in Principles of Fermentation Technowgy, Pergamon Press, Oxford, (1981). 11. Biotechnowgy - Principles and Applications, (Editors), J. J. Higgins, D. J. Best, J. Jones Blackwell Scientific Publications, London, (1985).

12. 13. 14. 15.

Chemical Weeko/, Chemical Weekly, Chemical Weekly, Chemical Weeko/,

July 16, 1996. Aug. 23, 1994. Sept. 10, 1996. Sept. 20, 1994.

BIOTECHNOLOGY IN INDIA 13.1 SCENARIO BEFORE THE YEAR ,3()QO -

ROLE OF DBT

Mter saying so much about 'Biotechnology', its {'8"entials, its relevance, its impact on development in different fields of mankind's interests, a further interest is inevitable in knowing our position in this area of science. If biotechnology is the third technological revolution (1st and 2nd being atomic energy and compute,r technology) of this century, we are equally concerned about it as anybody else. Although the needs and desires will not vary much throughout the world in an approach towards harnessing scientific development for human progress, the different stages of development, our existing problems and resources make us think differently bw: judiciously about the field of biotechnology. Problems of a developing country like India are different and so naturally, the priorities of research and exploitation of biotechnology are going to be different. Amongst the few problems are: (i) drought; (ii) rapid population growth; (iii) less healthy animal resources; (iJJ) food shortage; (v) import offertilizers; (vi) import of oil and petroleum products; (vii) poor hygiene; (viii) prevalence of many diseases and less health care etc. At the same time, we are endowed with some positive factors: (i) a strong agricultural base; (ii) ample solar energy; (iii) ample oil and minerals; (iJJ) perhaps the largest technological force in the world; (v) marine resources; (vi) Expertise in computer field etc. Thus, our priorities will be based on the analysis of our needs and resources. Biotechnologies which are available can be considered to be of two types: (i) high technology, huge investment-based processes; and (ii) low technology, less investment-based processes. Even if we can afford low technology less investment-based processes, by and large, there is no way without keeping pace with research in high technology areas. Thus, genetic engineering, use of DNA probes and monoclonal antibodies, synthesis of genes, advanced studies in molecular biology, modern protein chemistry developments, embryo transfer and artificial insemination technologies,

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membrane technology, new bioreactors, new plant breeding techniques, cell culture, tissue culture are all very much needed to be developed to their fullest capacities and should be up-to-date in our country. But, in applications, our efforts will be towards solving our unique problems. Thus crop productivity improvement and disease-resistant varieties, progress in biofertilizers, biopesticides will be the applications of our interest. In single cell protein (SCP), algal proteins and mushroom cultivation should get importance. In medicines, vaccines for diseases prevalent in our country, cheaper and safer vaccines and vaccination for all should be our goals. Fertility control vaccines for population control are needed here. The use of agriculture-based industries using biotechnology will be of our interest, so too chemicals from cell culture and tissue culture should get importance. Biomass can be available in ample quantity as sunlight is available throughout the year, so biomass for fuel alcohol and biogas production has its relevance in our development. Improvement of animal origin products will be to our benefit, since it can go hand-in-hand with our strong agricultural base. So breeding healthy animals to get increased output from them has to be paid attention to. There are many more examples to cite which make our ideas clear in applications of biotechnology in our country. Today, biotechnology can be said to be in an infant stage in our country. Although we have the largest technological force in the world, those who have adapted or learnt this sophisticated interdisciplinary science are comparatively few in number. Also there are very few private or public sector companies which have taken interest in the biological products and still negligible must be the efforts in the use of biotechnology. Government-funded research institutes and promotion of biotechnology activities by government administration has initiated some organised work. As we have seen in the chapter of worldwide status of biotechnology, big industries and universities have not started working in collaboration for its development as is the case in the United States. But initiatives from government will trigger that process too.

An Overview of Biotechnology Scenario in India: The field of Bitechnology has moved very fast allover the world in the last two decades. Biottechnological applications have great potential for developing countries for creating new jobs through value added products, and for generation of nonpolluting environmental friendly technologies. The Department of Biotechnology was set up in February 1986 as a separate department in the Ministry of Science and Technology. It was mainly meant to provide an administrative structure for planning, promotion and co-ordination of biotechnological programmes. The systematic impetus provided by the Department of Biotechnology (DBT) in R & D programmes has resulted in excellent progress in several frontline areas. Areas where biotechnology can find direct application are agriculture, health, environment and industry. DBT has initiated a system for contract reasearch through which a product or process will be developed in a time-bound manner for field testing, and subsequent large scale production. DBT spent Rs. 78 crores in 1993 out of which Rs. 75 crores were for plan Rs. 3 crores were for non-plan expenditure. Department of Biotechnology has been functioning on the advice of Scientific Advisory Committee (SAC-DBT), Standing Advisory Committee Overeseas (SAC-O), and through its task forces consisting of eminent scientists. Close linkages have been established with the concerned socio-economic ministries and efforts have been made to evolve joint mechanisms for identification and review of new R&D areas. A joint committee ofDBT-ICAR for example looks into common interests amongst the Ministry of Agriculture, ICAR and DBT. Scientific Advisory Committee ofDBT (SAC-DBT) - (1) advices on policy matters, (2) advices on new areas of thrust, (3) advices on implementation, monitoring, and (4) establishment of linkages with other sectors. 276 R&D projects were received in 1993 out of which 100 were approved for fmancial support. Interaction and collaboration is done with ICMR, lCAR, CSIR for respective fields.

252

Advances in Bwtechnology

Activities of DBT are: (1) Research and Development -

To identify specific Biotechnology related projects.

(2) Demonstration and Technology transfer - To generate technology and popularise in Biotechnological products and processes with manufacturers and users. (3) Infrastructure facilities -

Network at National level.

(4) Manpower development. (5) International co-operation. (6) Bioinformatics. (7) Safety and Regulatory arrangements. 1. Research and Development: Some of the significant results in R&D efforts in different areas include:

Complete protocols have been established for regeneration of cocoa and black pepper at the Kerala Agricultural University, Trissur. Protocol for plantlet regeneration of rubber has been established at the Rubber Research Institute, Kottayan. Field transfer and testing is being done. A process for preparing a non-toxinogenic oral vaccine for cholera has bee perfected at the Indian Institute of Chemical Biology (IlCB), Calcutta. Susceptibility of Plasmodium isolates from patients is being tested towards various anti-material drugs. The three dimensional strcture of a iron binding protein and Lactotransferrin has been determined at All India Institute of Medical Sciences (AIIMS), New Delhi. This protein has strong anti-bacterial properties and offers important applications in medical field and drug industry. PCR based system for diagnostic detection of Leishmania parasites in clinical samples including blood has been developed at the Indian Institute of Chemical Biology (IlCB), Calcutta. Wet workshops have evaluated the DNA probes and diagnostic tests developed at the National Institute of Immunology (NIl), New Delhi for detection of tuberculosis. Genome analysis, technology development and manpower training at the Indian Institute of Science (lISe), Bangalore has progressed well. The project has developed GENMAP software, analysed REPEAT motifs in yeast chromosome III, constructed indigenous STM for DNA imaging and also studied strcutural telomeric repeat sequences in genomes. Studies on characteristsation of molecular mutation in thalassemia chromosomes in North India have been carried out in the Post-Gradaute Institute of Medical Education and Research (PGIMER), Chandigarh. Technques for pre-natal diagnosis of thalassemia based on DNA analysis has been developed and successfully accomplished in 50 pregnancies in PGIMER, Chandigarh . . Diagnostic probes for haemoglobin E (HbE) by Amplification Refractory Mutation Systems (ARflS) technique and by restriction enzyme digest has been accomplished. 21 patients of Dunchenne Muscular Dystrophy (DMD) were examined for molecular characterisation and the advice offered to four families. In the Biochemical Engineering and Downstream processing area, good progress has been made for identification of species which degrade crude oil at National Environmental Engineering Research Institute (NEERI), Nagpur; the Tata Energy Research Institute (TERI), New Delhi; the National

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253

Institute of Oceanography, Goa and Goa University, Goa. The membrane absorptioh technology for the production and purification of immuno-biologicals has also been developed and standardised under the programme at National Chemical Laboratory (NCL), Pune. A model describing the bioleaching process which is presently being utilised by the Ministry of Mines has been developed through the R&D efforts of National Environmental Engineering Research Institute (NEERI), Nagpur. A project on xylanase from Bacillus and Streptomyces has been completed at the National Chemical Laboratory (NCL), Pune and a programme to improve the production ofxylanase from these organisms has been initiated using the site directed mutagenesis. Under the environmental biotechnology programme, ELISA and PCR techniques and DNA probes have been developed for detection of enteric pathogens in drinking water by the National Envrionmental Engineering Research Institute (NEERI), Nagpur. These are being field tested. A microbial strain has been developed for desulphurification of fossil fuels. Efforts are being made to install a pilot plant in collaboration with the user industry for desulphurisation of coal. Projects on carcass utilisation, mushroom cultivation, vermicompost, sericulture and poultry breeding have given benefits directly to SCjST population. The bovine Y chromosoe specific probes generated through PCR technique at the National Institute of Immunology (NIl), New Delhi have been tested for sex determination by other agencies like National Dairy Development Board (NDDB), Anand and were found to be 100% accurate. The ELISA based diagnQstic kits for new castle disease developed at the Tamil Nadu, Veternary and Animal Sciences University (lNVASU), Madras and Bharatiya Agro Industries Foundation (BAIF), Pune are under validation trials. The vaccines against viral diseases are also under trials. Two vaccines (beta hCG for female and oFSH for males) for fertility control have reached advanced stages of clinical trial. The project is undertaken by National Institute of Immunology (NIl) New Delhi and the Indian Institute of Science (IISc), Bangalore respectively. Also immunodiagnostic kits for hepatitis C and E are being developed at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad and E.coli diarhoea, rota viral diarhoea and cholera at AIIMS, New Delhi, IISc, Bangalore and CDRI, Lucknow under the Indo-US Vaccine Action Programme (VAP). Three national gene banks have been established for conservation of the valuable germplasm of medicinal and aromatic plants at the Central Institute of Medicinal and Aromatic Plants (CIMAP) Lucknow; The National Bureau of Plant Genetic Resources (NBPGR), New Delhi and Tropical Botanical Garden and Research Institute (TBGRI), Trivendrum, Biotechnological methods are used for conservation of germplasm from the regions of South Peniilsula, Himalayan regions and IndoGangatic plains. Immunodiagnostic techniques have been developed for detection of pebrine and nuclear polyhedrosis diseases of mulberry silkworm. Field trials and demonstrations of these tests are being conducted in silkworm seed production centre (grainages) and at the farmers' rearing houses in Karnataka, Andhra Pradesh and Tamil Nadu to establish their reproducibility and stability. Protocols for regeneration of Alnus nepalensis, Wrightia arhorea, Sapindus trifoliatus, Boswellia serrata, Bambusa vulgaris are being developed at Y.S. Parmar University, Solan and at the Regional Plant Resource Centre, Bhubaneshwar. Over 400 publications since 1987, 26 Ph.D. thesis and 6 patents are only indicative figures to illustrate the outcome of infrastructure of DBT.

254

Advances in Biotechnology

2. Demonstration and Technology Transfer: R&D projects are further used to generate technologies and ensure their validation and field trials through demonstration. The significant success is in the following activities - (a) Oilpalm plantation in the states of Andhra Pradesh, Karnataka and Maharashtra. (b) Tissue culture cardamom being planted in fields in Kerala, Karnatka, Tamil Nadu and giving 260 kg per ha. instead of 201 kg. per ha. for open pollinated seedings, thus showing .30% increase. (c) Large scale multiplication of forest tree species at Tissue Culture Pilot Plant facilities at NCL, Pune and TERI, New Delhi. Anogeissus pendula, Eucalyptus species, Populus deltoides, Tectoma grandis are the three species multiplied by tissue culture. Four lac plants have been sent for demonstration. (d) Biological control programme is being demonstrated over 11000 ha. field covering cotton, chickpea, sugarcane, tobacco, oil seeds and vegetable crops. Two pilot plant units are set at Madurai and Coimbatore to serve as model units for private entrepreneurs. At Central Tobacco Research Institute (CTRI) Rajmundry demonstration in tobacco nurseries was done on Farmers' day. (e) In the field of biofertilisers, technology packages are developed covering bioreactor design for production of blue green algae, media components and their concentrations and simple biossay method. 1500 demonstrations and 47 training programmes were conducted to popularise the use of biofertilizers. Immunoblot technique for identification of Rhizobium species, isolation of salt tolerant Rhizobium species are the other areas of work and promotion of technology. (f) Biotechnology department is planning to launch programmes in semi-intensive farming of marine/brakish and fresh water prawns. with adequate R&D support in feed development, disease and diagnostics, hatchery technology and environmental monitoring etc. (g) The major areas supported under the industrial biotechnology programmes are for products in human and animal health, agriculture, aquaculture, forestry and industrial products. Projects include development of diagnostic kits, r-DNA products, antibiotics, liposome intercalated drug delivery system, production of industrially important enzymes and polysaccharides, development of fish spawning agents and biotechnological enrichment of magl;lesium ore etc. Table 13.1 Examples of Technology Transfer

No.

Technology

Developed by

Transfe1Ted to

1.

F-Moc derivative of amino acid

Centre for biochemistry at New Delhi.

Atul Products, Gujarat.

2.

Detection of Hepatitis - B

National Instute of Immunology, New Delhi.

Lupin, Bhopal.

3.

Leprosy Immunomodulator

National Institute of Immunology, New Delhi.

Cadilla Lab, Ahmedabad.

4.

Lieishmaniasis detection kit

CDRL, Lucknow.

Span Diagnostics, Surat.

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Table 13.2 Infrastructure Facilities

Facility

No.

Institute/place

Remark

1.

National Facility for Marine Cynobacteria

at Tiruchirappalli

2.

National Plant Tissue Culture Repository

at National Bureau of Plant Genetic Resources New Delhi

extends service and material for research Molecular techniques used for germplasm characterisation and classification

3.

Animal House Facilities

at Central Drug Research Institute, Lucknow and at National Institute of Nutrition, Hyderabad

supplies animals to scientists and research institutes.

4.

Centre of Bitechnology

at New Delhi

Import and distribution of enzymes and biochemicals (within 6-8 weeks)

5.

National Facility for Animal Tissue and Cell Culture (NFATCC)

at Pune

maintenance/storage and supply of animal and human cells, obligate parasites, plasmids, vectors and genomic libraries. Total cell culture repository of 1127 cell lines

6.

Protein peptide synthesis facility and Gene structure and sequencing and synthesis of oligonucleotides

Indian Institute of Science Bangalore

7.

National facility on biochemical engineering research and process development

At IMTECH, Chandigarh

8.

National facility oliognucleotide synthesis

9.

CSIR Centre for Biochemicals

10.

Centre for reproductive biology and moTecular endocrinology \

for

at Indian Institute of Science, Bangalore and at Centre for Biochemistry, New Delhi and CCMB, Hyderabad has been supported for producing large number of oligonucleotides, linkers, restriction enzymes, plasmids and molecular size markers k at lISe, Bangalore

For basic fundamental research

256

Advances in Biotechnowgy Table 13.3 Current Consumption and Anticipated Future Demand of Biotech Products Consumption Rs. (in crom) Product Category 1. 2. 3. 4. 5. 6. 7. 8.

Human and animal health products Agricultural biotech products Industrial products Forest biotechnology Effluent treatment Composting, vermiculture Microbial leaching, biobeneficiation Others (oligonucleotides, plastics etc.) Total

1991

1995

2000 AD

333 044 138

515 187 240 010 010 005

002

005

1030 0580 0475 0050 0050 0010 0010 0010

517

972

2215

4. Manpower Development: This basically covers teaching and popularisation of biotechnology. According to 1994 DBT report, around 300 students are admitted in a year to post-graduate and post-doctoral programmes of Department in 2S selected institutions in the country, 200 mid-career scientists received training in various techniques and 40 scientists were selected for the award of Bitechnology Associateships (National and Oveseas). Training for technicians and industrial R&D personnel were conducted. Scholarships are awarded to selected top students of biology in All India CBSE examination (10 + 2 level). Industrial training is given to post-graduate students. A new course in down stream processing has been started at University Department of Chemical Technology (UDCT) Mumbai (Now University Institute of Chemical Technology (UICT) Mumbai). Different programmes are intermitantly reveiwed for improvement. The department is also trying to set up placement cells in various Biotechnology departments. 5. International Co-operation: The international bilateral co-operation especially with the countries like USA, FRG, Switzerland, Sweden, U.K., has been beneficial from the view point of training of scientists, acquisition of equipment and exchange of visits and information through joint workshops and meetings. The new programmes are identified with China, Poland, Cuba and Mongolia. The thrust is in high-tech areas of biotech products. The projects are in the areas of reproductive biology, immunology, bioreactor designs, biosensors, production of industrial enzymes, development of products and processes, vaccines and immuno-diagnostics for human health and animal use and enhanced agricultural productivity through genetic engineering. Multilateral co-operation is being developed through SAARC and with G-1S countries for biotechnology development. There are three projects going on in collaboration with Swiss. With SAARC countries collaboration is inthe form of workshop in diagnostics and advanced training in genetic engineering. With G-1S countries, collaboration is in setting of gene-banks for medicinal and aromatic plants. With US, the work is on the field of contraceptive development. 6. Bioinformatics: This is a unique information system established by the department which spans a subject-oriented scientific grid covering many scientific institutions and providing a singk reference for the biotechnology information in the country. The E-MAIL, facility has been introduced through ERNET which is facilitating access to INTERNET for worldwide biotechnology information service. The BTIS (Biotechnology Information System) centres have developed several value added databases and softwares for PC based sequence analysis. National Biotechnological Information Network is to share information, knowledge, knowhow, problem solving, technology and data capture. Network provides (1) Genetic material as hard data,

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(2) References as soft information, and (3) Management information. The creation or upgradation of instrumentation facilities in some laboratories in the country is done. These facilities are particularly useful for structural biology work and for projects and activities related to drug designing and drug targeting leading to development of better products. 7. Safety and Regulatory Agreements: The Biotechnology department has been closely interacting with Ministry of Environment and Forests through the Genetic Engineering Advisory Committee. Several discussions have been held with eminent scientists to develop procedures for speedy clearances of recombinant DNA products and also on ethical conSIderations especially in the field of Medical Biotechnology. Guidelines will be formulated for this. The Institutional Biosafety Committees are being further strengthened.

13.2 AN UPDATE ON BIOTECHNOLOGY IN INDIA The value of biotech products including conventional fermentation based microbial products produced in the country has been estimated at Rs. 890 crores at present. The health care area dominates the market with share of above 60%. Agricultural products including animal husbandry and agriculture account for around 15% and the balance 25% is held by industrial chemicals, alcohol and starch based products. Some of the important firms who have set up research and production units for different biotech products are Hindustan Lever, Rallies India, the Thappars, SPIC, lTC, A.V. Thomas & Co., Harrison Malaylam and Indo-American Hybrid Seeds. It is estimated that India's biotech industry items produced in the country would reach Rs. 3,500 crores by 2000 AD. In recent past there have been many biotech projects in India. Some of them are (a) Rathi Papains have been manufacturing and marketing papain for some years and have some

exports also. (b) Biocon Chemicals also has entered in the field of enzymes as a joint venture with a foreign

associate already in line. Enzymes for brewing - both for mash and and post-fermentation (to remove haze) are prime products. Firm has good R&D. Also glucose isomerase for High Fructose Corn Syrup (HFCS) is being developed. (c) Production of penicilins in joint venture of Max India with the world's largest producer

Novo Nordisk of Denmark was recently inaugurated in Haryana. This could be the base for 6 APA for semisynthetic penicillins. Enzyme developed by NCL is used by another party also. . (d) Cystine production project based on the use of human hair plenty available in India (and actually exported today) is coming up. (e) Another export-oriented programme for whole range of amino acids based on protein

hydrolysates through sophisticated methods of separation is promoted by J.F. Laboratories, France. Wide range of slaughter house waste, and oil cake proteins will be the base. (j) Aspartic acid, Aspartame production projects using the ample agro raw materials are also in the offing.

(g) A big yeast and yeast extract project is also coming up. (h) Wochardt is going to launch vaccine for hepatitis using genetic engineering technology of International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi. (i) Coimbatore based Stanes group is one of the leading producers of biofertilizers in South India. Fertilizers and Chemicals Travancore Ltd. (FACT), a government of India undertaking

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Advances in Biotechnology

will market Stanes group's biofertilizers based on Awspirilum, Awtobacter, Rhiwbium and phospate solubilizers. Among the achievements to be claimed from biotechnology progress in India are: A genetically-improved strain capable of tolerating initial high concentrations of sugar and final high concentrations of alcohol in-fermentation broth during alcohol fermentation has been developed by IMTECH, Chandigarh. This organism (yeast) can grow and utilize initial sugars at 26.5% concentration instead of 15% which is normally employed in alcohol fermentation. The whole downstream processing will change. Plant size can be smaller and much more alcohol can be produced from existing facilities with the use of the new strain. Process optimisation for production of biopesticides which are attractive alternatives to polluting chemical pesticides have been carried out at the Centre of Biotechnology at Anna University. Complete indigenous technique for strain improvement, process optimisation, automation is being developed. Several developments in biotechnological applications have taken place in the leather industry. To name just two, CLRI (Central Leather Research Institute) has developed a biotechnological enzymatic method for removing hair from animal skin. The indigenously developed product 'Clarizyme' has been found extremely good on sheep and goat. This method is cheaper and more effective compared to the lime and sulfide process used today. The quality of leather is also better. In other areas, microbial degradation system has been developed for cleaning of leather tannery effluents. Indian researchers at Jadhavpur University, West Bengal, have developed economical biotech process for producing lactic acid from potatoes. It is a two-step process: (1) enzymatic saccharification of potatoes using a-amylase and amyloglucosidase of Aspergillus oryzae. 75% conversion of starch to glucose occurs. (2) bacterial fermentation with Lactobacillus delbruckii. 69% yield on glucose supplied or 37.5% based on weight of potato starch is obtained. Cost reduction obtained is 21/2 times. Vaccines for fertility control, leprosy, malaria, effective antibiotic (against tuberculosis like Rifamycin S, tissue culture successes in eucalyptus, oil palm, teak, over 10 lakhs biogas plants to harness cheap energy sources - there is a lot to mention about and there is a lot in the offing.

13.3 SCENARIO AFTER THE YEAR 2000 -

ISSUES AND CHALLENGES

When we say India has bright future in biotechnology, it will be interesting to know who says it and in what perspective one says it. Such statements are now perceived as media hype. India has potential to make it big in biotechnology can be more realistic statement. Whether India can have bright future or not will depend on how India tackles the coming 2-3 crucial years of biotech on all fronts. We make an interesting list of opportunities (contract research, contract manufacturing, bioinformatics, clinical trials, alliances, setting up of new industry by 'American Indians') for India, to work in biotech sector but all that will be a partial analysis and if I may say so a 'shortsighted' approach. These opportunities are not decided by India or rather felt by India but are told to Indian players by (mostly) others. We are trying to consider these opportunities as the only priorities, where, we may make mistake. We have to grab these immediate opportunities (otherwise even that may be lost to competitors like ChinaJ Taiwan) but keep an eye on long-term objectives of becoming a global leader and not a universal server. Global partnership on various activities may be the beginning. Even biotech industry players have to define and first refine their ideas while working in this field. Biotech policy of nation and states should work intensely on the 'leader' factor rather than 'server' factor. In short the challenge is to work on two fronts - to work and grab widely discussed immediate opportunities and as a long-term goal, to establish as true 'innovator-expert' in this knowledge based sector.

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Growth of biotech sector based on indigenously developed products and not working on mere service model, working for our own problems through our own strengths is my dream, for development of biotechnology in India. I say it a dream because there are many challenges to work upon: (1) Getting 'public support for biotechnology policy (2) Creating conducive atmosphere of laws and regulations (3) Developing confidence of investors in biotech sector (4) Promoting commercialization of research from government-funded institutes (5) Pulling established industrial houses for business in biotech sector (6) Stupendous R&D efforts with long-term objectives (7) Moving up the value chain (from marketing-distribution activity to research for own products) (8) Supreme priority to quality consciousness (9) Eliminating state of confusion on ethical issues (10) Avoiding delays on procedures and permissions (ll) Firm stand on IPR issue, GE crops issue, which suits the nation's interests (Nothing out of compulsion!)

(12) Deciding the boundaries for entry of foreign biotech partners (13) Reducing dependence on import of technology (14) Satisfying the international expectations for promotion of right cooperation (15) Competition from China, Taiwan -like countries under the globalization atmosphere.

13.3.1 Value Chain Normally the value chain starts at research and ends with product sales. In biotech, research captures only small part of chain. It is true that as you try to go up the value chain for business the investment demands are high but so will be the returns and control on market. Also, efforts for revolution of technology throughout the value chain are important. In value chain management, integration of both research (stages of target identification to Lead optimization) and development (includes manufacturing, clinical trials and sales and marketing) is necessary and inevitable. In the world that is moving over knowledge economy, biotechnology domain relies upon new knowledge creation. Indian biotech industry should avoid sub-critical investments in low opportunity areas and instead must initiate intensive efforts for research. Indian biotech industry must pay attention to development of own new products to add in its portfolio. Indian biotech firms should focus both on segments that are high value and high growth oriented. Take an example of Perkin-Elmer. Perkin-Elmer built the machines that enabled the boom in genomics research-automated DNA sequencers. Genomics companies have done well, but PerkinElmer hasn't. The company is prepared to spend up to $ 1 billion over the next several years on external collaborations and acquisitions in an effort to climb the value chain and capture high-value applications of its tools for itself. Same way bioinformatics services may give some share of business to new start-ups in India while processing data for US and other developed countries, but that will be 'working at the lowest ~end of value chain' as far as field of Bioinformatics and Genomics is concerned. Indian capabilities have to move from structural genomics to functional genomics for climbing up the value chain.

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In the initial stage Indian biotech industry may not dare to go for high investments required for working at higher strata of value chain. So it is obvious that it will work only in value-added services such as bio-facilities, contract research, knowledge management and other infrastructure & support services to shorten timelines and establish their existence.

13.3.2 IPR Policy Healthy IPR framework is the outcome of the knowledge revolution. Sound IPR policies will playa key role in development of biotech sector. India is progressing toward full implementation of the WTO's TRIPS agreement, which lays down the framework for higher protection in all forms of IP on a global scale. Ability to maintain international standards and to overcome the IPR and regulatory issues remain an obstacle for many UK companies who might be looking to do business in the biotech sector in India. This was the opinion of UK biotech delegation on their recent visit to India. Japan External trade Organisation (JETRO) director general Hidehiro Ishiura said in his visit in Nov. 2001, "Global companies with serious interest on biotechnology and bioinformatics would find India an attractive destination." Japan still remained hesitant about investment and collaboration in India due to issues related to intellectual property rights (IPR). These concerns expressed by UK and Japan, are agreed by many experts, who feel that putting a tight patent regime in place shouJd be priority. That includes adhering to the GATT agreement India has signed, and its right enforcement. Under the Trade Related Intellectual Property Rights (TRIPs) agreement, India must implement patent protection on pharmaceuticals and biotech products by 2005. After 2005, we will not be able to produce any recombinant proteins that are patented. The nation's major biopharmaceutical companies are accelerating efforts to get bioequivalent versions of patented well-characterized recombinant proteins onto the market before 2005. Startups created in private sector to copy patented biologicals will be required to move into research, and low-cost generic producers will have to start discovery based activities.

13.3.3 Opportunities in Contract Research Internationally, the cost of R&D is constantly increasing and the pace of new discoveries is slowing down. The global spend on outsourced R&D is around $ 7 billion and this is expected to increase at 30 per cent per annum for the next five years. The trend to outsource low-cost research and development in biotechnology is increasing. This provides an immense opportunity for Indian companies to do contract research for overseas corporations. Indian companies with excellent technical manpower are, thus, well suited to take up contract research on the same lines as the IT subcontracting work. R&D productivity is going down. Fewer than 4% of the products generate sales of $ 500 million or more. Patent expiration of many of top drugs is a growing concern. R&D spends of the top 20 pharma companies touched $ 40 billion in 2000. Obviously, these companies would have to explore ways to cut R&D spending by outsourcing through small and medium-sized biotech companies. India can become a major center for contract research if we have a strong patent regime that would guarantee confidentiality of research. Some U.S. and European collaborations are tapping into Indian expertise. For example, the CCMB, Hyderabad recently won a contract from Onconova Therapeutics Inc. (Princeton, New Jersy) tQ crute transgenic fruit fly high throughput assay systems and use them to screen drug targets for

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anticancer effects. CCMB is providing world-class science at a cost 50-fold lower than Onconova would have to pay in the US Companies like Jubilant Biosys Ltd. (performing contract bioinformatics services, at client sites and in Bangalore, creating and curating databases, building customized tools, and performing contract research), Dr. Reddy's Laboratory (has established a research subsidiary in Atlanta 'Reddy US Therapeutics Inc.', as well as a contract research subsidiary that will focus on genomics) are other examples of companies exploiting contract research opportunities.

13.3.4 Missing Link between Research and Product Commercialization In my personal interactions with people in biotech sector, I have observed that there are many offers from funding agencies but there is scarcity of products and technologies to be promoted. India has invested a large amount of taxpayer's monies in building Indian Council of Agricultural Research (ICAR) and Council of Scientific and Industrial Research (CSIR) Institutes and Universities. India has 30 Agricultural Universities, 204 Universities and over 500 National Laboratories and Research Institutes of CSIR, ICAR and ICMR. Many of them have well-developed facilities for research in biotechnology. The Department of Biotechnology (DBT) and ICAR are requesting $ 560 million from the federal government for the tenth five-year plan. The DBT is seeking $ 90 million (Rs. 4050 million) for 2002-2003. Government-funded institutions that have absorbed nearly Rs. 19,000 millions during the last five years have largely dominated biotechnology R&D in India. The total expenditure in 1999-2000 has been to the tune of Rs. 4,590 million. On 28th Feb. 2001, the Indian Government announced a biotech R&D budget increase of Rs. 360 million ($ 8 millions) to Rs. 1.86 billion for public research institutes and universities for the coming financial year. What are the returns on investments and yearly expenses done for the government-run research programmes? How many products are developed to the level of commercialization? DBT's examples of 40 technology transfer from 1986 till date is not an impressive contribution. Some of the Government funded R&D institutions, which have necessary infrastructure, should take biotechnology products development as a priority goal in partnership with private sector. The Choice of the individual product to be developed is left to private sector. R&D institute can playa crucial role in development of right technology. Most of these sophisticated research facilities are not available to private sector due to complicated or restrictive regulations for private sector. Lack of coordination between government funding agencies results in support to similar projects in different biotech research institutes. The Central Government is planning to bring together all agencies involved in biomedical research funding, (DBT, CSIR, WHO, Department of Science and Technology, Indian Council of Medical Research) under a common platform to overcome this situation. This will avoid wastage of resources and finances. R&D efforts (two third or more of Government spending) were not successful in product development. The funding of individual project is less than $ 200,000 annually is sub-critical for biotechnology product development and has resulted in very poor development of technologies to produce competitive products. CSIR and ICAR institutions, and Universities should be persuaded to provide incubator facilities to 50 industries. If one industry focuses on one product then 50 products will be developed.

13.3.5 Status 2003 Asian countries as a w~ole do not have any significant position in overall global biotech activities. There is fast realization of need, and efforts in accordance to adopt modern biotechnology in this area.

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Unfortunately no country is having strengths to compete US or Europe. But individual Asian countries have niche areas where they have strengths. India, China, Japan Taiwan, Hong Kong, Singapore etc have a capacity to play role in service model of industries. In India, Government of India will focus on biotechnology and the Tenth 'Five Year Plan' acknowledges 'Biotechnology' as a driver in India's mission to achieve annual GDP growth of eight percent. Four critical issues, which will be dealt to ensure growth of biotech industry will be: resolving IPR issue, fostering partnerships with private sector, government's proactive role on funding research projects and setting fiscal regime that enables combining of benefits of research and monetary capital. The new initiatives by Indian Government for development of biotechnology can be hoped to bring expected growth of this sector. Many experts desire national policy on biotechnology. One observation is that 'healthcare industry' is discussed more, but it is unintentional. The healthcare sector becomes center of attention and discussion due to its immense power to financially support biotech research and growth. Agricultural sector does have reasonably good number of companies but tissue culture work done by them is mostly targeted to obtain stock plants for horticulture, floriculture. Contribution to genetically engineered crops and seeds from Indian biotech companies is insignificant today though potential is plenty. One reason for this may be uncertain policy and procedural delays in implementation from the government on issue of introduction of genetically engineered crops. Biotech market projections though differ are interesting. It is expected to be $ 6.7 billion to $ 15 billion by 2010 according to different estimates. Most of the start-up companies are trying to explore opportunities in the areas of short-term goals. The number of companies working in clinical trials, contract research, Genomics, Proteomics, and Bioinformatics is close to 100. Most of these companies have started after the year 2000. The number of companies doing basic innovative research and trying to develop own products is negligible. Analysis of profiles of industries shows that 67% of 400 companies in this report do not have any R&D activity, 16% have some research done while around 10% are strongly active in research. Unless Indian pharmas develop the culture of R&D and invest with conviction, it is difficUlt that they make any impact on the global scene. Vaccines production is perhaps the only field where we are almost self-sufficient from requirements point Qfview. Technically also our expertise in production of vaccines is on the move to achieve latest. Diagnostics field is the one where we see perhaps maximum number of companies. But, here too there are more importers and traders and diagnostics based on latest technologies of monoclonal antibodies or DNA probes are hardly developed indigenously. Biotech activity in food industry is almost negligible in the country and only handful of companies is active in this field. Also environmental biotechnology has not become a thrust area though expeJ;t:i~e in this field should have claim in India. Pollutant degrading organisms, reactors, consultancy, specific solutions to pollution problems could be the products and services in demand for anybody interestecf to work in this area. Frost and Sullivan study on Asian Biotech industry points out that in Asia only Japan and Singapore have wide spectrum of biotech activities in 5-7 areas. India has narrow spectrum with activities in two areas only. Many Indian bio-based companies are working in different sectors and on different products simultaneously and hardly you find specialized p~ayers. Those active in field of vaccines are working