Tuesday, November 30, 2010

Industrial Microbiology

Industrial Microbiology
Industrial Microbiology is a relatively new science, as is all of microbiology.
Industrial Microbiology deals with all forms of microbiology which have an economic aspect.
It deals with those areas of microbiology on which a monetary value can be placed which involves
a fermentation product or some forms of deterioration,
waste disposal etc.
Industrial microbiology is a very broad area for study.
Use of microbes to obtain a product or service of economic value constitutes industrial microbiology.
Any process mediated by or involving microorganisms in which a product of economic value is obtained is called fermentation.
The terms industrial microbiology and fermentation are virtually synonymous in their scope, objectives and activities.
The microbial product may be microbial cells (living or dead), microbial biomass, and components of microbial cells, intracellular or extracellular enzymes or chemicals produced by the microbes utilizing the medium constituents or the provided substrate.
The activities in industrial microbiology begin with the isolation of microorganisms from nature, their screening for product formation, improvement of product yields, maintenance of cultures, mass culture using bioreactors, and usually end with the recovery of products and their purification.

Industrial Microbiology includes the following areas

Soil and agricultural microbiology
Medical microbiology
Microbial physiology
Cytology and morphology
Marine microbiology
Food and dairy microbiology and

Disciplines not related to microbiology are also important in Industrial microbiology, such as-
Organic, inorganic and physical chemistry
Sales and law, particularly patent law.
Studying microbes helps us to understand the world around us:
Microbes are useful tools in research because of their rapid life cycle, their simple growth requirements, and their small size.
Due to this simplicity, microbes have been essential in understanding core questions in biology.
Attempts to classify microorganisms have lead to a classification system that divides all organisms into three domains of life, Archaea, Bacteria, and Eukarya.
Microbes provide tools for use in molecular biology. These tools have allowed scientists to make rapid progress in investigating many types of microorganisms.

Use of microorganisms in Industry:
Industrialist may wish to diversify their overall product line, or they may wish to employ microorganisms to bring about some change in a raw material, by-product, or product normally associated with company’s production activities.
Microorganisms may bring about deterioration or, in some other manner, modify a product in an unwanted manner so that an industrial concern is forced to consider the industrial aspects of microbial activity.
There are many facets to industrial microbiology.
Examples of particular areas of application include the sterilization, deterioration and quality control associated with the production and handling of food and beverage products.
Microorganisms are industrially employed as a means for prospecting for new oil reserves, and for obtaining better oil recovery from present reserves.
Roles of industrial microbiologists:
The industrial microbiologist is interested in ways of combating disease agents of plants, animals, and man.
He/she is concerned with the microbe’s ability to modify a soil environment for the growing of green plants, particularly with the relationships of microorganisms to soil fertility and with the ability of soil microbe to degrade man-made pesticides and other chemicals.
Industrial microbiology concerns itself
with the isolation and description of microorganisms from natural environments and
With the cultural conditions required for obtaining rapid and massive growth of these organisms in the lab and in large scale cultural vessels commonly known as fermentors.
The ability of microorganisms to convert inexpensive raw materials or substrates, to economically valuable organic compounds is of considerable concern to the industrial microbiologist.
Patents and Industrial Microbiology:
Patents are of importance to industrial microbiology in that they provide a certain degree of economic protection to an inventor for a new fermentation process or product.
Thus patents provide the impetus for the expenditure of the huge sums of money often required for the research and development associated with new fermentation processes.
Microbial Products of Potential Importance:
Products Example
1. Amino acids L-glutarnic acid, L-lysine
2. Antibiotics Streptomycin, penicillin, tetracyc1ines, polymyxin
3. Beverages Wine, beer, distilled beverages
4. Biodegradable plastic β-polyhydroxybutyrate
5. Enzymes Amylase, proteases, pectinases, invertase, cellulase
6. Flavouring agents Monosodium glutamate, nucleotides
7. Foods Cheese, pickles, yoghurt, bread, vinegar
8. Gases CO2, H2,CH4
9. Organic acids Lactic, citric, acetic, butyric, fumaric

In studying industrial microbiology, the student should concentrate on the economic aspects of how man makes use of or combats the activities of microorganisms.
But the student must not lose sight of the basic concepts of microbiology which have no immediately apparent money-making possibilities.

The term fermentation is derived from the Latin verb ferver, to boil, thus describing the appearance of the action of yeast on extracts of fruit or malted grain.
Fermentation is a process of energy production in a cell in an anaerobic environment (with no oxygen present).
In common usage, fermentation is a type of anaerobic respiration, however a more strict definition exists which defines fermentation as respiration in an anaerobic environment with no external electron acceptor.

Fermentation and industrial microbiology:
The production of alcohol by the action of yeast on malt or fruit extracts has been carried out on a large scale for very many years and was the first industrial process for the production of a microbial metabolite.
Thus, industrial microbiologists have extended the term fermentation to describe any process for the production of product by the mass culture of a microorganism.
What Causes Fermentation:
Spoiled wine threatening livelihood of vintners, so they funded research into how to promote production of alcohol, but prevent spoilage by acid during fermentation
Some believed air caused fermentation reactions, while others insisted living organisms caused fermentation
This debate also linked to debate over spontaneous generation.
Pasteur’s Experiments on fermentation
What causes fermentation?

Pasteur’s Experiments on fermentation (contd..)

Pasteur’s Experiments on fermentation (contd..)
Showed that anaerobic bacteria fermented grape juice into acids, which suggested a method for preventing spoilage in wine. To prevent this he developed a technique what came to be known as pasteurization (use of heat to kill contaminating bacteria to reduce spoilage of food and beverages) à industrial microbiology = biotechnology.
Pasteur, due to significant accomplishments working with microbes is considered the Father of Modern Microbiology.

Buchner’s Experiments on Acellular Fermentation
Showed, in 1897, that fermentation does not require the actual presence of living cells, but only cell-produced proteins called enzymes.
Buchner’s work begin the field of biochemistry and the study of metabolism (all chemical reactions within an organism).
Buchner’s (1860 –1917) Experiments on Acellular Fermentation

Industrial microbiology and development of pharmaceutical industry

Industrial microbiology refers primarily to bulk production of organic compounds such as antibiotics, hormones, vitamins, acids, solvents, and enzymes.
Industrial process usually occur on a much larger scale, produce a specific compound, and involve numerous complex stages.
The aim of industrial microbiology is to produce chemicals that can be purified and packaged for sale or for use in other commercial processes. Thousands of tons of organic chemicals worth several billion dollars are produced by this industry every year.
To create just one of these products, an industry must determine which microbes, starting compounds, and growth conditions work best, which requires an investment of 10 to 15 years and billions of dollars for research and development.
The microbes used by fermentation industries have traditionally been naturally occurring (wild) strains of bacteria or molds that carry out a particular action on a substrate.
Industrial microbiologists have several tricks to increase the amount of the chosen end product. First, they can manipulate the growth environment to increase the synthesis of a metabolite.
Another strategy is to select microbial strains that genetically lack a feedback system to regulate the formation of end products, thus encouraging mass accumulation of this product.

From microbial factories to industrial factories
Industrial fermentations begin with microbial cells acting as living factories. When exposed to optimum conditions, they multiply in the trillions and synthesize large volumes of a desired product.
Such mass microbial fermentations are the driving force of industrial microbiology. To produce appropriate levels of growth and fermentation, the microbes must be cultivated in a carefully controlled environment. This process is basically similar to culturing bacteria in a test tube of nutrient broth.
Many commercial fermentation processes have been worked out on a small scale in a lab and then scaled up to a large commercial venture.
An essential component for scale up is a fermentor, a device in which mass cultures are grown, reactions take place, and product develops.
Some fermentors are large tubes, flasks, or vats, but most industrial types are metal cylinders with built-in mechanisms for stirring, cooling, monitoring, and harvesting product.
Fermentors are made of materials that can made of materials that can withstand pressure, and are rustproof, nontoxic, and leak-proof.
For optimum yield, a fermentor must duplicate the actions occurring in a tiny volume (a test tube) on a massive scale.
Most microbes performing fermentations have as aerobic metabolism, and the large volumes make it difficult to provide adequate oxygen.
Fermentors have a built-in device called a sparger that aerates the medium to promote aerobic growth.
To increase the contact between the microbe and the nutrients, paddles located in the central part of the fermentor vigorously stir the fermentation mixture and maintain its uniformity.
The temperature of the chamber is maintained by cooling jacket.

Industrial microbiology and substance production
• The general steps in mass production of organic substances in a fermentor can be summarized as:
1. Introduction of microbes and sterile media into the reaction chamber
2. Fermentation
3. Downstream processing (recovery, purification, and packaging)
4. Removal of waste
Some products come from this process ready to package, whereas others require further purification, extraction, concentration, or drying.
The end product is usually in a power, cake, granular, or liquid form that is placed in sterilized containers.
The waste products can be drained off and can be used in other processes or discarded, and the residential microbes and nutrients from the fermentation chamber can be recycled back into the system or removed for the next run.
Microbial biotechnology
Biotechnology is better defined as process biotechnology. This is simply described as a discipline, which enables its exponents to convert raw materials to final products when either the raw material and/or a stage in the production process involve biological entities.
It involves the conjoint interaction of two identifiable sub-components; bioscience and biotechnology.
It is defined as “The applications of scientific and engineering principles to the processing of material by biological agents to provide goods and services

Microbial enzyme technology

Various microorganisms like bacteria, fungi and protozoa produce different kinds of enzymes. They produce extracellular enzyme to break those organic and inorganic compounds, which have high molecular weight. Simpler substances can easily be assimilated through cell membranes of microorganisms.
Bacillus mesentericus, B. polymyxa, B. macerans produce bacterial amylase and protease. Proteases are used to design cotton and silk.
Commercial production of amylase is carried out by yeast. Yeast also produce invertase. It is used for sweeting in confectionary.
Microbial genetic engineering
Genetic engineering is the most fundamental mechanics of biotechnologies. It involves exchange of genes, as well as the introduction into a cell of a gene belonging to another to bacteria, in particular the through in vitro genetic recombination.
Such technique has been applied to bacteria where genes of animal and human cells have been introduced and propagated.
The genetic recombination consists of an exchange of genes between two chromosomes.
Genetic recombination is the process capable of giving birth to cells or individuals in which two or more hereditary determinants, by which their former parents differed, are associated in new way.

Microbial Products and Processes
Why we use microbes in the industry?
 Microorganisms are used to produce useful products for the benefit of mankind.
 They are used to increase productivity.

Prerequisites for Using microbes to manufacture products

 Microbial products are found from various metabolic reactions of microorganisms.
 The overall reaction characterizing the industrial application of microbes can be summarized as follows:
Substrate (raw material) + Microorganisms (chemical factory)
New products

The organism:
It must be able to produce appreciable amounts of the products.
It should have relatively stable characteristics and the ability to grow rapidly and vigorously.
It should be nonpathogenic
The medium:
It must be cheap and readily available in large quantities.
Nutrient-containing waste have been found to be utilized practically as substrate. E.g: whey from dairy industry, waste liquors from paper industry.

The product:
An efficient and economic product should be produced.
Large-scale method and efficient recovery system of products should be developed.
Purification of desired end-product must be accomplished, because product is a heterogeneous mixture containing microbial cells, constituents of medium etc.

 Fermentation may mean---
 Fermentation (biochemistry), the process of energy production in a cell under anaerobic conditions (In a lack of oxygen)
 Fermentation (food), the conversion of carbohydrates into alcohols or acids under anaerobic conditions used for making certain foods
 Fermentation (wine), the process of fermentation commonly used in winemaking.
 Fermentation (tea), the name used in the tea industry for the aerobic treatment of tea leaves to break down and release certain unwanted chemicals
 Ethanol fermentation, a form of anaerobic respiration used primarily by yeasts when oxygen is not present in sufficient quantity for normal cellular respiration
 Industrial fermentation, the breakdown and re-assembly of biochemicals for industry, often in aerobic growth conditions
The major commercial products of microorganisms
Microbial cells: These may be used as food supplements or immunizing agents, e.g: yeast cells, vaccines etc.
Large or macromolecules: Enzymes synthesized by microorganisms.
Primary metabolic products: These are compounds essential for cell growth, e.g: vitamins.
Secondary metabolic products: These are compounds not required for cell growth, e.g: antibiotics
Classification of microbial products
Based on the intended use of the final products the various industrial processes used to produce these microbial products can be divided into several classes:
1. Production of Pharmaceutical chemicals:
 Most prominent in this case are the antibiotics and steroid drugs.
 Other substances such as insulin and interferon are now being produced by genetically engineered bacterium.
. Production of commercially valuable chemicals:
 This class includes solvents, enzymes and intermediate compounds (e,g. ethanol) for the synthesis of other substrates.
3. Production of Food Supplements:
 Mass production of yeasts, bacteria and algae from nutritionally enriched media provides a good source of protein and other organic nutrients useful as food supplements, e.g: bread, yogurt, spirullina etc.
4. Production of alcoholic beverages:
 Beer, wine, whisky etc are used as alcoholic beverages.
5. Production of Vaccines:
 The whole cell or some part or product of the cell is used for the preparation of vaccines.
6. Production of insecticides:
 Microbes are used as pesticides or biocontrolling agents, e.g: Bacillus thuringensis.
7. Application in Mining and Petroleum Industry:
 Microbes are used for petroleum recovery and other mining process.
8. Microbial activity for the treatment of waste materials:
 All kinds of materials such as leather, textiles, woods, metals etc are subject to deterioration by contamination with degrading microbes.

Metabolic products
 The microbes used by fermentation industries have traditionally been naturally occurring strains of bacteria and molds that carry out a particular metabolic action on a substrate.
At an increasing rate, however, these microbes are mutant strains of fungi and bacteria that selectively synthesize large amounts of various metabolic intermediates or metabo
 Industrial processes harvested following 2 basic metabolic products:
1. Primary metabolites are produced during the major metabolic pathways and are essential for microbe’s function.
2. Secondary metabolites are by-products of metabolism that may not be critical to the microbe’s function.

Characteristics of Secondary Metabolites
 Secondary Metabolites are not essential for growth and reproduction.
 The formation of secondary metabolites is extremely dependent on growth conditions, especially on the composition of the medium.
 Secondary Metabolites are often produced as a group of closely related compounds. For instance, a single strain of a species of Streptomyces has been found to produce over 30 related but different anthracycline antibiotics.
It is often possible to get dramatic overproduction of Secondary Metabolites, whereas primary metabolites, linked as they are to primary metabolism, usually can not be si
Differences between Primary and Secondary Products
 In general, primary products are compounds such as amino acids and organic acids synthesized during the logarithmic phase of microbial growth.
Secondary products are compounds such as vitamins, antibiotics and steroids synthesized during stationary phase
Strategies to increase the amount of the chosen end products
 Industrial microbiologists can manipulate the growth environment to increase the synthesis of a metabolite. For e.g: adding lactose to glucose as the fermentation substrate increases the production of penicillin by Penicillium.
They can select microbial strains that genetically lack a feedback system to regulate the formation of end product, thus encouraging mass accumulation


Standards of materials used in sophisticated fermentor design

• All materials coming to contact with the solutions entering the bioreactor or the actual organism culture must be corrosion resistant to prevent trace metal contamination of the process.
• The materials must be non-toxic so that slight dissolution of the material or components does not inhibit culture growth.
• The materials of the bioreactor must withstand repeated sterilization with high pressure stream.
• The bioreactor stirrer system, entry ports and end plates must be sufficiently rigid not to be deformed or broken under mechanical stress.
Visual inspection of the medium and culture is advantageous, transparent materials should be used wherever possible.

The parts of a fermentation process

Regardless of the type of fermentation an established process may be divided into 6 basic parts:
1) The formulation of media to be used in culturing the process organism during the development of the inoculum and in the production fermenter.
2) The sterilization of the medium, fermenters and ancillary equipment.
3) The production of an active, pure culture in sufficient quantity to inoculate the production vessel.
4) The growth of the organism in the production fermenter under optimum conditions for product formation.
5) The extraction of the product and its purification.
The disposal of effluents produced by the process

 Batch culture
 Fed-batch
 Continuous culture
Batch culture
In a batch culture the microbes are inoculated into a fixed volume of medium and as growth takes place nutrients are consumed and products of growth (biomass, metabolites) accumulate.
The nutrient environment within the bioreactor is continuously changing, thus , in turn, enforcing changes to cell metabolism.
Eventually, cell multiplication ceases because of exhaustion or limitation of nutrients and accumulation of toxic excreted waste products.
In industrial usage, batch cultivation has been operated to optimise organism or biomass production then to allow the organism to perform specific biochemical transformation such as end-product formation (e.g: aa, enzymes) or decomposition of substances (sewage treatment).
Prolonging the life in a Batch culture
There are means of Prolonging the life in a Batch culture and thus increasing the yield by various substrate feed methods:
Gradual addition of concentrated components of the nutrient, e.g: carbohydrates, so increasing the volume of the culture (fed-batch)-used for industrial production of baker’s yeast.
Addition of medium to the culture (perfusion) and withdrawal of an equal volume of used free cell-free medium-used in animal cultivation.
Advantages of batch and fed-batch culture techniques in Industry
Products may be required only in relatively small quantities at any given time.
Market needs may be intermittent.
Shelf-life of certain products is short.
High product conc. is required in broth to optimise downstream processing operations.
Some metabolic products are produced only during the stationary phase of the growth cycle.
Instability of some production strains require their regular renewal.
Continuous Culture
The practice of continuous culture gives near-balanced growth with the little fluctuation of nutrients, metabolites, cell numbers and biomass.
This practice depends on fresh medium entering a batch system at the exponential phase with a corresponding withdrawal of medium plus cells,
Continuous methods of cultivation will permit organisms to grow under steady state (unchanging) conditions in which growth occurs at a constant rate and in a constant environment.
In industrial practice continuously operated systems are of limited use and include only single cell protein and ethanol productions and some forms of waste-water treatment processes.

Type of culture Operational characteristics Application
Solid Simple, cheap, selection of colonies from single cell possible, process control limited Maintenance of strains, genetic studies, production of enzymes, composting
Film Various types of bioreactor, trickling filter, rotating disc, packed bed, sponge reactor, rotating tube Waste water treatment, monolayer culture, bacterial leaching, vinegar production

Type of culture Operational characteristics Application
Submerged homogeneous distribution of cells; batch Spontaneous reaction, various types of reactor, continuous stirred tank reactor, air lift, loop. Deep shaft etc. agitation by stirrers, air, liquid process control for physical parameters possible. Standard types of cultivation antibiotics, solvents, acids etc
Fed-batch Simple method for control of regulatory effects, e.g. glucose repression Production of Baker’s yeast

Type of culture Operational characteristics Application
Continuous One-stage homogeneous Proper control of reaction, excellent for kinetic and regulatory studies, higher costs of experiment, problem of aseptic operation, the need for highly trained operators. Few cases of application in industrial scale; production of SCP, waste-water treatment.

Some examples of commercial products used in vaccines for active immunization
Disease Nature of immunizing agent
Tuberculosis Live attenuated cells of Mycobacterium bovis
Measles Live attenuated Rubeola virus
Poliomyelitis Live attenuated strains of poliovirus
Rabies Killed rabies virus
Whooping cough Killed cells of Bordetella pertusis
Typhoid fever Killed cells of Salmonella typhi
Diptheria Toxoid prepared from exotoxin of Corynebacterium diptheriae
Tetanus Toxoid prepared from exotoxin of Clostridium tetani
Meningococcal meningitis Capsular polysaccharides from Neisseria meningitisdis

Some antibiotics produced by microbes
Antibiotic Microorganisms
Amphotericin B Streptomyces nodosus
Bacitracin Bacillus licheniformis
Chlorotetracycline Streptomyces aureofaciens
Kanamycin S kanamyceticus
Nystatin S. noursei
Penicillin Penicillin chrysogenum
Polymixin B Bacillus polymyxa

Some examples of enzymes
Enzymes Source Application
Glucose isomerase Bacillus spp, Streptomyces spp Production of high fructose corn syrup
Lipase Rhizopus spp Use in fat removal
Cellulase Trichoderma reesii Digestive aid
Amylase Many bacteria and fungi Starch digesting agent
Renin Fungi Coagulation of milk to make cheese: Dairy product
Protease Many bacteria and fungi Proteolytic enzyme

Human proteins
 Genetic engineering has expanded the roles of microbes in the pharmaceutical industry to produce human proteins.
 By using recombinant DNA technology human DNA sequences that code for various proteins can be produced commercially.
 Recombinant DNA tech provides a means of producing relatively large amount of human proteins for use as prophylactic drugs and diagnostic reagents.
 Examples: insulin, growth hormones, interferons, interleukin-2 etc.
Industrially important microorganisms: Screening and selection of microorganisms for useful products
Industrial microorganisms are organisms which have been selected carefully so that they manufacture one or more specific products.
Even if industrial microbe is one which has been isolated by traditional techniques, it becomes a highly modified organism before it enters large-scale industries.
Industrial microorganisms are metabolic specialists, capable of producing specially and to high yield metabolic products.
Where do Industrial Strains Come from?
The ultimate source of all strains of industrial microorganisms is the natural environment.
But through the years, as large-scale microbial processes have preferred, a number of industrial strains have been deposited in Culture Collections.
There are numbers of culture collections which serve as the repositories of microbial cultures.

Culture collections that supply cultures of Industrial microorganisms
Abbreviation Name Location
ATCC American Type Culture Collection US
CDDA Canadian Department of Agriculture Canada
CIP Collection of Institute Pasteur France
IAM Institute of Applied Microbiology Japan
NCTC National Collection of Type Cultures UK
DSM Deutsche Sammlung von Mikroorganismen Germany

Properties of Industrially Important Microorganisms

A microorganism suitable for industrial use must produce the substance of interest.
The organism must be available in pure culture.
Must be genetically stable.
Must grow in large-scale culture.
It must be possible to maintain cultures of the organism for a long period of time in the lab and in the industrial plant.
The culture should preferably produce spores or some other reproductive cell from so that the organisms can be easily inoculated into large fermentors.
The organism must be capable of growing vigorously after inoculation into seed stage vessel.
It must produce the desired product in a relatively short period of time.
The organism must be able to grow in a relatively inexpensive liquid culture medium obtainable in bulk quantities.
It must be able to produce a desirable product preferably in a single one easily recovered and preferably with the absence of any toxic byproducts.
An industrial organism should not be harmful to humans or economically important animals and plants.
The organism should be preferably capable of protecting itself from contamination.
The most favorable industrial organisms are those of large cell size, since larger cells settle rapidly from a culture or can be easily filtered out with relatively inexpensive filter materials.
An industrial microbe should be amenable to genetic manipulation.
Fungi, yeasts and filamentous bacteria re preferred as industrial microorganism.
The Isolation of Industrially Important Microorganisms
The diversity of microorganisms may be exploited still by searching for strains from the natural environment able to produce products of commercial value.
The first stage is isolation.
Isolation involves obtaining either pure or mixed cultures followed by their assessment to determine which carry out the desired reaction or produce the desired product.
Isolation methods utilizing selection of the desired characteristic
Isolation methods depending on the use of desirable characteristics as selective factors are essentially types of enrichment culture.
Enrichment culture is a technique resulting in an increase in the number of a given organism relative to the number of other types in the original inoculum.
Prior to the culture stage it is often advantageous to subject the environmental source to conditions which favor the survival of the target organisms.
For e.g. air-drying the soil will favor the survival of actinomycetes.
Enrichment Liquid Culture:
Enrichment Liquid Culture is frequently carried out in shake flasks.
The growth of the desired type from a mixed inoculum will result in the modification of the medium and therefore changes the selective force which may allow the growth of other organisms.
The selective force may be re-established by inoculating the enriched culture into identical fresh medium.
Such sub culturing may be repeated several times before the dominant organism is isolated by spreading a small inoculum of the enriched culture onto solid medium.
Enrichment Cultures Using Solidified Media:
Solidified Media have been used for the isolation of certain enzyme producers and these techniques usually involve the use of a selective medium incorporating the substrate of the enzyme which encourages the growth of the producing types.
For e.g. isolation of Bacillus spp producing alkaline protease. Soils of various pHs were used as the initial inoculum and, to a certain extent, the number of producers isolated correlated with alkalinity of soil sample. The soil samples were then pasteurized to destroy vegetative cells and then inoculated into agar media at pH 9-10, containing a dispersion of an insoluble protein. Colonies which produced a clear zone due to the digestion of protein were taken to alkaline protease producers.
Screening procedures For Industrial Microorganisms
Screening procedures may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from a large microbial population.
Different types of techniques are used for screening procedure depending on the source, microorganisms, products etc.
The aim of the screening is the collection of low No. pf organism from a large No. of sample.
Screening is of two types:
Primary screening
Secondary screening
Primary screening
Primary screening allows the detection and isolation of microbe that posses potentially industrial applications.
The primary screening may have yielded only a very small number that have any real commercial value; because it determines which microbes are able to produce a compound without providing much idea of the production or yield potential for the organisms.
Secondary screening
Following primary screening, a secondary screening procedure is required to further test the capabilities and to gain information about target organisms.
Criteria of Secondary screening
Secondary screening is conducted on agar plates, in flasks or small fermentors containing liquid media or as a combination of these approaches.
Liquid cultures are better than agar plate media.
Secondary screening can be quantitative and qualitative in its approach.
Qualitative approach tells us the spectrum of microbes which is sensitive to a newly discovered antibiotic and quantitative approach tells us the yield of antibiotic.
Secondary screening should yield the types of in formations which are needed in order to evaluate the true potential of a microorganism for industrial usage.
For example, it should determine what types of microorganism are involved and whether they can be classified at least to families or genera.
Secondary screening should determine whether the microbes are actually producing chemical compounds not previously described or alternatively, for fermentation processes that are already known, it should determine whether a more economical process is possible.
It should be reveal whether these are pH, aeration or other chemical requirements associated with particular microorganism, both for the growth of organism and for the formation of chemical products.
Secondary screening should also detect gross genetic instability in microbial culture.
It should show whether certain medium constituents are missing or possibly are toxic to growth of the organism or to its ability to accumulate fermentation products.
It should also show something of the chemical stability of the product and of the products solubility in various organic solvents.
Secondary screening should reveal whether a product resulting from a microbial fermentation occurs in the culture broth in more than one chemical form, and whether it is an optically or biologically active material.
Secondary screening should revel whether microorganism are able to chemically alter or even destroying their own fermentation products.
Molecular Screening Methods
Early screening strategies tended to be empirical, labor intensive and had relatively low success rates.
New screening methods have been developed which are more precisely targeted to identify the desired activity.
The progress in molecular biology, genetics and immunology has contributed extensively to the development of innovative screens.
The major contributions are:
• The provision of test organisms that have increased sensitivities, or resistances, to known agents. For e.g. the use of super-sensitive strains for the detection of β-lactam antibiotics.
• The cloning of genes coding for enzymes or receptors that may be used in inhibitor or binding screens makes such materials more accessible and available in much lager amounts.
• The development of reporter gene assays.
• Molecular probes for particular gene sequences may enable the detection of organisms capable of producing certain product groups.
• The development of immunologically based assays such as ELISA.

The Preservation of Industrially Important Microorganisms
The isolation of a suitable organism for a commercial process may be a long and very expensive procedure.
Thus, preservation techniques have been developed to maintain cultures in a state of ‘suspended animation’ by storing either
at reduced temperature or
in a dehydrated form.
Storage at reduced temperature
Storage on agar slopes:
Cultures grown on agar slopes may be stored at 5°C or -20°C and sub-cultured at approx. 6 monthly intervals.
The time of sub-culture may be extended to 1 yr if the slopes are covered with sterile medicinal grade mineral oil.
Storage Under Liquid Nitrogen:
The metabolic activities of m.o may be reduced by storage at very low temp (-150° to 196° C) which may be achieved using a liquid nitrogen refrigerator.
Storage in a dehydrated form
Dried cultures:
Dried soil cultures have been used widely for culture preservation, particularly for sporulating mycelial microorganism.
Moist, sterile soil may be inoculated with a culture and then allowed to dry at room tempt for approximately 2 weeks.
The dry soil may be stored in a dry atmosphere or preferably in a refrigerator.
Lyophilization or freeze drying involves freezing of a culture followed by its drying under vacuum, which results in the sublimation of the cell water.
The technique involves growing the culture to the max stationary phase and resuspending the cells in a protective medium such as milk, serum or sodium glutamate.
A few drops of the suspension are transferred to an ampoule, which is then frozen and subjected to a high vacuum until sublimation is complete, after which the ampoule is sealed.
The ampoules may be stored in a refrigerator and the cells may remain viable for 10 yrs or more.
Strain Improvement
The initial source of an industrial microorganism is the natural environment, but the original isolate is greatly modified in the lab.
As a result of this modification, progressive improvement in the yield of a product can be anticipated.
For e.g. production of penicillin from the fungus Penicillium chrysogenum. When penicillin was 1st produced on a large scale, yields of only 1-10 μg/ml were obtained. Over the yrs, as a result of strain improvement coupled with changes in the medium and growth conditions, the yield of penicillin has been increased to about 50,000 μg/ml!
The introduction of new genetic techniques has led to further yield increases.
The process of strain improvement involves the continual genetic modification of the culture.
Genetic modification may be achieved by selecting natural variants, by selecting recombinants.
Selection of natural variants may be results in increased yields but it is not possible to rely on such improvements, and techniques must be employed to increase the chances of improving the culture.
These techniques are the isolation of induced mutants and recombination.
The most dramatic examples of strain improvement come from the applications of recombinant DNA technology which has resulted in microorganism producing compounds which they were not able to produce previously.
The advances in these techniques have resulted in very significant improvements in the production of conventional fermentation products.
Stages of Strain Improvement
Process for strain improvement comprises of several stages.
Data provided by Customer analysis to plan further research
Strain and Technology parameters required for "start point" determination
Strain mutability evaluation includes period for probationary mutagenesis series when great variety of mutagen factor combinations affects the strain. All data for obtained mutants during this preliminary mutagenesis is considered thoroughly for further stage.
Screening and selection among great deal of mutant varieties is a very demanding stage:
experts' deep knowledge in combination with many years of experience and scientific intuition makes it successful
Induced mutagenesis as the basis of Laboratory Know-How represents combination of classical methods for chemical and physical impact on the strain
Technology parameter optimisation
Process scale-up
Recovery Technology Improvement: under Customer request
Use of Microorganisms in Different Industries: Biocatalysts
The chemical industry:
using biocatalysts to produce novel compounds, reduce waste byproducts and improve chemical purity.
The plastics industry: decreasing the use of petroleum for plastic production by making "green plastics" from renewable crops such as corn or soybeans.
The paper industry:
improving manufacturing processes, including the use of enzymes to lower toxic byproducts from pulp processes.
The textiles industry:
lessening toxic byproducts of fabric dying and finishing processes. Fabric detergents are becoming more effective with the addition of enzymes to their active ingredients.
The food industry: improving baking processes, fermentation-derived preservatives and analysis techniques for food safety.
The livestock industry: adding enzymes to increase nutrient uptake and decrease phosphate byproducts.
The energy industry: using enzymes to manufacture cleaner biofuels from agricultural wastes.

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