Industrial Microbiology

Industrial Microbiology - 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 (Casida, Jr., 1968). 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 services generated by microorganisms range from the degradation of organic wastes, detoxification of industrial wastes and toxic compounds, to the degradation of petroleum to manage oil spills, etc. Industrial microbiology also encompasses activities like production of biocontrol agents, inoculants used as biofertilizers, etc.

Obviously, the scope and activities of industrial microbiology are too extensive to be covered in any detail in a book like this scope; therefore, the coverage in this chapter remains generalized and rather elementary.

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.

Microbial Products of Potential Importance - Product / Activity ExamplesProducts  1. Amino acids L-glutarnic acid, L-lysine 2. Antibiotics Streptomycin, penicillin, tetracyc1ines, polymyxin3. Beverages Wine, beer, distilled beverages4. Biodegradable plastic β-polyhydroxybutyrate5. Enzymes Amylase, proteases, pectinases, invertase, cellulase6. Flavouring agents Monosodium glutamate, nucleotides7. Foods Cheese, pickles, yoghurt, bread, vinegar8. Gases CO2, H2,CH4

9. Organic acids Lactic, citric, acetic, butyric, fumaric10. Organic solvents Acetone, ethanol, butanol, amyl alcohol11. Others Glycerol, fats, steroids, gibberellins11 a. Vitamins B12, riboflavin, A12. Recombinant proteins Insulin, interferon, subunit vaccines13. Substrates A wide range of compounds used for chemical syntheses of valuable products.

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Cells/Biomass  14. Biomass Food and feed yeast, other organisms used as single cell protein (SCP) 15. Cells Biofertilizers, biocontrol agents, bacterial insecticides, mycorrhyzae16. Vaccines A variety of viral and bacterial vaccinesActivities  

Biotransformation Steroids, antibiotics D-sorbitol

Degradation Disposal of biological and industrial wastes, detoxification of toxic compounds, petroleum

Solubilization /accumulation

Improved recovery of oil and metals, discovery of new oil reserves, removal of toxic metals

FERMENTORS

The fermentation processes maintain the growth of industrial microorganism in an environment which stimulates the synthesis of the desired commercial product. This is carried out in a fermentor which is essentially a large vessel in which organisms are maintained at the required temperature, pH, oxygen and substrate concentration. The fermentors are quite complicated in design and variable in size. Industrial fermentors are designed to provide the best possible growth and biosynthetic activities of the organism. The design of the fermentor depends upon the purpose for which it is to be utilized.

Definition:- Fermentor can be defined as a large vessel used to culture microorganisms on a large scale frequently for the production of some commercially valuable product. The microorganism involved in the process is known as fermentor.

Bioreactors - A bioreactor is a device in which a substrate of low value is utilized by living cells or enzymes to generate a product of higher value. Bioreactors are extensively used for food processing, fermentation, waste treatment, etc. On the basis of the agent used, bioreactors are grouped into two broad classes:

(i) those based on living cells and,

(ii) those employing enzymes.

But in terms of process requirements, they are of the following types:

(i) aerobic,

(ii) anaerobic,

(iii) solid state, and

(iv) immobilized cell bioreactors.

All bioreactors deal with heterogeneous systems having two or more phases, e.g., liquid, gas, solid. Therefore, optimal conditions for fermentation necessitate efficient transfer of mass, heat and momentum from one phase to the other.

A bioreactor should provide for the following:

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(i) agitation (for mixing of cells and medium),

(ii) aeration (aerobic fermenters; for O2 supply),

(iii) regulation of factors like temperature, pH, pressure, aeration, nutrient feeding, liquid level, etc.,

(iv) sterilization and maintenance of sterility, and

(v) withdrawal of cells/medium (for continuous fermenters). Modem fermenters are usually integrated with computers for efficient process monitoring, data acquisition, etc.

The size of fermenters ranges from 1-21 laboratory fermenters to 500,000 1 or, occasionally, even more; fermenters of upto 1.2 million litres have been used. Generally, 20-25% of fermenter volume is left unfilled with medium as "head space" to allow for splashing, foaming and aeration. The fermenter design varies greatly depending on the type of fermentation for which it is used.

Solid State Fermentation - In such fermentations, microbial growth and product formation occur at the surface of solid substrates. Examples of such fermentations are mushroom cultivation, mold ripened cheeses, starter cultures, etc. More recently, this approach has been used for the production of extracellular enzymes, certain valuable chemicals, fungal toxins, and fungal spores (used for biotransformation).

Traditional substrates are several agricultural products, rice, wheat, maize, soybean, etc. The substrate provides a rich and complex source of nutrients, which mayor may not need to be supplemented.

Such substrates selectively support mycelial organisms, which can grow at high nutrient concentrations and produce a variety of extracellular enzymes, e.g., a large number of filamentous fungi, and a few bacteria (Actinomycetes and one strain of Bacillus).

According to the physical state, solid state fermentations are divided into two groups:

(i) low moisture solids fermented without or with occasional/continuous agitation, and

(ii) suspended solids fermented in packed columns, through which liquid is circulated. The fungi used for solid state fermentations are usually obligate aerobes.

Solid state fermentations on large scale use stationary or rotary trays. Temperature and humidity controlled air is circulated through the stacked solids. Less frequently, rotory drum type fermenters have been used.

Solid state fermentations offer certain unique advantages, but suffer from some important disadvantages. However, commercial application of this process for biochemical production is chiefly confined to Japan.

Anaerobic Fermentation - In anaerobic fermentation, a provision for aeration is usually not needed. But in some cases, aeration may be needed initially for inoculum build up. In most cases, a mixing device is also unnecessary; while in some cases initial mixing of the inoculum is necessary. Once the fermentation begins, the gas produced in the process generates sufficient mixing.

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The air present in the headspace of the fermentor should be replaced by CO2, H2, N2 or a suitable mixture of these; this is particularly important for obligate anaerobes like Clostridium.

The fermentation usually liberates CO2 and H2, which are collected and used, e.g., CO2 for making dry ice and methanol, and for bubbling into freshly inoculated fermenters.

In case of acetogens and other gas utilizing bacteria, O2free sterile CO2 or other gases are bubbled through the medium. Acetogens have been cultured in 400 I fermenters by bubbling sterile CO2 and 3, kg cells could be harvested in each run.

Recovery of products from anaerobic fermenters does not require anaerobic conditions. But many enzymes of such organisms are highly O2, sensitive. Therefore, when recovery of such enzymes is the objective, cells must be harvested under strictly anaerobic conditions.

Aerobic Fermentation - The main feature of aerobic fermentation is the provision for adequate aeration; in some cases, the amount of air needed per hour is about 60-times the medium volume. Therefore, bioreactors used for aerobic fermentation have a provision for adequate supply of sterile air, which is generally sparged into the medium.

In addition, these fermenters may have a mechanism for stirring and mixing of the medium and cells. Aerobic fermenters may be either of the

i) stirred tank type in which mechanical motor driven stirrers are provided or

(ii) of air lift type in which no mechanical stirrers are used and the agitation is achieved by the air bubbles generated by the air supply.

Generally, these bioreactors are of closed or batch type, but continuous flow reactors are also used such reactors provide a continuous source of cells and are also suitable for product generation when the product is released into the medium.

Basic factors involved in fermentor design:- The main function of a fermentor is to provide a controlled environment for the growth of microorganisms to obtain a desired product. In designing and constructing a fermentor a number of factors must be considered:

1) The vessel should be capable of being operated aseptically for a number of days and should be reliable in long-term operation and should meet the requirements of contain ment regulations.

2) Adequate aeration and a should be provided to meet the metabolic requirements of microorganisms. However, the mixing should not cause damage to the organism.

3) Power consumption should be as low as possible.

4) A system of temperature control should be provided.

5) A system of pH control should be provided.

6) Sampling facilities should be provided.

7) Evaporation losses from the fermentor should not be excessive.

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8) The vessel should be designed to require minimal use of labour in operation, harvesting, cleaning and maintenance.

9) The vessel should be constructed to ensure smooth internal surfaces, using welds instead of flange joints whereever possible.

10) The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant.

11) The cheapest materials which enable satisfactory results should be used.

12) There should be adequate service provisions for individual plant.

The first two points are the most critical. The design of a fermentor will involve co-operation between experts in microbiology, biochemistry, chemical engineering, mechanical engineering and costing. Although many different fermentors have been described, very few have proved to be satisfactory for industrial aerobic fermentations. The most commonly used ones are based on a stirred upright cylinder with sparger aeration

Typical Fermentor: Industrial fermentors are designed to provide the best possible growth and biosynthesis

conditions of the microbial cultures. Since most industrial fermentations utilize pure cultures the fermentor should have provisions for the control of or prevention of contaminating microorganisms. If the fermentation is to be carried out aerobically, then provision must be made for rapid sterile air circulation in the fermentor. There must be provision for the removal of C02 formed during fermentation. There must be provision for stirring or mixing the medium during microbial growth for uniform distribution of microorganisms, air and nutrients. There must be provision for intermittent adding of antifoam agents. There must be provision for

Picture: A typical Fermentor with three multibladded impellers.

temperature and pH control. The fermentor should have provision for the withdrawal of cultures during fermentation aseptically. There must be provision for the introduction of inoculum into the fermentor.

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Finally, there must be a drain at the bottom of the fermentor for removing fermentation broth from the fermentor.

The capacity of the fermentor ranges from a few hundred to several thousand gallons. Thus there are different types of fermentors available. The capacity of laboratory fermentor is about 12 to 15 litres. The pilot plant fermentors have the capacity of 100 to 1000 gallons. Large fermentors range from 10,000 to 1,00,000 gallons capacity. There are industrial fermentors whose capacity ranges from 0.25 million gallons to 0.5 million gallons.

pH control:- The pH of the medium is to be maintained at optimum level. For this purpose, samples are withdrawn from the fermentor for pH determination followed by addition of alkali or acid to the fermentation medium. Nowadays pH control is achieved by acid or alkali addition, which in turn is controlled by autotitrator. The autotitrator, in turn, is connected to a pH probe.

Temperature control:- Various microorganisms utilized in the fermentation process differ in their temperature requirements (optimum) for growth and biochemical activities. Growth temperature may or may not coincide with the optimum temperature for the fermentation. Hence control of temperature is very essential. Besides, during fermentation microorganisms evolve heat which can accumulate to a considerable degree in the fermentor. Hence fermentor must be cooled in some manner to maintain optimum temperature during fermentation. Temperature control is achieved either by spraying cold water on the surface of the fermentor or by passing

cold water through the jacket around the fermentor. This is supplemented by the use of internal coils inside the fermentor through which cold water is circulated. Sometimes fermentation medium requires high temperatures, in such cases heat may be applied by passive steam through the coils or jacket of the fermentor.

Picture :Four types of Agitators :(A) Disc Turbine,(B) Vaned Disc,(C) Opened turbine,and (D) Marine turbine.

Agitation:- In the fermentor there must be provision for stirring or mixing the medium during fermentation for uniform distribution of nutrients, air, microbes and temperature. The mixing or stirring process is called agitation and it is achieved by using agitation device. The agitating device consists of a strong and straight shaft to which impellers are fitted. An impeller, consists of a circular disc to which blades are fitted. Different types of blades are available and are used according to the requirements. The shaft passes through a bearing in the lid of the fermentor it is rotated with the help of an electric motor mounted externally at the top of the fermentor. The speed of the agitator can be controlled by adjustable pulleys and belts connected with the motor. The height of the impeller blades above the bottom of the fermentor is adjustable according to one’s desire. The liquid medium is thrown up towards the walls of the fermentor while rotating the impellers. This result in the formation of a vorter which is eliminated by using four equally placed baffles attached to the walls of the fermentor.

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Aeration:- Most of the aerobic fermentations require air. Even in anaerobic fermentations air is required for the growth and development of the microbes in the initial stages. Most aerobic fermentations require that sterile air under pressure be introduced into the fermentation vessel. The cheapest means for sterilization of air is to pass through a sterile filter composed of glass wool, carbon particle or some other finely divided material that will trap microorganisms present in air. Beyond this filter, the air is carried through sterile piping to the bottom of fermentor beneath the impeller blades. Here the air passes through into a sparger which may be

designed in various ways but which often consists of a pipe with minute holes that allow air under pressure to escape as tiny bubbles into the liquid medium.

Spargers with hole smaller than this require too great air pressure for economical bubble for motion. Spargers in fermentors for growth of mycelium forming organisms often utilize ¼ inch holes to prevent ¼ plugging of the holes by hyphal growth. The air bubble from the sparger are picked up and dispersed through the medium by the action of impeller blades mounted above the sparger. The smaller the air bubbles the greater is the bubble surface area covered in the medium. But, smaller air bubbles require greater air pressure to pass through fine holes of the sparger. Since sterile air is a costly item for an industrial fermentation, the size

of air bubbles must be adjusted to give the greatest possible aeration without increasing the overall cost of the fermentation. In some large fermentors the impellers are not utilized for aeration. Instead the medium is stirred by the direct rush of air bubbles from the sparger at the bottom of the fermentor. These fermentors are specially designed for the above purpose.

There are various ways of injecting air into the fermentor.

1) Impeller air injection: - In this method air is fed to the impeller by means of a hollow shaft and injected into the medium through the holes present in the impellers.

2) Two phase injection: - A two phase mixture of air and medium is fed into the fermentor in the form of suspension.

3) Air lift injection: - In this method air is used to circulate the contents of fermentor, with tubes provided on the external surface of the fermentor or internal tubing.

4) Sparger air injection: - This is the most commonly used method. It is a universal practice to introduce the air through a sparger placed close to the underside of the impeller. In this method, single get multi fed and porous spargers are used. The porous spargers are unpopular owing to the fact that there is a tendency of microorganisms blocking the holes.

Antifoams (Foam control):

In most microbiological processes, foaming is a problem. Aeration and agitation of a liquid medium can cause the production of foam. Foaming may be due to a component in the medium or some factor produced by the microorganisms. The most common cause of foaming is due to proteins in the medium, such as corn steep liquor, pharmamedia, peanut meal, soybean meal, yeast extract or meat extract. These proteins may denature at the air-broth interface and form a skin which doesn’t rupture easily. The foaming can cause removal of cells from the medium which will lead to autolysis and further release of microbial cell and further release of microbial cell proteins will probably increase the stability of the foam. If foam is not controlled, numerous changes may occur and physical and chemical changes may be created. These include reduction in the working volume of the fermentor due to oxygen exhausted gas bubbles circulating in the systems, changes in the bubble size, lower mass and heat transfer rates, invalid process data due to interference at sensing electrodes and incorrect monitoring and control. The biological problems include deposition of cells in upper parts of the fermentor, problems of sterile operation with the air filter exits of the fermentor becoming wet and there is a danger of microbial infection and the possibility of siphoning leading to the loss of product.

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In 1973 Hall and coworkers recognized fine patterns of foaming in fermentations:

1) Foaming remains at constant level throughout the fermentation. Initially it is due to the medium and later due to microbial activity.

2) A steady fall in foaming during the early part of the fermentation, after which it remains constant. Initially it is due to the medium but there are no later effects caused by the microorganisms.

3) The foaming falls slightly in the early stages of the fermentation then rises. There are very slight effects caused by the medium but the major effects are due to microbial activity.

4) The fermentation has a low initial foaming capacity which rises. These effects are due solely to microbial activity.

5) A more complex foaming pattern during the fermentation which may be a combination of two or more of the previously described patterns.

If excessive foaming is encountered there are three ways of approaching the problem:-

1) To try and avoid foam formation by using a defined medium and a modification of some of the physical parameters (pH temperature, aeration and agitation). This assumes that the foam is due to a component in the medium and a metabolite.

2) The foam is unavoidable and antifoam should be used. This is the more standard approach.

3) To use mechanical foam breaker.

Antifoams are surface active agents, reducing the surface tension in the foams and destabilizing protein films by

a) Hydrophobic bridges between two surfaces,

b) Displacement of absorbed protein and

c) Rapid spreading on the surface of the film.

An ideal antifoam should have the following properties.

1) Should easily disperse and have fast action on existing foam.

2) It should be active at low concentrations.

3) Should be active preventing new foam formation.

4) Should not be metabolized by the microorganism.

5) Should be nontoxic to the microorganism.

6) Should be nontoxic to humans and animals.

7) Should not cause any hazards during handling.

8) Should not create any problem during extraction and purification of the product.

9) Should be cheap.

10) Should have no effect on oxygen transfer.

11) Should be heat sterilizable.

Some of the ideal anti foaming agents which meet most of these above requirements have been found suitable in different fermentation processes are as follows:-

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a) Esters.

b) Alcohols: Stearyl and octy decanol.

c) Silicones.

d) Fatty acids and their derivatives, particularly glycerides which include cotton seed oil, linseed oil, soybean oil, olive oil castor oil sunflower oil, rape seed oil and cod liver oil.

e) Sulphonates.

These antifoams are generally added when foaming occurs during the fermentations. Because of many antifoam are of low solubility they need a carrier such as lard oil, liquid paraffin or castor oil, which may be metabolized and affect the fermentation process.

Unfortunately, the concentrations of many antifoam agents which are necessary to control fermentations will reduce the oxygen transfer rate by as much as 50%, therefore anti foam additions must be kept to an absolute minimum. If the oxygen transfer is severely affected by anti foam addition then mechanical foam breakers may have to be considered as possible alternative.

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Microorganisms in Industry and Raw Materials

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Isolation and Screening of Microorganisms - The success of an industrial fermentation process chiefly depends on the microorganism strain used. An ideal producer or economically important strain should have the following characteristics.

1. It should be pure, and free from phage.

2. It should be genetically stable, but amenable to genetic modification.

3. It should produce both vegetative cells and spores; species producing only mycelium are rarely used.

4. It should grow vigorously after inoculation in seed stage vessels.

5. Should produce a single valuable product, and no toxic by-products.

6. Product should be produced in a short time, e.g., 3 days.

7. It should be amenable to long term conservation.

8. The risk of contamination should be minimal under the optimum performance conditions.

Isolation of Microorganisms - The first step in developing a producer strain is the isolation of concerned microorganisms from their natural habitats. Alternatively, microorganisms can be obtained as pure cultures from organisation, which maintain culture collections, e.g., American Type Culture Collection (ATCC).

Rockville, Maryland, U.S.A., Commonwealth Mycological Institute (CMI), Kew, Surrey, England, Fermentation Research Institute (FERM), Tokyo, Japan, U.S.S.R. Research Institute for Antibiotics (RIA), Moscow, U.S.S.R., etc.

The microorganisms of industrial importance are, generally, bacteria, actinomycetes, fungi and algae. These organisms occur virtually everywhere, e.g., in air, water, soil, surfaces of plants and animals, and plant and animals tissues. But most common sources of industrial microorganisms are soils, and lake and river mud.

Often the ecological habitat from which a desired microorganism is more likely to be isolated will depend on the characteristics of the product desired from it, and of process development. For example, if the objective is to isolate a source of enzymes, which can withstand high temperatures, the obvious place to look will be hot water springs.

A variety of complex isolation procedures have been developed, but no single method can reveal all the microorganisms present in a sample. Many different microorganisms can be isolated by using specialized enrichment techniques, e.g., soil treatment (UV irradiation, air drying or heating at 70120°C, filtration or continuous percolation, washings from root systems, treatment with detergents or alcohols, preinoculation with toxic agents), selective inhibitors (antimetabolites, antibiotics, etc.), nutritional (specific C and N sources), variations in pH, temperature, aeration, etc.

The enrichment techniques are designed for selective multiplication of only some of the microorganisms present in a sample. These approaches however take a long time (20-40 days), and require considerable labour and money.

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The main isolation methods used routinely for isolation from soil samples are: sponging (soil directly), dilution, gradient plate, aerosol dilution, flotation, and differential centrifugation. Often these methods are used in conjunction with an enrichment technique.

Screening of Microorganisms for New Products - The next step after isolation of microorganisms is their screening. A set of highly selective procedures, which allows the detection and isolation of microorganisms producing the desired metabolite, constitutes primary screening. Ideally, primary screening should be rapid, inexpensive, predictive, specific but effective for a broad range of compounds and applicable on a large scale.

Primary screening is time consuming and labour intensive since a large number of isolates have to be screened to identify a few potential ones. However this is possibly the most critical step since it eliminates the large bulk of unwanted useless isolates, which are either non producers or producers of known compounds.

Computer based databases play an important role by instantaneously providing detailed information about the already known microbial antibiotic compounds.

Rapid and effective screening techniques have been devised for a variety of microbial products, which utilize either a property of the product or that of its biosynthetic pathway for detection of desirable isolates. Some of the screening techniques are relatively simple, e.g., for extracellular enzymes and enzyme inhibitors.

However for most microbial products of high value, the screening is usually complex and tedious, and often may involve two or more steps, e.g., for antimicrobials. In some cases, it may be desirable to concentrate on a group of organisms expected to yield new products.

For example, the search for new antibiotics now focuses on rare Actinomycetes, i.e., Actinomycetes other than those belonging to the genus Streptomyces. Suitably designed specialized screening techniques may be used to detect compounds having various pharmacological activities other than antibiotics.

Strain Improvement - After an organism producing a valuable product is identified, it becomes necessary to increase the product yield from fermentation to minimise production costs. Product yields can be increased by

(i) developing a suitable medium for fermentation,

(ii) refining the fermentation process and

(iii) improving the productivity of the strain.

Generally, major improvements arise from the last approach; therefore, all fermentation enterprises place a considerable emphasis on this activity. The techniques and approaches used to genetically modify strains, to increase the production of the desired product are called strain improvement or strain development.

Strain improvement is based on the following three approaches:

(i) Mutant selection,

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(ii) Recombination, and

(iii) Recombinant DNA technology.

Inoculum Development - The preparation of a population of microorganisms from a dormant stock culture to an active state of growth that is suitable for inoculation in the final production stage is called inoculum development. As a first step in inoculum development, inoculum is taken from a working stock culture to initiate growth in a suitable liquid medium.

Bacterial vegetative cells and spores are suspended, usually, in sterile tap water, which is then added to the broth. In case of nonsporulating fungi and actinomycetes the hyphae are fragmented and then transferred to the broth inoculum development is generally done using flask cultures; flasks of 50 ml to 12 litres may be used and their number can be increased as per need. Where needed, small fermenters may be used.

Inoculum development is usually done in a stepwise sequence to increase the volume to the desired level. At each step, inoculum is used at 0.5-5% of the medium volume; this allows a 20-200-fold increase in inoculum volume at each step. Typically, the inoculum used for production stage is about 5% of the medium volume.

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Media for Industrial Fermentations

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Culture Media - Inoculum preparation media are quite different from production media. These media are designed for rapid microbial growth, and little or no product accumulation will normally occur. Many production processes depend on inducible enzymes.

Basic requirements must be met by any such medium. All microorganisms require water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic. On small scale it is relatively simple to devise a medium containing pure compounds, but the resulting medium, although supporting satisfactory growth, may be unsuitable for use in large scale processes.

For large scale production one must normally use sources of nutrients to create a medium which will meet as many as possible of the following criteria.

1) It will produce the maximum yield of product or biomass per gram of substrate used.

2) It will permit the maximum rate of product formation.

3) There will be minimum yield of undesired products.

4) It will be of consistent quality and be easily available throughout the year.

5) It will cause minimal problems during media making and sterilization.

6) It will cause minimal problems in other aspects of the production process particularly

aeration, agitation, extraction, purification and waste treatment.

The growth medium used for the cultivation of a particular strain of micro organism and the subsequent production of either microbial cells or a biochemical product is called production or fermentation medium. In fermentation industry we have to design a suitable production medium. This is done by trial and error method. The composition of fermentation medium may be simple or complex which depeids on the microorganism and its fermentation. Simple and complex media are further divided into two types “synthetic” and “crude”. The synthetic medium contains all constituents in definite proportions which are required by the organism for its growth and biochemical activities. The synthetic media have certain advantages

1) Since the amount and chemical structure of each component is known, the composition of the medium can be altered according to the need. individual components can either be added or deleted. These considerations allow the designing of the medium to obtain desired end products.

2) The media can be designed either for growth or product yield.

3) Foaming is not a problem as a medium does not contain proteins.

4) The recovery and purification of fermentation product becomes simple.

Regarding disadvantages 1) The media are expensive, because of the cost of the pure ingredients used. 2) The yield derived from these media is relatively low.

Non synthetic or “crude” medium: - The crude medium contains ill defined sources of nutrients, as such they provide an excess of both nutrients and growth factors. The medium does not contain metals, inorganic salts, or organic compounds toxic to the organism or to its product formation. Moreover the crude carbon and nitrogen sources are in the form that the organism can use. For example, the medium should not provide a pentose sugar for an organism that can only use a hexose sugar, protein for an organism that does not have proteolytic enzymes, or starch for an organism that lacks amy1.

Crude media, and to some extent the synthetic media should meet the following requirements in addition to providing nutrients for growth and product formation.

1) Buffering capacity.

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2) Lack of foam formation.

3) Showing the growth of the contaminating organisms.

4) Promotion of vigorous aeration and agitation.

5) Recovery of end products without resorting to complex processes.

6) Withstanding the sterilization without affecting the nutritive constituents.

7) Allowing the growth of organism in its proper morphological state.

8) The media must be economically feasible for particular fermentation. The nutrients used for the medium must be inexpensive.

The crude medium is preferred for industrial fermentation because of the following reasons.

1) Crude medium usually allows much higher yield of fermentation products than synthetic media.

2) Crude media components are inexpensive.

3) For crude medium the by-products of agriculture can be used.

4) These contain large quantity of usable proteins and carbohydrates.

5) Most of the agricultural wastes are renewable and hence no shortage of fermentable substrate.

6) There is no need of addition of inorganic nutrients as the crude medium itself contains them.

7) Crude media are in themselves complex mixture of nutrients supplying carbon and nitrogen compounds as well as microbial growth factors.

8) Crude antifoam agents can be added to control foam without changing the nutritive balance of the medium.

9) The carbon sources are usually used in a crude form.

The crude sources of simple sugars include beat and cane molasses whey, sulfite wastes liquor, cell fruits, cannery wastes, polysaccharides like starches supplied by corn, wheat, rye, rice, potatoes, sweet potatoes and other agricultural products. Crude proteinaceous animal and plant materials contain nitrogen sources of fermentation of medium. Examples of these sources include corn steep liquor, casein, distiller’s solubles, cereal grains, peptones, fish meal stick liquor, meat scorps, soya bean meal and yeast extracts, cotton seed meal, liniseed meal, and peanut oil meal. Cellulosic by products can be used as carbon source such as wood waste; oat hulls, corn cols and straw.

The crude raw materials that can be used in the production medium are divided into different categories, such as saccharine materials, starchy materials, cellulosic materials,

vegetable oils, molasses, cheese whey, sulphite waste liquor, corn steep liquor, cotton liviseed, peanut meal, soy bean meal.

Saccharine Materials: Molasses: - Beet and cane molasses is the by product of the sugar industry. The molasses are the concentrated syrup or mother liquor recovered at any one of several steps in the sugar refining process. There are several types of molasses prepared from sugar cane the cheapest and the most used sugar sources, for industrial fermentations. After removal of sucrose from the sugar cane juice by crystallization, certain amount of liquor containing 52%of sugar is left out which is known as black strap molasses. When it is used for fermentation medium it is considered to contain 50% of fermentable sugars. There is another type of molasses called refinery black strap molasses which is more or less similar to that of black strap molasses. High test or invert molasses are those molasses which contain 70 to 75% of sugars. In this the whole cane juice is partially hydrolyzed to monosaccharides with heat and acid and then concentrated

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without removing sugar. Thus it contains much of the original sugar of the sugarcane juice. Beet molasses resembles the sugarcane molasses. “Hydrol’ is a molasses resulting from the manufacture of crystalline dextrose from corn starch. It contains 60% sugar. The molasses in general contains sugar as a source of carbon and organic acids like aconitic acid, malic, citric, lactic, formic, acetic and propionic acids. The nitrogen containing compounds are mainly amino acids; a few vitamins like niacin, pantothenic acid, riboflavin and biotin. It also contains same salts like Ca, Mg and phosphorous compounds. In India at present black strap molasses of can is utilized in the large scale production of ethyl alcohol, country liquors, and others liquors like rum, brandy, gin and whisky. Fruit juices are also utilized as saccharine material in the industrial production medium.

Starchy Materials: - Polysaccharides such as starches are also utilized for the fermentation medium. Starch from corn, wheat, oat, rye, rice, potatoes and sweet potatoes and other agricultural products serve as source of carbon in the fermentation medium. The starches of the above materials require pretreatment before being used as raw material for fermentation medium. These starches are to be converted into fermentable sugars by enzymes or dilute acids or combination of both.

Cellulosic Materials: - These are complex carbohydrates. These require pretreatment before being used as raw materials for the fermentation.

Sulfite waste liquor: - It is the spent sulfite liquor from the paper- pulping industry. During the manufacture of paper-pulp, wood is subjected to hydrolysis with the help of calcium bisulphite under heat and pressure (digestion process). At the end of the digestion process spent liquid is left and it is referred to as sulfite waste liquor. This liquid is a waste and is to be disposed off with treatment. Regarding its chemical composition, it is a dilute sugar solution having about 2.1 % sugars and can be used as dilute fermentation medium in some fermentation industries. For example, it is chiefly used in the industrial production of ethyl alcohol (using S.cerevisiae). The sulfite waste liquor obtained from the paper pulp industry cannot be used directly in the fermentation industry. It is necessary to remove the free sulfur dioxide or sulfurous acid present in the waste liquor, since these are toxic to microorganisms. They may be removed by steam stripping or precipitation with lime.

Wood Molasses: - Wood waste residues hydrolyzed by acid provide sources similar to that of sulfite waste liquor. This may produce 65% to 85% fermentable sugars. For the production of wood molasses wood waste residues are subjected to sulphuric acid of 0.5% con. at a temperature between 150 to 185°C using continuous process. The syrup so obtained contains 4 to 5% reducing sugars. It is then subjected to concentration to give a kind of wood molasses. Wood molasses prepared from conifers contains about 85% hexoses and 15%pentoses and that of broad leaved plants about 65% hexoses and 35% pentoses. Now it is possible to hydrolyze all the cellulose to glucose. Rice straw and other related agricultural materials can serve as good source of cellulose. Rice straw has been used as a fermentation medium in the production of silage and single cell proteins, mushroom cultivation etc.

Nitrogenous Materials

Corn Steep Liquor (CSL):- Corn steep liquor is that water extract from the steeping of corn during the manufacture of starch, gluten, and other corn products. This spent water is subjected to concentration. Corn steep liquor was used in the commercial production of penicillin. Now it has been widely used in the fungal antibiotic media. It is also used with manufacture of food stuffs.

Soybean Meal: - The material left after dealing of the soybean seeds is called soybean meal. It is a complex nitrogenous source and is used as ingredient for fermentation media.

Parma Media: - It is a finely ground yellow powder prepared from the embryo of cotton seeds. It contains 56% proteins, 24% carbon 5% oil and 5% ash. The ash contains Ca, Fe Cl & Phosphorus.

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Distillers Solubles:- During the manufacture of alcohol using grain or maize, alcohol is distilled from fermented broth, leaving the residues. The solids are eliminated from the residue leaving the effluent. The effluent is subjected to concentration and dried. And it is called “Distillers Solubles”. Other nitrogenous materials used in the production media are groundnut meal, fish meal, peptones, yeast extracts etc.

In all such cases, the appropriate inducers must be included either in all the stages or at least in the final stages of inoculum development. This will ensure the presence of the concerned inducible enzymes at high levels for the production to start immediately after inoculation.

Contamination - The inoculum used for production tanks must be contamination free. But the risk of contamination is always present during inoculum development. Therefore, every effort must be made to detect as well as prevent contamination.

Sterilization - Sterilization is the process of inactivating or removing all living organisms from a substance or surface. In concept, it is regarded as absolute in that all living cells must be inactivated / removed, usually in a single step at the given time. But in practice, the success of sterilization procedures is only a probability. Therefore, the probability of a cell escaping inactivation/filtration does exist although it is usually very small.

When a closed system is sterilized once, it remains so indefinitely since it has no openings for the entry of microorganisms. But most fermentation vessels are open systems; such systems are initially sterilized and must be kept sterilized by ensuring the removal of living cells at their entry points, e.g., the cotton plug of a culture flask.

Common Contaminants - The most common contaminants of different industrial processes are considerably different. Some examples are given below.

1. In canning industry, Clostridium butyricum is the chief concern. This obligate anaerobe can grow in sealed cans, and produce heat resistant spores and a deadly toxin. However, it is not a problem for catsup (too acidic), jam and jellies (too high sugar concentration) and milk (stored at low temperature).

2. Organisms like lactobacillus are a problem in production of wine.

3. In antibiotic industry, potential contaminants are many, e.g., molds, yeast, and many bacteria, including Bacillus.

4. The most dreaded contaminants of fermentation industry are phages. The only effective protection against phages is to develop resistant strains.

Sterilization Procedures - Sterilization involves either inactivation or removal of living organisms. This may be achieved by

(i) Heating,

(ii) Irradiation.

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(iii) Chemicals or

(iv) Filtration; these are briefly discussed below.

Heating. It is the most commonly used and the least expensive sterilizing agent. Dry heat is used in ovens and is suitable for sterilization of solids, which can withstand the high temperatures needed for sterilization, e.g., laboratory glassware, talc, etc. Steam, i.e., moist or wet heat, is used for sterilization of media and fermenter vessels. An autoclave uses steam for sterilization (at 121°C and 15p.s.i.). the period of time at this temperaturepressure depending on medium volume, e.g., 12-15 min for 200 mi. 17-22 min for 500 ml, 20-25 min for 1 I and 30-35 min for 2 l. But sterilization of oils will require a few hours, and concentrated media (10-20% solid) must be agitated for effective sterilization.

Autoclaves can also be used to sterilize laboratory vessels, small volumes of media and even small fermenters. Large fermenters are sterilized by either a direct injection of steam or by indirect heating by passing steam through heat exchange coils or a jacket. The steam should always be saturated. Media sterilization may be achieved in a continuous flow sterilization system either by direct steam injection or by indirect steam heating, and then filled in a sterile fermenter. Alternatively, the medium may be filled in the fermenter and steam-sterilized with the latter. Heat killing in most part is due to protein inactivation. In general, moist heat is far superior to dry heat. Bacterial spores are the most heat resistant, e.g., spores of thermophilic bacteria can survive steam at 30p.s.i. at 134°C for 1-10 min and dry heat at 180°C for up to 15 min.

Radiation. High energy X-rays are used for sterilization of a variety of labware and of food. In general, vegetative cells are much more susceptible than bacterial spores (Clostridium spores can resist nearly 0.5 M rad). But Deinococcus radiodurans vegetative cells can survive 6 M rad.

Viruses are usually similar to bacterial spores but some viruses, e.g., encephalitis virus require up to 4.5 M rad. In practice, 2.5 M rad is used for sterilizing pharmaceutical and medical products. X-rays cause inactivation by inducing single and double strand DNA breaks, and by producing free radicals and peroxides, to which -SH enzymes are particularly susceptible.

Chemicals. The chemicals used for sterilization cause inactivation by oxidation or alkylation; these are formaldehyde, H2O2, ethylene oxide, propylene oxide etc. H2O2 (10-25% w/v) is being increasingly used in the sterilization of milk and of containers for food products.

It is a powerful oxidizing agent, kills both vegetative cells and spores and is very safe. Ethylene oxide is used for sterilizing equipments, which are likely to be damaged by heat, and is very effective, but highly toxic and violently explosive if mixed with air.

Filtration. Aerobic fermentation requires a very high rate of air supply often equaling 1 vol of air (equal to medium volume) every minute. Air contains both fungal spores and bacteria, which are ordinarily removed by filtration using either a depth filter or a screen filter.

Depth filters are made from fibrous or powdered materials pressed or bonded together in a relatively thick layer; the materials used are fiberglass, cotton, mineral wool, cellulose fibers, etc. in form of mats, wads or cylinders.

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Modem depth filters are cylinders of bonded borosilicate microfibers. Depth filters allow higher filtration rates and efficiencies than screen filters, but are not suitable for filtration of moist air.

Screen filters are membranes of cellulose esters or other polymers with pores of 0.45 µm or smaller (bacterial contaminants are 0.5 µm or larger). Usually, a microfibers profiler is used with such filters to remove gross contamination.

All filters themselves must be sterilized before they can be used to sterilize the air. Filters are also used to sterilize the effluent gases from fermenters, especially in case of pathogenic microorganisms.

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Stock Cultures

Stock Cultures: It is extremely important to maintain microorganisms for extended periods in viable conditions, and in situation which did not alter their desired product formation capacity. This condition is also true for strains used in biological assays. Thus, microbial species procured from various culture collection centers are maintained in viable conditions and known as stock-culture collection. The stock culture generally retain all the characteristics initially described. Stock culture collection centres have been established through out the world to help microbiologists in obtaining cultures for various studies. These centres also help in classifying a newly isolated organism. There are two types of stock cultures: working stocks and primary stocks.

(a) Working Stocks: These stocks are used frequently and they must be maintained in vigorous and uncontaminated conditions on agar slants, agar stabs, spore preparations, or broth culture, and one held under refrigeration. They must be checked constantly for possible changes in growth characteristics, nutrition, productive capacity and contamination.

(b) Primary Stocks: The cultures that are held in reserve for presently practical or new fermentation for comparative purposes, for biological assays, or for possible later screening programmes. These are not maintained in a state of physiological activity. Transfers from these cultures are made only when a new working stock culture is required, or when the primary stock culture is sub cultured to avoid death of the cells. Thus, primary stock cultures are stored in such a manner as to require the least possible numbers of transfers over a period of time. Further, these are stored at room temperature, are maintained in sterile soil, or in agar or broth overlayed with sterile mineral oil.

Agar and broth culture without mineral oil also are refrigerated. The culture in milk of agar are maintained frozen at low temperature. Finally, primary stock cultures are lyophilized or frozen-dried, and stored at low temperature. The culture of Blakeslea trispora used in 13 - carotene production, cannot be stored at refrigeration temperature because they die relatively quickly. However, at room temperature transfers are being made to fresh medium when the cultures become nearly dried out.

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Culture Preservation

Methods of Preservations

A number of methods are used for maintaining organisms in a viable condition over a long period of time. Different microbes behave differentially using a specific condition of growth. Therefore, a method useful for one species may not be applicable -to another. Some of the methods are as given below:

(a) Agar Slant culture: Agar slants are prepared in vitro. After inoculation slants are incubated for a period of 24h and then stored in a refrigerator. These cultures require periodic transfer after six months.

(b) Agar slant culture covered with oil: The agar slants are incubated after inoculation until profuse growth appears. These are then covered with sterile mineral oil to a depth of 1 cm above the tip of the slanted surface. Transfers are made by removing a loopful of growth, touching the loop to the glass surface to drain off excess oil in the medium and then preserving the initial block culture.

(c) Saline suspension: High concentration of sodium chloride is used as inhibitor of bacterial growth. Bacteria are suspended in 1% salt solution in screw cap tubes to prevent evaporation. The tubes are stored at room temperature and transfers are made on agar slants.

(d) Preservation at very low temperature: The organisms are suspended in a nutrient broth containing 15% glycerol, or in skimmed milk containing 7.5% glucose. The suspensions are frozen and stored at - 15°C to -30°C. The ready availability of liquid nitrogen (- 196°C) has provided another means of preservation of stock

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cultures. In this procedure, the cultures are frozen with a protective agent (glycerol or dimethyl sulfoxide) in sealed ampules. The frozen cultures are kept in liquid nitrogen flask.

(e) Preservation by drying in vacuum: The organisms are dried over CaCl in a vacuum, then stored in the refrigerator. The organism survives longer than when air dried.

(f) Lyophilization or freeze drying: The microbial suspension is placed in small vials.

A thin film is frozen over the inside surface of the vial by rotating it in a mixture of dry ice

or alcohol or acetone at a temperature of —78°C. The vials are connected to a high vacuum line. This dries the organism while still frozen. Finally, the ampules are sealed off in a vacuum with a small flame. These cultures can then be stored for several years at 4°C. To revive microbial cultures, it is merely necessary to break up the vial aseptically to which suitable sterile medium is added. After incubation, growth appears which allows them for further transfer. The process permits the maintenance of a large number of cultures without variations in the characteristics of the cultures which generally reduces the danger of contamination.

Stock Culture Collection Centres

(a) American Type Culture Collection, 12301, Parkiawn Drive,

Rockville, Maryland, USA.

(b) Indian Collection of Industrial Microorganisms, National

Chemical Laboratory, Pune.

(c) Institute of Pasteur, Paris (France).

(d) Institute of Microbial Technology, Sector 39-A, Chandigarh.

(e) National Collection of Type cultures, Central Public Health Laboratory, Colinadate, Avenue, London.

(f) Microbiological Type Culture Collection, 4-54, Juso Nishinocho, Higashiyodogowaku, Osaka, Japan.

(g) Commonwealth Mycological Institute, Ferry Lane, Kew, Furrey, England.

(h) Centre de collections de types Microbeins, 19, Rue Cesar-Roux, Lausane, Switzerland.

(i) Central Bureau Voor Schimmel cultures, Javaloan, 20, Baarn, Neetherlands.

(j) National collection of Industrial Bacteria, Department of Scientific and Industrial Research, Tony Research Station, P0 BOX 31, 135, Abbey Road, Aberdeen, Scotland.

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Methods of Culture Maintenance

There are three methods for culture maintenance which seem to be generally used in the fermentation industries: (a) drying organisms on soil or some other solid, (b) storing organisms on agar slants, and (c) removing the water from the cells or spores by lyophilization and storage of dried product.

(i) Preservation of culture by drying. These are different methods of drying of cultures given low:

(a) Dried on Silica Gel: The higher survival rate was noticed at 4°C in comparison to storage room temperature.

(b) Dried on soil: About 50% of the total cells remain viable after 20 years of storage. It is : that 92-96% cells remain viable after 4 years.

2. Maintenance of Cultures by Storage with Limited Metabolic Activity.

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(a) Storage on Agar Slants: Recent studies have suggested that storage under oil for 10 months lid not change carbohydrate assimilation pattern for Mucor racemosus, Cunnighamella echinuata, Penicillium cyclopium and Aspergillus niger.

(b) Storage of spores in water: Long term viability has been noted when spores of various fungi suspended in sterile distilled water and stored in a refrigerator. Similar success has been obtained with the bioassay organisms such as Saccharomyces cerevisiae and Sarcinia lutea suspended in weak buffer and stored in a refrigerator for more than a year.

(c) Storage at frozen temperature: Yamasato et al (1973) studied the viability of 259 strains belonging to 32 genera suspended in 10% glycerol and stored at -53°C for 16 months. About 10% of the Gram - positive bacteria and 3% of the Gram-negative bacteria lost viability quickly. Honey was suggested as a better adjuvant for frozen storage than glycerol.

Preservation by storage of cells or spore suspension in liquid nitrogen has been widely used since the initial advantages described by Sokolski et. al (1984). Daily and Higgens (1973) reported the inclusion of 10% glycerol with 5% of lactose, maltose, or raffmose in the suspending solution increased the viability of spores, vegetative cells and Streptomycete mycelial fragments.

(iii) Preservation by Lyophilization: As stated earlier, in addition to that the sterile glass ampoules are suspended in a carrier or protective agent such as sterile bovine serum or skim milk, apid1y frozen at low temperature, and dried in a high vacuum, the ampules are then sealed and stored in a refrigerator. .If properly prepared and stored, most lyophilized cultures will remain viable for more than 10 years.

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