Vitamins Production by Fermentations - Microbial production is the only source of vitamin Bl2 whilst of an the other water-soluble vitamins now available commercially only riboflavin (vitamin B2) is manufactured to any significant extent, microbiologically.

There are reports that ascorbic acid is also produced by microbial fermentation but the details are not available. None of the fat soluble vitamins is produced industrially by microbiological methods but one compound, β-carotene which is converted by animals to vitamin A, can be prepared by microbial synthesis.

Processes for Microbial Synthesis of Vitamin B12 - It seems probable that the only primary source of vitamin Bu in nature is the metabolic activity of the microorganisms. It is synthesized by a wide range of bacteria and Streptomycetes, though not to any extent by yeasts and fungi. While over 100 fermentation processes have been described for the production of vitamin Bl only half a dozen have apparently been used on a commercial scale.

Production of Vitamin B12 (Cyanocobalamine)

The steps involved in production of Vitamin B12 are:

a) Selection of microorganisms

b) Inoculum preparation

c) Fermenting media

d) pH

e) Temperature and fermentation process

f) Aeration and agitation

g) Addition of Antifoaming agents

h) Recovery

a) Selection of microorganisms:- The organisms involved in vitamin B production are Sterptomyces griseus, Bacillus megaterium, B. coagulance, Pseudornonas dentrificans, Propionibacterium sherman/i and a Pseudomonas spp. Normally, the vitamin B is produced on large scale by using submerged fermentation method. Fermentation is usually completed within 3 to 5 days. B

b) Inoculum preparation:- For every commercial fermentation the preparation of inoculum is essential prior to the addition of the culture into the fermenting medium. Here also one can prepare the stock culture from the selected strains. The pure culture of Streptomyces olivaceus is inoculated and grown in 100-250 ml of inoculum medium (usually Bennet’s agar medium is used) contained in flasks. Thus seeded flasks are to be kept on mechanical shakers during incubation for proper aeration. After the growth these flasks are utilized for inoculating into fermenting medium. Usually 50% of the inoculum is sufficient for the initiation of fermentation.

C) Fermenting medium:- The fermenting medium for vitamin B12 usually consists of carbohydrates, proteins, a source of cobalt and other salts. In the composition, cobalt plays very important role in giving high yields of vitamin B12 . The composition of vitamins B12 fermentation medium is as follows:

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Composition of Vitamin B12 medium:

Distillers solubles :4.0%

(Soya meal, bean meal)

Dextrose: 5.0-10.0%

CaCO3 : 0.5%

C0Cl3.6H20: 1.5tolOp.p.m.

pH 7.0

After sterilization of the medium in the fermentor, the fermentation medium is ready for inoculation with the seed lot cultures to initiate the production of vitamin 812.

d) pH:- Once the fermentation is started there is rapid consumption of sugar in the first 24 hours. After 2 to 4 days, lysis of mycelium begins resulting in the rise of pH. The stabilization of the mash is practiced by reducing the pH to about 5 with sulphuric acid and adding small amounts of reducing agent.

e) Temperature and fermentation process:- A temperature of 27°C is satisfactory during fermentation in the production flasks. The duration of the fermentation is about 3 to 4 days, or until mycelium lysis begins to occur. Thus, most of the vitamin 812 remains within microbial cells until autolysis sets in, and therefore the recovery of the vitamin from the fermentation broth is simplified by harvesting before autolysis has become serious.

f) Aeration and Agitation:- The growth of the Streptomyces strain mainly depends upon the rate of aeration and the speed of agitation. Aeration rates higher than the optimum results in foaming. The optimum rate of aeration is about 0.5 volume air/volume medium/minute.

g) Addition of antifoaming agents:- In this fermentation the formation of foaming is a very serious problem. There are several antifoaming agents available which suppress foam formation. They include: soyabean oil, corn oil, lard oil, and silicones.

h) Recovery:- As we know already, during the fermentation period, most of the vitamin B is present in the mycelium. It is released into the medium at the end of the fermentation period. But, a large amount of vitamin B is still present in the mycelia. Heating the mixture to boiling at pH5 or below releases the vitamin B from the mycelium.

The mycelium with other solids is filtered or centrifuged. Then the filtered broth is treated with cyanide to bring about the conversion of cobalamin to cyanocobalamin. The adsorption of cyanocobalamin from the solution is practiced by passing it through an adsorbing agent packed in columns. Adsorbing agents such as charcoal, bentonite, fuller’s earth and ion exchange resins are used. After removal of cyanocobalamin from the adsorbent, it is treated with water-acetone to form an organic solvent of vitamin B then it is concentrated and crystallized.

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Production of Riboflavin (Vitamin B2)

(a) Media preparation and biosynthesis of Riboflavin.

For Riboflavin production, basic medium consists of corn steep liquor 2.25%, commercial peptone 3.5%, soybean oil 4.5% but it can be supplemented further by addition of different peptones, glycine, distiller’s solubles, or yeast extract. The glucose and inositol increase the production of riboflavin.

The medium should be kept at 26-28°C at pH 6.8 for 4-5 days incubation. After inoculation the submerged growth of Ashbya gossypi is supported by insufficient air supply. The excess air inhibits mycelial production and reduces the riboflavin yield. The fermentation progresses through three phases.

(b) First phase: In this phase, rapid growth occurs with small quantity of riboflavin production. The utilization of glucose occurs resulting into decrease in pH due to accumulation of pyruvate. By the end of this phase, the glucose is exhausted and growth ceases.

(c) Second phase: Sporulation occurs in this phase. The pyruvate decreases in concentration. Ammonia accumulates because of an increase in deaminase activity. The pH reaches towards alkalinity.

(d) Third phase: There is a rapid synthesis of cell-bound riboflavin (FMN and FAD). This phase is accompanied by rapid increase in catalase activity subsequently cytochromes disappear.

As the fermentation completes, the autolysis takes place which releases free riboflavin into the medium as well as retained in the nucleotide form. It is also observed that certain purines also stimulate riboflavin production without simultaneous growth stimulation.

The riboflavin is present both in solution and bound to the mycelium in the fermentation broth. The bound vitamin is released from the cells by heat treatment (lh, 120°C) and the mycelium is separated and discarded. The riboflavin is then further purified. The crystalline riboflavin preparation of high purity have been produced using Saccharomyces fermentation with acetate as sole C source.

Uses: It is essential for the growth and reproduction of both humans and animals and, thus, it often is recommended as a feed additive for the animal nutrition. The riboflavin deficiency in rats causes stunted growth, dermatitis, and eye damage. Ariboflavinosis is a disease in humans caused by riboflavin deficiency.

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Production of Penicillin

Penicillium species required the production medium which contains lactose (1%), Calcium carbonate (1%), corn steep liquor 8.5%, glucose (1%), sodium hydrogen phosphate (0.4%) phenyl acetic acid (O.5g). The pH is kept between 5 and 6 and temperature for incubation is 23-25°C. Aeration and agitation are necessary.

Fermentation: Penicillin is produced by Penicillum chrysogenum Q-176, a fungus that can be grown in stirred fermenters. The inoculum under aerobic condition (seed) can be produced when there is glucose in sufficient amount in the medium. If particular penicillin is produced specific precursor (substance added prior or simultaneously with the fermentation which

incorporated without any major change in the molecules) is added in the medium for e.g. phenyl acetic acid or its derivatives such as ethanol amide to get penicillin G. The antifoam agents such as vegetable oil (corn or soybean oil) is added to the medium before sterilization.

The spore suspension is inoculated in flasks, each containing 15 g barley seeds. These flask are vacuum dried, to which sterilized quartz is added.

The preparation of inoculum takes place on barley seeds. The flask containing 15 g barks seeds are to be mixed with mother culture, and incubated at 25°C for 7 days. The spores develops on barley seeds are suspended in distilled water to make spore suspension. After testing the antibioc activity, the seeds containing flasks are ready for seeding in fermenter. Three phases of growth can be differentiated during cultivation of Penicillium chrysogenum.

(a) First phase. In this phase, growth of mycelium occurs; yield of antibiotic is quite lo Lactic acid present in corn steep liquor is utilized at a maximum rate by the microorganism. Lactose is used slowly. Ammonia is liberated into the medium resulting into rise in pH.

(b) Second phase. There was intense synthesis of penicillin in this phase, due to rapid consumption of lactose and the ammonium nitrogen (NH3 N). The mycelial mass increases; the pH remains unchanged.

(c) Third phase. The concentration of antibiotic decreases in the medium. The autolysis of mycelium starts liberation of ammonia and slight rise in pH.

Recovery: When the fermentation cycle (7 days) is completed, the whole batch is harvest for recovery. Its activity disappears on evaporation to dryness, hydrolyzed to penicilloic acid. Penicillin has tendency that it remains in aqueous phase at normal pH and in solvent phase at acidic pH. This property of penicillin is used in recovery of potassium penicillin from natural solution. Once the fermentation is completed the broth is separated from fungal mycelium and processed by absorption, precipitation and crystallization to yield the final product. This basic product can then be modified by chemical procedures to yield a variety of semi synthetic penicillins such as ampicillin, amoxycillin, etc.

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5

ALCOHOL FERMENTATION

BEER

Beer is made of malt, as well as other sources of starch such as rice and corn, water, hops and yeast as the fermentation agent. The purpose of the brewing process is the conversion of the starch contributed by the malt and the adjuncts corn or rice to maltose and dextrins. This conversion is accomplished by the enzymes present in the malt. The solution of carbohydrates obtained (called wort) is then boiled with hops cooled and fermented to beer containing alcohol, carbon dioxide, and residual dextrins. The finished beer has as major constituents water, dextrins, alcohol and carbon dioxide and as minor though important constituents, unfermented sugars, proteins and aromatics such as hops-resins etc.

Beer production:- The ingredients used to produce beer are malt, adjuncts, water, hops, yeast and a few other additions.

Malt and Malting:- Malt is prepared from barley, wheat, oat and it is cleaned of dust and other foreign materials. The actual process of malting barley is carried out in malt house. Malt contains the enzymes which degrade the starch of the malt itself and additional starchy adjuncts, such as corn starch and rice starch, to dextrins and maltose. The dextrins remain to give body to the beer, and the maltose is fermented to the alcohol and carbon dioxide. The malt is also the source of the beer proteins which are important for foam and of the flavor typical of the beer.

For processing into malt, the barley is cleaned and soaked in water so that water is then drained off and sprouting continued for 5 to 7 days, until the desired growth of the embryo has been obtained. During this time, the malt has to be turned and is thus aerated frequently to prevent local overheating and achieve uniformly controlled growth. At the end of the period necessary for optimum growth (measured by the length of the growth of the germ or acrospore and the hardness of the endosperms), the sprouts are killed by killing the malt, duration and killing steps are very important for colour, aroma, and level of enzymes. After killing, the sprouts and rootlets are removed and the malt is aged for several weeks. It is then ready for use in the brewery.

During malting, certain enzymes are increased and others are newly formed: -amylase is increased, and ct-amylase is newly formed. a- amylase is important for attack on the starch (Iiquefaction), and - amylase for final sugar formation. This conversion starts in the malting process, increasing the proportion of lower carbohydrates, and it is even more important for production of the work during mashing. The overall composition of the malt is as follows:

Starch: 59%

Sugars: 10%

Gums: 10%

Cellulose: 5%

Protein: 10%

Fat: 2.5%

Ash: 2%

Brewing process:- The malt is transferred to the brewery for grinding and other raw materials used for brewing include adjuncts (rice or corn) as additional sources of starch and hops.

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The hops are plant belonging to the family Cannabidaceae. This is a climbing herbaceous dioecious plant. Only pistillate flowers which are cone like are used in brewing. Staminate flowers are destroyed to make the pistillate flowers seedless. Chemically hops contain 50% of hydrocarbons and the remainder are esters, alcohols and carbonyl compounds. Other than this, it contains hop-resin and hop-oil. The hop resins are contained in small yellow aggregates below the petals. They are the major flavor contributors, being responsible for the characteristic bitterness in beer. These resins also have preservative properties. Hops also contains hop-oil responsible for the strength of the beer. Brewers usually use hop flowers (many people mistake them as leaves because of their green colour) directly and some may use hop extracts.

The other raw material of importance, often not thought of as such, is water. Water should have the right hardness and calcium content for enzyme activity and must not have off flavours. The mineral content of the water influences the final beer flavour.

After getting the malt from the malt house, the ground malt is mixed with water to let the amylases convert the molecules. The brewers use mash tub for holding the mash for certain period of time at a selected temperature to assure maximum conversion. Meanwhile, the adjuncts are boiled in the cooker to achieve gelatinization. The pregelatinized adjuncts are added to the main bulk of the brew in the mash tub where they go through final stages of conversion until no free starch remains and the desired ratio of dextrin and maltose is obtained. The total mash consisting of adjuncts and malt mash, including the barley husks is heated at about 72°C, when enzyme activity ceases. It is then passed through the lauder tub or mash filter. During filtration, the spent grain solids and the insoluble barley husks are removed. This filtered solution, now is called wort. The wort is then boiled with hops in the brew kettle for 1.5 hour. This solubilizes the valuable hop constituents, while undesirable proteins and tanning are coagulated and at the same time the wort is sterilized.

From the brew kettle the wort passes via the hop strainer, where the insoluble part of the hops (about 70 percent of the originat hop dry weight) is removed, to the hot wort settling tank. After air cooling, the brew is

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now moved to the fermentors. The yeast is added (Saccharomyces cerevisiae var ellipsoides) and the wort is fermented to beer. Fermentation takes place at a low temperature (6-12°C) for a period of 8 to 10 days. At the end of the fermentation the yeast settles and the beer is removed and stored in tanks for further clarification, aging and carbonation. Keg beer is sold unpasteurized in the package of sterile filtered prior to filling. The final beer (lager beer contains:

Alcohol: 3.8%

Dextrins: 4.3%

Proteins: 0.3%

Ash: 0.3%

CO2 : 0.4%

It also contains an appreciable amount of vitamins such as riboflavin.

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WINE

Wine is produced by the normal alcoholic fermentation of juices of ripe grapes. It can also be produced by direct fermentation of sugars(Glucose or Fructose). Wine is also produced from peaches, oranges, cherries, etc. Accordingly they are called peach wine, orange wine, cherry wine etc.

Fermentation practices differ from winery to winery and also with the type of the wine to be produced. But general procedure is more or less the same. Wines are endless in their varieties. Based on the colour two basic types are recognized mainly red wine and white wine. In making red wine the grapes are crushed and steamed and the skins and seeds are left in the must. White wine is made from white grapes or the grapes in which the skins and seeds are removed. Dry wines are those which contain more sugars. Sparkling wines are those which contain CO2. It is made by secondary fermentation in closed containers. Still wines are those which do not contain CO2. Fortified wines contain added alcohol in the form of Brandy.

WHITE WINE

The quality of wine depends on the type of the grapes, its maturity and health. The grapes are first separated from the stems and crushed. The crushed grapes provide the must. Rapid processing is to be carried out to inhibit the growth of undesirable microorganisms especially the acetic acid bacteria. Besides this oxidative, enzyme catalyzed processors occur on prolonged standing of the must which may affect the colour and the quality of wine. But certain rest period is required to facilitate the flow of free run juice. The must should be treated immediately with SO in order to inhibit oxidative enzyme action and also to prevent growth of undesirable microorganisms. Sometimes must is directly sulfited. Sulfating is necessary to prevent bacterial fermentation of the malic acid of the must. Next step is to make must free from particulate matter by centrifugation, filtration or sedimentation which occurs on standing. Removal of sediment reduces the susceptibility of the must to oxidation and improve the colour and aroma of the wine. Alternatively oxidation of enzymes can also be stopped by treating the must at high temperatures for short time (85°C). This also kills the microorganisms. In this case inoculation with fermenting yeast is absolutely essential. But even if the must is not heated it is desirable to inoculate the must with pure culture of yeast. If the fermentation is brought about in large fermentors increase of temperature becomes a problem. If the temperature goes above 38°C the fermentation stops because of inactivation and loss of viable yeasts. Besides these fermentation brought about at higher temperatures leads to loss of aroma and quality of wine. Therefore temperature should not exceed 20°C.

At the end of the fermentation the wine is removed from the fermented broth (lees) as soon as possible otherwise the yeast cells begin to autolize. Autolyses leads to the malolactic fermentation. After separation of wine from broth the wine is clarified, stabilized then bottled after a storage period which generally varies from 3 to 9 months but may be extended upto 2 years if high quality of wine is required. During the storage some chemical reactions such as oxidation and esterification take place. The ethanol content of the wine is between 8 to 15% by Volume.

Must Composition

CarbonI 5-25%

Acids (Taxtoric & Malic) 0.9% to 1.5%

Tannins: 0.02-0.04%

Pectins : 0.12-0.15%

Ash: 0.3%

Aroma: Traces,

Nitrogen: Traces,

Higher alcohol: Traces,

Aldehydes: Traces.

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Temperature: - Temperature affects yeast, and consequently the course of wine fermentation. The maximum temperature for S.cervisiae is near 40°C - 45°C and minimum temperature is 0°C. Within the 15°C to 35°C range higher the temperature faster is the fermentation. The outdoor temperature in various wine production areas differs greatly so that it is sometimes necessary to cool the must so that its temperature does not exceed 35°C during fermentation. In northern countries where the temperature is very low the must is sometimes heated. For the production of white wine temperature between 18°C and 20°C is recommended in Germany whereas in California temperatures between 10°C - 16°C is recommended. For red wine temperature between 22°C and 30°C is recommended.

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Sugar concentration: - Some musts have higher sugar concentrations. For example must made from some berries such must have higher osmotic pressures which has negative effect on yeast cells because both growth and fermentation is lowered. At higher sugar levels alcohol formation decreases as shown below.

Sugar concentration of must mg/per litre Ethanol concentration in two months

370 8.6

420 6.3

470 5.9

550 3.4

750 0

Another disagreeable side effect of higher concentration of sugars in must is the increase formation of acetic acid by yeasts.

Carbon dioxide: - At relatively low CO2 concentration of 0.6 to 1.8 g.per litre, the growth c’ yeast is inhibited. The effect of CO2 concentration on yeast depends upon pH also. Inhibition increased at lower pH values.

Sulphur dioxide: - The biological effect of SO2 is that it inhibits the activity of undesirable microorganisms of the must which affect the course of fermentation and quality of the wine negatively. This group of microorganism includes the acetic acid bacteria, lactic acid bacteria as well as some yeasts supplies naturally present in the must. The chemical effect of SO2 is defined to bind acetaldehyde which is formed during fermentation and which has undesirable properties. The use of SO2 is of great importance in fighting acetic acid bacteria, for musts which are low in total acidity it is often desirable to use SO 2 to inhibit malo-lactic fermentation by lactic acid bacteria to prevent further reduction of the acid level. Finally it is required to support certain yeast species to retain good wine quality.

Yeasts: - The grape must carries with it the yeast flora which is required for the alcoholic fermentation. In many wineries in USA, Canada, South Africa & Australia pure yeast cultures are used commercially. However in majority of countries the spontaneous fermentation by naturally occurring yeasts is largely practiced. Grape berries are the habitat of various yeast species while grape juice which is obtained aseptically from berries contains yeast cells numbering about 1O5 to 106 cells per ml. After crushing the freshly pressed must contains 1O5 to 106 yeast cell per ml. The number of Saccharomyces species found in fermentation of grape musts is very large; S. cerevisae comprises the major portion in all wine production areas.

Use of pure culture yeasts:- The wine industry has also introduced the use of pure cultures from specially selected and grown musts in which the natural flora has been killed by pasteurisization. These are many advantages if pure cultures are used for fermentations. The wine of pure cultures of yeasts can be carried out with yeasts in several forms.

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Vinegar Production

There are mainly two steps in vinegar production. In the first stage yeast converts sugar into ethanol anaerobically while in the second step ethanol is oxidized to acetic acid aerobically by Acetobacter and Gluconobacter. This process is called acetification.

Vinegar is an alcoholic fermentation product and if enough acetic acid is present, it is a legal vinegar. The Saccharomyces cerevisae var. ellipsoideus carried out the conversion of sugar into ethanol in the first step, while the species of Acetobacter or Gluconobacter convert ethanol into acetic acid.

Substrate: Vinegar is produced by using fruit juices, starchy vegetables, malted cereals such as barely, rice, wheat, corn, sugar cane syrups, molasses, honey, alcohol etc.

From the fruit juice such as apple juice, the vinegar produced is known as cider vinegar.

If it is produced by using Ale then it is called Alegar. While malted grain made malt vinegar and ethanol produced sprit vinegar.

Method: Mainly there are two methods: (a) slow or “let home” process; (b) quick or “French orleans” process.

(a) Slow process: In the slow process, the alcoholic liquid (substrate) is not allowed to move during acetification and fermented juice or malt liquors are used for acetic acid production.

Apple juice is specially used for alcoholic production by batch fermentation process. The barrel is partially filled with the fermented juice and it is allowed to undergo the process of acetification. The vinegar bacteria “food” is supplemented from the previous batch of vinegar. A film of vinegar bacteria called “mother of vinegar” should grow on the surface of the liquid which indicates that ethanol is oxidised into acetic acid and formed vinegar.

Demerits of this process are the absence of productive strain, and poor yield of ethanol gives inferior quality of vinegar.

(b) Quick process: In this process about 1/4 barrel is filled with raw vinegar from previous run supplemented with active vinegar bacteria. This is to be acetified so as to check competing microbes. Now the barrel is filled by alcoholic hydrolysate up to half filling and keep watch, examine if bacteria are growing in a film on the top of the liquid. This process generally takes weeks to months at 21 to 29°C. The recovery is made in part by withdrawing and replacing by equal quantity of alcoholic liquor. This is continuous fermentation.

Major difficulty in this process is dropping of the gelatinous film of vinegar bacteria which results in retardation of the acetification.

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To avoid this a raft or floating frame work is provided to support the filter. Another important process is called quick generator process.

(C) Generator process: This involves the movement of ethanol liquid during acetification. This ethanol liquid is trickled over the film of vinegar bacteria. A simple generator or cylindrical tank made up of wood is used.

In starting a new generator, the slime of vinegar bacteria must be established before vinegar is produced. Hence, in the beginning the middle section of the tank is filled with raw vinegar that contained active vinegar bacteria. To inoculate the shaving with the desired bacteria an alcoholic liquid acetified with vinegar is slowly trickled through the generator to build up bacterial growth on the shaving and then is recirculated.

The fringe generator is also recommended for vinegar production which contains a large cylindrical airtight tank equipped with a sprinkler at the top. The cooling coils are fitted in the lower part of the middle section. The recirculation of the vinegar is done from the bottom collection chamber. The generator gives high yield of acetic acid and leaves little residue i.e. alcohol.

(d) Makin’s process: A fine mist of a mixture of vinegar bacteria and nutrient alcoholic solution is sprayed through jet nozzles into a chamber. The mist is kept in circulation by filtered air for a while and is allowed to fall to the bottom for circulation, collection, etc.

(e) Submerged process: A stirred medium containing 8-12% of ethanol is inoculated with Acetobacter acetigenum and is held at 24-29°C. The bacteria grow in a suspension of fine air bubbles fermenting liquid.

(f) Finishing: It is the slow process, the vinegar produced is less harsh while in quick process improved vinegar is produced as far as its taste, and body and flavor are concerned. The unit of vinegar is in grams. While it is commercially and internationally defined in terms of grain which is 10 times that of gram.

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Defects: If it is (storage vessel) of steel or iron the ferric ion is oxidised into ferrous ion. The tannin, phosphate or proteins are formed which snake the vinegar hazy in appearance. If the barrel is of tin or copper, cloudiness develops which results into darkening of vinegar.

The animal pests such as mites, fruit flies, vinegar eel (Anguillula aceti) attack the film of acetic acid bacteria which cause it to sink and deteriorate vinegar. Some times Leuconostoc, Lactobacillus contaminate the vinegar imparting off flavor.

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Citric Acid Production

(i) Fermentation: Aspergillus niger has been the choice for the production of this primary metabolite citric acid for several decades. A large number of other microorganisms (fungi and yeast) such as Aspergillus clavatus, A. wentii, Penicillium luteum, P. citrinum, Mucor pyriforms, Candida lipolytica, C. oleophila, C. guillermondis, Hensenula spp. Torulopsis spp., Pichia spp., Debaromyces daussenii etc. have also been used for citric acid production in industries. The advantages of using yeast, rather than A. niger are the possibility of using very high initial sugar concentrations together with a much faster fermentations. This combination gives a high productivity run to which must be added the reported insensitivity of the fermentation to variations in the heavy metal content of the crude carbohydrates.

Yeasts are used for citric acid production using carbohydrate and n alkanes. In all the processes, a variety of carbohydrates such as beet molasses, cane molasses, sucrose, commercial glucose, starch hydrolysate etc. used in fermentation medium. The starchy raw material is diluted to obtain 20-25% sugar concentration and mixed with a nitrogen source (ammonium salts or urea) and other salts. The pH of the medium is kept around 5 when molasses is used and at pH 3 when sucrose used. The fermentation is carried out by any of the processes:

(a) Koji process or solid state fermentation. It is a Japanese process in which special strains of Aspergillus niger are used with the solid substrate such as sweet potato starch.

(b) Liquid surface culture process. In this case, A. niger floats on the surface of a solution.

(c) Submerged fermentation process: It is the process in which the fungal mycelium grows throughout a solution in a deep tank’

(a) Koji process: Mold is used in the preparation called Koji to which wheat bran was substituted in the sweet potato material. The pH of the bran is adjusted between 4 and 5, and additional moisture is picked up during steaming so as to get the water content of the mash around 70-80%. After cooling the bran to 30-60°C, the mass is inoculated with a koji which was made by a special strain of A. niger which is probably not as possible to the presence of ions of iron as the culture strains used in other process. Since bran contains starch which on saccharification by the amylase enzyme of A. niger induces citric acid production. The bran after inoculation, is spread in trays to a depth of 3-5 cm and kept for incubation at 25-30°C. After 5-8 days, the koji is harvested and citric acid is extracted with water.

(b) Liquid surface culture process: In this case aluminum or stainless steel shallow pans (5-20 cms deep) or trays are used. The sterilized medium usually contains molasses and salts. The fermentation is carried out by blowing the spores of A.niger over the surface of the solution for 5-6 days, after which dry air is used. Spore germination occurs within 24 hours and a white mycelium grows over the surface of the solution, Eight or ten days after inoculation, the initial sugar concentration (20-25%) reduced to the range of 1-3%. The liquid can be drained off and any portion of mycelial mat left becomes submerged and inactivated. The small quantity of citric acid is produced during the growth phase. This is called primary metabolite. The mycelium can also be reused.

During the preparation of fermentable sugar from molasses, sucrose is the main carbohydrate along with some glucose as well as protein, peptide, amino acids, and inorganic ions. This is to be subjected to heat; so it contains saccharic acids and related compounds in traces. The initial sugar concentration is about 20-25%. The removal of metallic ions or reduction in quantity of undesirable ions in sucrose syrup by adsorption with a combination of CaCO colloidal silica, tricalcium phosphate and starch are other important steps. The iron is also precipitated by addition of calcium ferrocyanide.

Initially, the pH remains in the range of 5-6, but on spore germination, pH approaches the range of 1.5-2 as ammonium ions are removed from the solution. It is important to mention that at initial pH of 3-5 some oxalic

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acid is also produced. The presence of iron also favours oxalic acid production, and of yellow or yellow green pigments in the mycelium sometimes secreted into the culture solution and are difficult to remove during product recovery and purification.

(c) Submerged culture process: This process is quite economical. In this case, the organism (Aspergillus japonicus) which is a black Aspergillus is slowly bubbled in a steam of air through a culture solution of 15 cm depth. Since the organism shows subsurface growth and produces citric acid in the culture solution, the yields are inferior in comparison to liquid surface culture fermentation.

Continuous culture techniques are not considered suitable for use in citric acid product.

Recovery: The culture filtrate used to be hazy due to the presence of residual antifoam agents, mycelia and oxalate. The Ca(OH) slurry is added to precipitate calcium citrate.

After filterations, the filtrate is transferred and treated with H2SO4

to precipitate Ca as CaSO4 This is subjected to the treatment with activated carbon. It is demineralized by successive passages through ion exchange beds and the purified solution is evaporated in a circulating granulator or in a circulating crystallizers. The crystals are

removed by centrifugation. The remaining mother liquor is returned to the recovery stream. The solvent extraction can also be performed by adding 100 parts tri-n-butyl phosphate and 5-30 parts n-butyl acetate or methyl isobutyl ketone which are to be mixed with the filtrate. The solvent is then extracted with water at 70-90°C. Citric acid is further concentrated, decolourized and crystallized.

Uses: Citric acid is sold in the market as an anhydrous crystalline chemical, as crystalline monohydrate, or as a crystalline sodium salt. Citric acid is used in the soft drinks, jams, jelly, wines, candies and frozen fruits. it also used in artificial flavours. In medical applications, it is used in blood transfusion and as effervescent product. In cosmetic industries, various astringent lotions have citric acid where it is used to adjust pH and acts as sequestrant and in hair dressing and added in hair setting fluids. It also acts as synergists with antioxidants for oils and delay browning in sliced peaches.

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17

Ethyl Alcohol Production

Ethyl Alcohol Production by Microbiological Processes - Production of ethyl alcohol from sugary materials is one of the oldest known microbiological processes. Alcohol is an important solvent and raw material used in a variety of chemical industries. Although to day industrial alcohol is also produced synthetically from ethylene, production of alcohol by fermentation of cheap sugary materials such as molasses by yeast, is still an important industry.

For ethyl alcohol production, selected strains of Saccharomyces cerevisiae are employed since all the strains are not equally efficient. The alcohol tolerance and sugar tolerance are important criteria used in the selection of yeast strains. Strains tolerant to high sugar and alcohol concentration are desired. The raw material generally used is either crude cane molasses or best molasses which contain about 50 per cent fermentable sugars.

The production process involves the dilution of molasses to a suitable sugar concentration (15-16 per cent sugars), addition of a small quantity of nitrogen source (urea, ammonium sulfate or ammonium phosphate), adjustment of pH to about 5.0, and the, addition of an actively growing yeast culture.

The fermentation is .carried out in big deep tanks of steel or stainless steel. The fermentation is allowed to continue for about 24-36 hours at 25°C-30°C after which the cells are allowed to settle. The fermented mash is then distilled and passed through rectifying columns to recover ethyl alcohol.

A large amount of carbon dioxide is also produced during the fermentation which is purified and compressed. The yield of ethyl alcohol is about 50 per cent of the fermentable sugar concentration. Further purification of the ethyl alcohol is done by fractional distillation. In Some distilleries, the yeast is recovered and used as animal feed while in most, it is discarded into the effluents, a procedure that is very undesirable

In recent years because of the possibility of using ethyl alcohol as a fuel supplement and a chemical feed stock, there is increased interest in increasing production but at a cheaper and economical rate. For this, a variety of improvements in the traditional batch fermentation have been described in literature. Among these, the one that has attracted attention is the cell recycle technique which does not involve much additional expenditure.

Basically, the technique involves the reuse of cell mass that is produced during the fermentation. It has been found that by doing so, about 5-10 per cent of the substrate which would have been otherwise used for cell growth is saved ill addition to a great saving in the cost of inoculum and time. By using recycling technology, fermentation time has been drastically reduced from 24-36 hours in a batch fermentation to as low as 5-6 hours.

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Beer Manufacturing- Beer is made by the yeast fermentation of grains to ethanal and carbon dioxide. There are five major steps in the manufacture of beer or ale from grain. These are malting, mashing, fermenting, maturing, and finishing. Malting and mashing are concerned with the conversion of starch into fermentable form such as maltose or glucose.

The chief raw material is malt, which is germinated barley that has been dried and ground. It contains starch, proteins, and high concentration of amylases and proteinases. Amylases convert the starch into ferm entable sugar. Mould amylase derived from Aspergillus oryzae is sometimes used for the same purpose. Ground malt is mashed in warm water to bring about the digestion of starch and proteins

The aqueous extract contains dextrins, maltose, and other sugars, protein breakdown products, minerals and various growth factors. This is a rich nutrient medium and is called beer wort. The beer wort is filtered arid hops are added, Hops are the flowers of Humulus lupulus. They are added for flavour, colour, and for aroma and for mild antibacterial activity to prevent the growth of spoilage bacteria.

A large inoculum of selected strain of Saccharomyces cerevisiae is added to the wort to bring about a vigorous fermentation. Yeasts are Classified as 'top yeasts' or 'bottom yeast'. Top yeasts float on the surface of a fermenting mixture and are employed in making ale. Bottom yeasts settle in the fermentation tank and are

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used in making beer. Beer fermentation takes place at 6 to 12°C., whereas ale fermentation is complete in five to seven days at 14 to 23C. The alcoholic content of beer is between 3 to 6 percent that of ale is somewhat higher

The fermented wort is refrigerated at 0°C for two weeks to several months to remove the harsh flavour and other undesirable characteristics. Some of the harshness attributed to higher alcohols disappears as they are oxidized or esterified during aging.

Finishing process consists of carbonation, cooling, filtering and dispensing into barrels, bottles, and cans. Bottled or canned beer is usually pasteurized at 60°C for 20 minutes to kill yeasts and other microorganisms. As an alternative, the beer may be passed through a, filter to remove microorganisms, and then aseptically dispensed into sterile cans.

The composition of American lager beer is as follows: It also contains appreciable amounts of vitamins, particularly, riboflavin. In addition, there are a number of minor constituents, some of which are important for flavour and aroma.

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Wine Manufacturing- Wine is the product made by the normal alcoholic fermentation of the juice of sound, ripe grapes and the usual cellar treatment. Beverages produced by the alcoholic fermentation of other fruits and certain vegetable products are also called wines, for example, peach wine, orange wine, cherry wine.

Wine making is a much simpler, process. It can be made by a direct fermentation of, sugars, i.e. glucose and fructose, instead of starch which requires hydrolysis to yield sugars. Many fruits have the wine yeast Saccharomyces cerevisiae var. elliposideus on them. All that necessary is to crush the fruits. An, alcoholic fermentation starts spontaneously.

The characteristic qualities of famous wines are attributed in part to strains of yeast found in certain localities. However, undesirable moulds, wild. Yeast, and bacteria are also likely to be present and the fermentation may not give a predictably good product. Many wine makers now destroy natural yeasts by adding sulphur dioxide to the raw juice.

The grapes are crushed carefully and the juice is collected. To the raw juice or must, sulphur dioxide is added as sodium metabisulphite. The must is then inoculated with a starter. Culture selected strain of S. cerevisiae var. elliposideus. At the start the must is aerated slightly to promote vigorous yeast growth. Once the fermentation sets in, the rapid production of carbon, dioxide maintains anaerobic condition.

The temperature of fermentation is usually 25 to 30°C and the process may extend from few days to 2 weeks. The yield of ethanol varies from 7 to 15 percent (by volume). The wine is placed in large casks to settle, clarify and age for two to five years to develop a good flavour and aroma. Wines are endless in their varieties and differ in S0 many attributes that it is difficult to classify them.

According to colour the two most basic types are red and white wine. In, making cedwines the grapes are crushed and stemmed but, the skin and seeds are left in the must. White Wines are made from white grapes or from the juice of grapes from which the skins have been removed.

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Dry Wines are those which contain too little sugar to be detected by taste. In sweet wines the sugar content is high enough to be detected by taste. Sparkling wines contain carbon dioxide. They are made effervescent by secondary fermentation in closed containers, generally in the bottle itself. Still wines are those which do not contain Carbon dioxide. Fortified wines contain added alcohol in the form of brandy.

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Vinegar Manufacturing - Vinegar is manufactured by more rapid methods, using the generator (German process). Generators are of various sizes and shapes. They may be as large as 15 feet in diameter and 20 feet high. The generator is equipped with a false perforated bottom, through which air enters and supports beechwood shavings.

Near the top of the generator, there is a false top or, perforated plate over which is arranged a rotating sprinkler, or sparger. This produces a uniform distribution of vinegar stock (vinegar plus alcohol containing substrate) over the shavings. The generator current to the liquid flow. The acetic acid bacteria grow as a thin film over the wooden shavings.

A large area of cells is thus simultaneously exposed to the medium and to air. The bacterial oxidation of alcohol to acetic acid evolves heat. Cooling coils are necessary to keep the temperature within the favourable range of 25 to 30 °C. Several passages through a generator are required to produce vinegar of desired strength. This is accomplished by recirculation of by the use of several generators in series.

In recent years vinegar is produced in acetalors by submerged fermentation. This allows a much stricter control of aeration and temperature. It is reported that the same quantity of alcoholic substrate may be fermented 30 times faster by this process. The vinegar is used in pickling, in preserving meats and tables, and in the manufacture of salad dressing and catsup.

Wine vinegar is the aristocrat of vinegars and commands a premium price. Commercial development of such products as orange grape, fruit, tangerine, and orange peel vinegar may take place in future: properly processed, these vinegar will compete with, wine vinegars.

Vinegar fermentation, however, is not a competitive process for the production of acetic acid.

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Organic Acid Production - Many organic acids such as acetic, lactic, citric, gluconic, itaconic, fumaric etc., are produced by microbial- fermentations. Acetic acid is produced by the oxidation of ethyl alcohol by bacteria such as Acetobacter species and the production of vinegar from fruit juices is perhaps one of the oldest organic acid fermentations known. The commercial production of vinegar is still an emperical process which involves a preliminary fermentation of the fruit juice to produce ethyl alcohol and its secondary fermentation into acetic acid, under aerobic conditions. Various species of Acetobacter have the ability to oxidize alcohol to acetic acid. The rate and final amount of acetic acid produced depends to a great extent on the oxygen supply, the initial a alcohol concentration and the strain of the bacterium. Thus, for increasing efficiency of acetic acid production various types of vinegar generators are used.

In one type of generator wooden shavings packed in a column are saturated with the mother vinegar (old vinegar containing acetic bacteria) and fresh fermented alcoholic solution is then circulate.

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Acetic acid bacteria develop as a thin film on the wooden shavings and carry out the oxidation of ethanol to acetic acid. Solution containing about 5-6 per cent alcohol are converted into vinegar in less than five days.

These days, the process of acetic fermentation is further improved by the use of stirred tank fermentors or column fermentors which are vigorously aerated. In these, the desirable acetic acid level can be obtained in a few hours.

Citric Acid Fermentations -Citric acid, which is a key intermediate of the TCA cycle is produced by fungi, yeast and bacteria as an overflow product due to a faulty operation of the citric acid cycle. The ability of fungi to produce citric acid was first discovered by Wehmer in 1893 and today all the citric acid commercially produced comes from the mold fermentation. Among the organisms used for citric acid production, A. niger has been the mold of choice for several decades. A variety of carbohydrate sources such as beet molasses, cane molasses, sucrose, commercial glucose, starch hydrolysates etc., have been used for citric acid production. Among these, sucrose, cane and beet molasses have been found to be the best. For citric acid production the raw material is diluted to 20-25 per cent sugar concentration and mixed with a nitrogen source and other salts. The pH of the medium is maintained around five when molasses is used and at a lower level (pH 3.0) when sucrose is used

The fermentations are carried out either under surface, submerged, or solid state conditions. In the surface culture method, shallow aluminium or stainless steel pans are filled with the growth medium, inoculated with the fungal spores and allowed to ferment. In the submerged culture method the mold is cultured in fermentors under vigorous stirring and mixing, while in solid state fermentation, the mold is grown over carrier material such as bagasse etc., which is impregnated with the fermentation medium.

The production of citric acid by A.niger is largely influenced by the concentration of trace metals such as iron, manganese, copper and zinc in the medium. An appropriate concentration of these elements is essential for good acid production. However, an excess is detrimental. To optimize the level of these trace metals, the raw materials are treated with either ferrocyanide, charcoal, chelating agents or catien exchange resins. Addition of methanol at 3-4 % concentration has been found to enhance the yield of citric acid. This fermentation is an aerobic fermentation and, therefore, adequate aeration is essential for successful citric, acid production

 In recent years, the production of citric add by yeast is gaining importance because a variety of yeasts such as Candida, Hansenula etc. have been found to produce citric acid from carbohydrates and hydrocarbons. Strains of Candida lipolytica appear promising and good yields of citric acid from various raw materials has been reported. The mechanism by which these yeasts produce citric acid appears to be slightly different from the mechanism by which the fungi produce citric acid.

After the fermentation is over, calcium citrate is, precipitated from the fermented broth by the addition of calcium hydroxide. It is then filtered washed and treated with sulphuric acid to precipitate calcium sulphate. The solution containing citric acid is then purified by treatment with ion exchange resins, charcoal etc., and finally crystalized. Citric acid is used in food, beverage, textile, pharmaceutical and detergent industries. It is also increasingly used in the removal of toxic and corrosive gases in air. The variety of uses that it has, have increased the demand for this organic acid. Besides fungi and yeast, the possibilities of using bacteria to produce citric acid is also being explored.

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Vitamins Production by Fermentations - Microbial production is the only source of vitamin Bl2 whilst of an the other water-soluble vitamins now available commercially only riboflavin (vitamin B2) is manufactured to any significant extent, microbiologically.

There are reports that ascorbic acid is also produced by microbial fermentation but the details are not available. None of the fat soluble vitamins is produced industrially by microbiological methods but one compound, β-carotene which is converted by animals to vitamin A, can be prepared by microbial synthesis.

Processes for Microbial Synthesis of Vitamin B12 - It seems probable that the only primary source of vitamin Bu in nature is the metabolic activity of the microorganisms. It is synthesized by a wide range of bacteria and Streptomycetes, though not to any extent by yeasts and fungi. While over 100 fermentation processes have been described for the production of vitamin Bl only half a dozen have apparently been used on a commercial scale.

These arc as follows :

1) Recovery of vitamin BH as by-product of the streptomycin and aureomycin antibiotic fermentations.

2) Fermentation processes using Bacillus megatherium, Streptomyces olivaceus and other species, Propionibacterium freudemeichii, and P. shermanii.

The processes using the Propionibacterium species are the most productive and are now widely used commercially. Both batch and continuous processes have been described.

It is important Ito select microbial species which make the 5, 6 dimethyl benzimidazolylcobamid exclusively several manufacturers have been led astray by organisms that gave high yields of the related cobamides including pseudo-vitamin Bu (adeninylcobamide). The, natural form of the vitamin is Barker's Coenzyme where a deoxyadenosyl residue replaces the cyano group found in the commercial vitamin.

Practically all of the cobamides formed in the fermentation are retained in the cells, and the first step is the separation of the cells from the fermentation medium. Large high speed centrifuges are used to concentrate the bacteria to a cream, while filters are used to remove Streptomycete cells. The vitamin B12 activity is released from the cells by acid, heating, cyanide or other treatments. Addition of cyanide solutions decomposes the coenzyme form of the vitamin in and results in the formation of the cyanocobalamin.

The cyanocobalamin is adsorbed on ion exchange resin IRC-50 or charcoal, and is eluted. It is then purified further by partition between Phenolic solvents and water. The vitamin is finally crystallized from aqueous-acetone solutions. The crystalline product often contains some Water of crystallization.

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Gibberellic Acid Fermentation - Gibberellic acid and related gibberellins are important growth regulators of plants. Commercial production of these acids helps in boosting agriculture.

This acid is formed by the fungus. Gibberella fujikuroi (imperfect state, Fusarium moniliforme) and can be produced commercially using aerated submerged cultures. A glucose-mineral salt medium, incubation at 25°C and slightly acidic pH are used for fermentation. It takes normally 2-3 days.

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Antibiotic Manufacturing- The manufacturers of antibiotics maintain pilot plant facilities whose purpose is to upgrade yields and to bring about improvements in processing procedures. Studies are continually being made on strain improvement, inoculum conditions, fermentation conditions, and various combinations of these factors. For example, improved mutant strains almost always require adjustments in fermentation conditions in order to achieve the high yields in fermenters that are obtainable in shaken flasks.

Since all of the antibiotics are made by aerobic fermentations, there are a number of similarities in the processes used for their production. Many of the specific details regarding the plant scale production of a given antibiotic, with certain exceptions, remain trade secrets. However, the general outline of these methods is fairly well known to those connected with the industry.

Penicillin Production - The mould from which Fleming isolated penicillin, in was later identified as Penicillium notatum. A variety of moulds belonging to other species and genera were later found to yield greater amounts of the antibiotic and a series of closely related penicillins.

The naturally occuring penicillins differ from each other in the side chain (R group). Penicillin was produced by a surface culture method early in World War II. Sub-merged culture methods were introduced by 1943 and are now almost exclusively employed. Penicillin production needs strict asceptic conditions. Contamination by other microorganisms reduces the yield of penicillin.

This is caused by the widespread occurrence of penicillinase producing bacteria which inactivate the antibiotic. Secondly, penicillin production also needs tremendous around of air. In all methods, deep tanks with a capacity of several thousand gallons arc filled with a culture medium. The medium consists of cornsteep liquor, lactose, glucose, nutrient , salts, phenyl acetic acid or a derivative and calcium carbonate as buffer.

The medium is inoculated with a suspension of conidia of Penicillium chrysogenum. The medium is constantly aerated and agitated, and the mould grows throughout as pellets. After about seven days, growth is complete, the pH rises to 8.0 or above, and penicillin production ceases. When the fermentation is complete, the masses of mould growth are separated from the culture medium by centrifugation and filtration.

The complex process of extracting the penicillin from the clear fluid then begins. The method involves various extractions with organic solvents and recrystallization. Penicillin is assayed to determine its potency before being bottled and sold.

The potency of a batch of penicillin is determined by a biologic assay in which the unknown is compared with a standard preparation of crystalline sodium penicillin G. Cylender-plate method is used to determine the potency of penicillin. The test consists of adding nutrient, .agar, previously inoculated, with a specified strain of Staphylococcus, to a sterile Petri dish. Stainless steel cylender open at both the ends are placed on the agar.

The cylinders are filled with suitable dilutions of the working standards of penicillin and of the unknown sample. The plates are incubated at 37°c for 16 to 18 hours. The diameters of the zones of inhibition of the bacterial growth are measured. The antibiotic activity of the unknown sample is determined by comparing its zones of inhibition with those of the standard penicillin.

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Antibiotic Preparation - Antibiotics are metabolites having preferential antimicrobial activity. Therefore, they are widely used for curing of human ailments caused by microorganisms. But some of the antibiotics have antitumour activity as well.

The antibiotic penicillin was discovered by Fleming in 1929, but its commercial production could commence only during early 1940s. Although more than 7000 antibiotic compounds have been isolated, only 100 or so are being used to treat human, animal and plant diseases.

Antibiotic compounds are used either in their natural form or as semisynthetic derivatives the latter are usually produced by isolating the antibiotic nucleus and its chemical modification. Antibiotics are produced by both fungi and bacteria but over 50% of them are obtained from Streptomyces alone.

Inoculum. A high yielding strain is a prerequisite for antibiotic production. Therefore, constant strain improvement is an integral part of commercial production activities. Inoculum development begins on specific solid media, and subsequently specific liquid media are used.

The inoculum is prepared usually in the form of a spore suspension, which is transferred into the fermenters. As a rule, the number of stages between the preserved material and the final inoculum stage is kept to the minimum to minimise the risk of the organism losing its high yield potential.Fermenter. Antibiotics are generally produced in stainless steel fermenters (30,000-200,000 1 medium volume) used in the batch or fed batch mode. Agitation is mostly by impellers, but air-lift system is also used. Water cooling is often used to maintain the temperature between 24-26°C for most antibiotic producers.

Generally, the fermenter is maintained at above atmospheric pressure; this reduces contamination risk and enhances O2 supply in the medium. Sterile air is supplied as per need, and for some processes, e.g., penicillin production, materials need to be added throughout the process. In most cases, it is critical to prevent contamination, and suitable cleaning procedures between fermenter runs must be adhered to.

The final stage fermenter is preferably used for antibiotic production for the longest possible period. But the initial stages of fermentation are designed for considerable microbial growth; typically, these are carried out in seed stage fermenters of smaller size.

One or more seed-stages may be used, depending on the process and the strain, to produce the maximum amount of biomass in the correct physiological state for high antibiotic production when introduced in the final stage fermenter.

Production Medium. Antibiotic production employs a variety of media, a different one for each stage of operation. A considerable research effort is directed at the developing seed-stage and production media to reduce costs and to enhance yields. A typical production medium has about 10% (w/v) solids.

Generally, yields are much higher on complex media. In some cases, a suitable precursor for the antibiotic is also provided, e.g., for penicillin G production phenylacetic acid or phenoxyacetic acid is used as precursor.

Since the antibiotics are secondary metabolites, the production medium is so designed that a key nutrient becomes limiting at a critical stage to initiate the secondary metabolism in the organism. The nutrient to be

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made limiting depends on the process, e.g., glucose for penicillin production and phosphate for several antibiotics produced by Streptomyces.

In most production processes, the production fermenter is run in a fed batch mode in which a nutrient, e.g., glucose, is added continuously throughout the fermentation to enhance the duration of antibiotic production. In such a case, small volumes of broth are usually withdrawn to check increase in volume of the broth in the fermenter.

Antifoaming agents are added at the appropriate stage of fermentation to minimise foaming; silicones, vegetable oils, etc. are used for this purpose. Selected microbiological, physical and chemical features are monitored during the fermentation in order to achieve a proper control on the process.

The nutritional status of broth is monitored regularly to adjust nutrient and precursor (if any) feeding in fed-batch systems. Microprocessors and computers are commonly used to record all the parameters and process changes, and to control various operations like sterilization, etc.

At the end of final production stage incubation, the broth contains only a low concentration (3-35% of the total solutes in the broth) of the antibiotic. The initial step in antibiotic recovery is separation of cells from the broth; this is usually achieved by filtration or centrifugation.

But in some cases, the whole broth is used for extraction. Antibiotic isolation and purification employs solvent extraction, ion exchange, ultrafiltration, reverse osmosis, precipitation and crystallization.

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