Fermentation Technology

Fermentation technology is the oldest of all biotechnological processes. The term is derived from the Latin verb fevere, to boil- the appearance of fruit extracts or malted grain acted upon by yeast, during the production of alcohol.Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat.A fermenter is the set up to carry out the process of fermentation. The fermenters vary from laboratory experimental models of one or two liters capacity, to industrial models of several hundred liters capacity, which refers to the volume of the main fermenting vessel.A bioreactor differs from a fermenter in that the former is used for the mass culture of plant or animal cells, instead of microorganisms. The chemical compounds synthesized by these cultured cells, such as therapeutic agents, can be extracted easily from the cell biomass. The design engineering and operational parameters of both fermenters and bioreactors are identical. With the involvement of microorganisms as elicitors in some situations, the distinction between the two concepts is being gradually obliterated.The fermentation process requires the following:

1. A pure culture of the chosen organism, in sufficient quantity and in the correct physiological state.

2. Sterilized, carefully composed medium for growth of the organism.

3. A seed fermenter, a mini-model of production fermenter to develop an inoculums to initiate the process in the main fermenter.

4. A production fermenter, the functional large model and

5. Equipment for (a) drawing the culture medium in steady state (b) cell separation (c) collection of cell free supernatant

(d) Product purification and (e) effluent treatment.

Step 1 to 3 constitutes the upstream and step 5 constitutes the downstream of the fermentation process.

Fermenters / bioreactors are equipped with an aerator to supply oxygen in aerobic processes, a stirrer to keep the concentration of the medium uniform and a thermostat to regulate temperature, a pH detector and similar control devices.

Fig: Cross section of fermenter for Penicillin production

Fig: Flow sheet of a multipurpose fermenter and its auxiliary equipment

Types of Culture Systems

A. Batch Processing or Culture:

At the onset of the stationary phase, the culture is disbanded for the recovery of its biomass (cells, organism) or the compounds that accumulated in the medium ( alcohol, amino acids) and a new batch is se t up. This is batch processing or batch culture.

The best advantage of batch processing is the optimum levels of product recovery. The disadvantages are the wastage of unused nutrients, the peaked input of labour and the time lost between batches.

B. Continuous Processing or Culture:

The culture medium may be designed such that growth is limited by the availability of one or two components of the medium. When the initial quantity of this component is exhausted, growth ceases and a steady state is reached, but growth is renewed by the addition of the limiting component.

A certain amount of the whole culture medium (aliquot) can also be added periodically, at the time when steady state sets in. The addition of nutrients will increase the volume of the medium in the fermentation vessel. It is so arranged so that the increased volume will drain off as an overflow, which is collected and used for recovery of products. At each steps of addition of the medium, the medium becomes dilute both in terms of the concentration of the biomass and the products. New growth, stimulated by the added medium, will increase the biomass and the products, till another steady state sets in and another aliquot of medium will reverse the process.

This is continuous culture or processing. Since the growth of the organism is controlled by the availability of growth limiting chemical component of the medium, this system is called a chemostat. The rate at which aliquots are added is the dilution rate that is in effect the factor that dictates the rate of growth.

The events in a continuous culture are:

1. The growth rate of cells will be less than the dilution rate and they will be washed out of the vessel at a rate greater than they are being produced, resulting in a decrease of biomass concentration both within the vessel and in the overflow.

2. The substrate concentration in the vessel will rise because fewer cells are left in the vessel to consume it.

3. The increased substrate concentration in the vessel will result in the cells growing at a rate greater than the dilution rate and biomass concentration will increase and

4. The steady state will be re-established.

Hence, a chemostat is a nutrient limited self-balancing culture system, which-may be maintained in a steady state over a wide range of sub-maximum specific growth rates.

The continuous processing offers the most control over the growth of cells.

Commercial adaptation of continuous processing is confined to biomass production and to a limited extent to the production of potable and industrial alcohol.

The steady state of continuous processing is advantageous as the system is far easier to control. During batch processing, heat output, acid or alkali production and oxygen consumption will range from very low rates at the start to very high rates during the late exponential phase. The control of the environmental factors of the system becomes difficult. In the continuous processing, the rates of consumption of nutrients and those of the output chemicals are maintainable at optimum levels. Besides the labour demand is also more uniform.

Continuous processing may suffer from contamination, both from within and outside. The fermenter design, along strict operational control, should actually take care of this problem.

The production of growth associated products like ethanol is more efficient in continuous processing, particularly for industrial use.

Continuous culturing is highly selective and favors the propagation of the best adapted organism in culture.

A commercial organism is highly mutated such that it will produce very high amounts of the desired product. But physiologically such strains are inefficient and give way in culture to inferior producers - a kind of contamination from within.

C. Fed- Batch Culture or Processing :

In the fed-batch system a fresh aliquot of the Medium is continuously or periodically added. without the removal of the culture fluid. The fermenter is designed to accommodate the increasing volumes. The system is always at a quasi-steady state.

Fed-batch achieved some appreciable degree of process and product control.

A low but constantly replenished medium has the following advantages:

1. Maintaining conditions in the culture within the aeration capacity of the fermenter;

2. Removing the repressive effects of medium components such as rapidly used carbon and nitrogen sources and phosphate;

3. Avoiding the toxic effects of a medium component and

4. Providing limiting level of a required nutrient for an auxotrophic strain.

Production of baker’s yeast is mostly by fed-batch culture, where biomass is the desired product. Diluting the culture with a batch of fresh medium prevents the production of ethanol, at the expense of biomass; the moment traces of ethanol were detected in the exhaust gas.

The production of penicillin, a secondary metabolite, is also by fed-batch method. Penicillin process has two stages: an initial growth phase followed by the production phase called the ‘idiophase’. The culture is maintained at low levels of biomass and phenyl acetic acid, the precursor of penicillin, is fed into the fermenter continuously, but a low rate, as the precursor is toxic to the organism at higher concentrations.

PRODUCTS OF FERMENTATION PROCESSES

The growth of micro-organisms or other cells results in a wide range of products. Each culture operation has one or few set objectives. The process has to be monitored carefully and continuously, to maintain the precise conditions needed and recover optimum levels of products. Accordingly, fermentation processes aim at one or more of the following:

01. production of cells (biomass) such as yeasts;02. extraction of metabolic products

such amino acids, proteins (including enzymes), vitamins alcohol, etc., for human and/or animal consumption or industrial use such as fertilizer production;

03. modification of compounds (through the mediation of elicitors or through biotransformation); and

04. production of recombinant products:

A. MICROBIAL BIOMASS

Microbial biomass produced commercially as single cell protein using such unicellular algae as species of Spirulina for human or animal consumption, or viable yeast cells needed for the baking industry, which was also used as human feed at one time. Bacterial biomass is used as animal feed. The biomass of Fusarium graminearum is also produced for a similar use.

B. MICROBIAL METABOLITES

1. Primary Metabolites: During the log or exponential phase organisms produce a verity of substances that are essential for their growth, such as nucleotides, nucleic acids, amino acids, proteins, carbohydrates, lipids etc. or by products of energy yielding metabolism such as ethanol, acetone, butanol etc. This phase is described as tropophase. And the products are usually called primary metabolites.

Ethanol, Alcoholic beverages Saccharomyces cerevisiae Food

Citric acid Aspergillus niger Industrial

Acetone, butanol Clostridium acetobutyricum Solvents

Vitamin B12 Pseudomonas denitrificans Nutritional

Xanthun gum Xanthomonas campestris Industrial

2. Secondary metabolites: Organisms produce a number of products other than the primary metabolites. The phase, during which products that have no obvious role in metabolism of the culture organisms are produced, is called the idiophase, and products are called secondary metabolites.

In reality, the distinction between the primary and secondary metabolites is not a straightjacket situation. Many secondary metabolites are produced from intermediates and end products of secondary metabolism. Some like those of the Enterobacteriaceae do not undergo secondary metabolism.

Penicillin Penicillium chrysogenum Antibiotic

Erythromycin Streptomyces erythreus Antibiotic

Streptomycin Streptomyces griseus Antibiotic

Cephalospori Cephalosporium acrimonium Antibiotic

n

Griseofulvin Penicillium griseofulvin Antifungal / Antiviral

Gibberellin Gibberella fujikuroi Plant growth regulator

Secondary metabolites may be repressed in certain cases. Glucose represses the production of actinomycin, penicillin, neomycin and streptomycin; phosphate represses streptomycin and tetracycline production. Hence, the culture medium for secondary metabolite production should be carefully chosen.

C. PRODUCTION OF ENZYMES

Industrial production of enzymes is needed for the commercial production of food and beverages. Enzymes are also used in clinical or industrial analysis and now they are even added to washing powders (cellulase, protease, lipase). Enzymes may be produced by microbial, plant or animal cultures. Even plant and animal enzymes can be produced microbial fermentation. While most enzymes are produced in the tropophase, some like the amylases (by Bacillus stearothermophilus) are produced in the idiophase and hence the secondary Metabolites.

Amylases Aspergillus oryzae

Cellulase Trichoderma reesii

Invertase Saccharomyces cerevisiea

Lipase Saccharomycopsis lipolytica

Proteases Bacillus species Glucamylas

e Aspergillus niger

D. FOOD INDUSTRY PRODUCTS

A very wide range of innumerable products of the food industry, such as sour cream, yoghurt, cheeses, fermented meats, bread and other bakery products, alcoholic beverages, vinegar, fermented vegetables and pickles etc. are produced through microbial fermentation processes. The efficiency of the strains of the organisms used and the processes are being continuously improved to market quality products at more reasonable costs.

E. RECOMBINANT PRODUCTS

Recombinant DNA technology has made it possible to introduce genes from any organism into micro-organisms and vice versa, resulting in transgenic organ and the latter are made to produce the gene product. Genetically manipulated Escherichia coli, Saccharomyces cerevisiae, other yeasts and even filamentous fungi are now being used to produce interferon, insulin, human serum albumin and several other products.

F. BIOTRANSFORMATION

Production of a structurally similar compound from a particular one, during the fermentation process is transformation or biotransformation or bioconversion. The oldest instance of this process is the production of acetic acid from ethanol.

Immobilized plant cells may be used for biotransformation. Using alginate as the immobilizing polymer, digitoxin from Digitalis lanata was converted into digoxin, which is a therapeutic agent in great demand. Similarly, codeinone was converted into codein and tyrosine from Mucuna pruriens was converted into DOPA.

G. ELICITORS

It is possible to induce production or enhance production of a compound in cultures by using elicitors, which may be microorganisms. For example, Saccharomyces cerevisiae was an efficient elicitor in the production of glyceollin (GIycine max) and berberine (Thalictrum rugosum). Rhizopus arrhizus trebled diosgenin production by Dioscorea deltoidea. The production of morphine and codeine by Papaver somniferurm was increased 18 times by Verticillium dahliae

GENETIC IMPROVEMENT OF FERMENTATION PROCESSES

The genome of the organism ultimately controls its metabolism. Although improved fermenter engineering design and optimal cultural conditions can quantitatively enhance the microbial products, this will only be up to a limit. Genetic-improvement of the organism is fundamental to the success of fermentation technology. Mutation and recombination are the two ways to meet this end.

A. MUTATION

A certain amount of mutational change in the genome occurs as a natural process, though the probability is small. Exposing a culture of a microorganism to UV light, ionizing radiation or certain chemicals enhances the rate of occurrence of mutations. But it is a tremendous task for the industrial geneticist to screen the very large number of randomly produced mutants and to select the ones with the desired qualities.

The synthesis of a number of products of cell metabolism is controlled by a feed-back inhibition. When a compound reaches a particular level of accumulation; its synthesis is stopped. Synthesis starts again when the level of the compound falls below the specific level. If a mutant is produced, in which the-feed-back signaling is suppressed, the product is synthesized continuously. By such a manipulation, a high producing strain of Corynebacterium glutamacium was developed to recover very high quantities of lysine. Such strains that do not produce controlling end products are called auxotrophs.

B. RECOMBENATION

Recombination is defined as any process that brings together genes from different sources.

A strain of Brevibacterium flavum is a high producer of lysine but is limited by its poor capacity to absorb glucose. Another strain of the bacterium, which is an efficient absorber of glucose but which does not produce lysine, was used to develop a recombinant strain, through protoplast fusion. The new strain utilizes high levels of glucose and yields higher levels of lysine.

A gene for the synthesis of phenylalanine was transferred to a chosen strain of Escherichia coli, which was a non-producer, but a good experimental and production tool.

Transformation of a high cephalosporin producing strain of cephalosporium acremonium with a plasmid containing the gene REXH has significantly increased the titer.

A number of human proteins, such as insulin, human growth hormone, bone growth factor, alpha, beta and gamma interferon's, interleukin-2, tumor-necrosis factor, tissue-plasminogen activator, blood clotting factor, epidermal growth factor, granulocyte colony stimulating factor, erythropoietin etc. are being produced through recombinant microorganisms.

C. DNA MANIPULATION

In vitro DNA technology was used to increase the number of copies of a critical pathway gene (operon), as for example the production of threonine in Escherichia coli, at rates 40 to 50 times higher than usual.

Fermentation technology is a very vibrant and fast growing area of biotechnology, absorbing an ever increasing processes and products. With a longer history than any area of biological sciences, fermentation technology has a longer and brighter future, in the service of mankind, covering such important areas as food and medicine.

PRODUCTION OF VITAMINS BY FERMENTATION PROCESS

Vitamins are the essential components of the human diet which are required for a normal physiological behavior of the body. These products are now a days being produced by the microorganisms using the fermentation principles. Various vitamins have been identified till date and their production strategies designed, however.

Production by A. gossypii: A. gossypii is the causative organism of a disease of cotton bolls (especially in South Africa), which produces rooting and staining. It may also cause infection to beans, citrus fruits, coffee, okra and tomatoes. Since the organism is highly pathogenic, it is essential to sterilize all fermentation residues and cultures before they are discarded.

Nutrient requirement: order to achieve maximum yield of riboflavin commercial glucose and sometimes sucrose and maltose are used as the source of carbon. Peptone and animal stick liquor are used as nitrogen source and corn-steep liquor plant protein source. Biotin, thiamin, and meso-inositol are also required for the optimum growth of microorganism.

Stock culture:

A. gossypii may be transferred at weekly intervals on a medium containing:

• Peptone 0.5%• Yeast extract 0.3%• Malt extract 0.3%• Commercial glucose 1.0%• Agar 2.0%

incubation temperature 27-30°C

Inoculum development:

Riboflavin: Riboflavin also known as vitamin B2 was first isolated from milk and synthesized in 1935. It occurs as intense yellow color due to the presence of complete isoalloxazine ring system. Chemical structure of riboflavin is shown as follows. Riboflavin is hygroscopic and sensitive to alkalies and is decomposed by ultraviolet and visible light. It is essential for growth and reproduction in mammalians. Riboflavin may be produced by a number of microorganisms including yeast-like microbes, and bacteria. The most important of these are Eremothecium ashbyii, Ashbya gossypii, and certain Clostridium species.

A loopful of a 24 h old culture of A. gossypii NRRL Y-1056 placed in 100 ml of the

following medium in a 500 ml flask and incubated for 24 h on a reciprocating

shaker at 26 to 30°C

• Glucose 2.0%• Peptone 0.5%• Corn-steep liquor 1.0%• Water to 100 ml

The pH of this medium before sterilization for 30 min. at 121°C was 6.5. The contents of the flask were used o seed 6 liter of sterilized medium of the following composition contained in a 9 liter glass bottle:

• Glucose 2.0%• Corn-steep liquor 1.0%• Animal-stick liquor 0.5%• Water to 6 liter

The pH of the medium before sterilization for 45 minutes at 121°C was 6.5. The organism was grown for 24 h with aeration provided by passing sterile air through a perforated tube located at the bottom of the bottle. This vulture can be used to inoculate 200 or 300 gallons of sterilized medium in a fermenter.

Production:

For maximum yield of riboflavin the following composition of media is recommended and used:

• Glucose 2.0%• Corn-steep liquor 1.8-2.1%• Animal-stick liquor 1.0%• An antifoam agent Small amount

The medium has been sterilized at pH 4.5 and at a temperature 135°C for 5 minutes. An inoculum of 0.5 to 1.0% has been used for seeding fermenters. Sufficient aeration for adequate mixing of the medium but not hampering growth should be provided. An aeration rate being maintained at about 0.25 vol. of air per vol of medium per min. is satisfactory. Fermentation is carried out at a temperature of 28 to 30°C for 96 to 120 h. The yields have been 500 to 600 μg/ml of riboflavin, The fermentation liquor thus produced may be evaporated to a syrup and then dried Oil drum dryers to yield a concentrate containing 2.5% of riboflavin.

Production by E. ashbyii: Riboflavin is produced industrially from E. ashbyii, a yeast like organism belonging to the Ascomycetes. Various patents describe the methodologies for riboflavin production. The methods described by Rudert (1945) for its production front E. ashbyii from subsequently carbohydrate-free media is based on the total weight of nutrients, the medium contains 10-90 % of proteinaceous materials, a metabolizable lipid and nutrient such as peptone or a combination of salts (0.05%, KH2PO4 0.07% MgSO4 .7H20), 0.107 NaCl and 0.01 % FeSO4.7H2O).

In carrying out production, the following procedure is illustrative of Rudert's invention:

The media are adjusted to an initial pH of 5.5 to 7.5 and dispensed in containers to give a depth of 0.5 inch, sterilized at 20 psi for 45 minutes, cooled to 30°C, and inoculated with 0.7% of an active culture of E. ashbyii. During production, the temperature is maintained between 20 and 34°C and the medium is aerated with 1.5 to 2 Cu ft of sterile air/ min/sq. ft of mesh surface. At the end of 50 to 90 h, the conversion is completed. The dried residue contains 200 to 6000 µg/g of riboflavin.

Gaden, Petosiava and Winckar ( 1954) reported on the production of riboflavin from citrus molasses, using E. ashbyii, NRRL 1363. The citrus molasses were clarified by settling followed by decantation.

The inoculum was prepared by growing the microorganism for 24 h in a sterilized medium containing:

• Clarified citrus molasses 1.5%• Yeast extract 0.30%• Peptone 1.0% pH adjusted to 6.6 to 6.8

The use of an initial concentration of citrus molasses equivalent to approximate 6.0% reducing sugar, fortified by the addition of a commercial enzymatic yeast hydrolysate at a concentration of 0.3% has been recommended for the production of highest yields of riboflavin. The pH of the production medium was adjusted between 6.5 before sterilization, which generally resulted in a pH of about 6.4 to 6.8 after sterilization. This production tin was inoculated with 4% by volume of inoculum. Yields of as much as 729 µg/,ml of riboflavin were led on 7-9 days of incubation at 27°C to 30°C in shaker flasks.

Production by Candida species: Holder (1943) found that Candida guilliermondia grew and produced riboflavin satisfactorily in media lining dextrose, mannose, levulose or sucrose. Aspargine and glycine were found to be suitable and very inexpensive sources of nitrogen for riboflavin production by C. guilliermondia (A.T.C.C. 9058). Most satisfactory results were obtained when the pH was adjusted to 5 to 6 and temperature was maintained 0°C. An increased yields of riboflavin were obtained by adding small amount of sterile potassium cyanide to the medium under vigorous fermentation. The fermentation time was 6-7 days. Tanner and Van Lanen (1947) have patented a method for producing riboflavin from C. flareri. The method briefly consists of fermentation under aerobic conditions at 30°C for 7 days using other suitable Candida species, in a medium containing a fermentable sugar, an assimilable source of nitrogen, non iron salts, biotin and less than 10.3 μg of iron/100 ml. The preferred species of candida C. flareri and C. guilliermondia. Recovery of riboflavin: Riboflavin may be recovered from production substrates by a variety of procedures, many of them are patented. Presztesy (1944) patented a procedure for extracting riboflavin with butanol, followed by the use of other vents, such as petroleum ether and acetone. McMillan (1945) patented a chemical precipitation method in which a soluble reducing agent and a finely divided diatomaceous earth were used. Hines (1945a) described a method wherein riboflavin was absorbed on fuller's earth, silica gel, or other adsorbent and eluted with an aldehyde, ketone or alcoholic solution of an organic base. Another procedure by Hiner (1945b) related to the conversion of riboflavin to a less soluble form by the action of reducing bacteria, such as Streptococcus faecalis. while (1947) has patented a method for securing crystalline riboflavin from the precipitate produced by the reduction of this vitamin to a less soluble form by either reducing bacteria or using some chemical reducing agent.

PRODUCTION OF ANTIBIOTICS (IS0LATiON OF FERMENTATION PRODUCTS)

Industrial fermentation industry, across the globe, commendably received its ever outstanding impetus for the most coveted strategic expansion as well as profits with the wonderful advent and ex-ploitation of antibiotics as potential well known chemotherapeutic agents’. During the World War II the actual demand for penicillin almost reached its peak to save the lines of millions of wounded soldiers; and later on followed by streptomycin and a host of other antibiotics in the domain of global scenario of pharmaceutical industry. These developments instantly triggered off extensive and intensive research programmers most articulately designed to look for useful microorganisms that are capable of producing highly effective, viable, and good antibiotics; and oriented a tremendous push towards the adequate research and development for producing antibiotic substances on a commercial scale. Thus, several altogether newer cultural procedures were devised, developed, and the state-of-the-art technique of suberged- agitated- aerated fermentation using deep-tank fermentors came into being with obvious high rate of success.

The Penicillins

Penicillins, the ß-lactam antibiotics, have indeed enjoyed the legendary of a long history of application as chemotherapeutic agents since 1929 by the epoch making discovery of Alexander Fleming and, even today, they legitimately command the reputation for being prescribed more than 50% of all the known antibiotics across the globe. The most genuine and remarkable combination of unique very effective bactericidal property and desirable levels of extremely low toxicity are solely attributed by the ensuing bacterial cell wall biosynthesis. In reality, the relatively low cost of these therapeutic agents is exclusively based upon the tremendous acclaimed enhancement in fermentation yields which have been gainfully accomplished through years of dedicated researches as: strain improvement, fermentation optimization procedures, and above all meticulous refinement of downstream processing. It would be worth while to lay proper emphasis upon the current status of knowledge with regard to the genetics and molecular biology of penicillin biosynthesis.

Production of Benzylpenicillins [Penicillin G]

Alexander Flemings originally isolated strain of Penicillium notatum (Straub) afforded actually very low yield of penicillin. Vigorous search for improvement of strain revealed the isolation of P. chrysogenum which distinctly gave much higher yields of penicillin. Importantly, the newer strains of Penicillium could even produce upto 180 folds higher yields in comparison to the original isolate that are solely based upon the novel phenomenon of 'mutation' or the so-called 'genetic engineering' methodologies.In actual practice, penicillin is commercially produced in submerged vat cultures employing a highly purified and selected strain of P. chrysogenum, whereby the ultimate yield of the targeted product (penicillin) has been enhanced almost three folds i.e., from 10 mcg/ml to 30 mcg/ml. Interestingly,

these modified, researched, purified strains of Penicillium do exhibit a number of marked and pronounced characteristic features, such as: high-titre values, improved growth, immense tolerance to the side-chain precursors, acetyltransferase activity, ability to store intracellular requirement(s).

Inoculum: Penicillium notatum (i.e., Fleming's initial/original strain) together with other 'early isolates' afforded exclusively low yields of penicillin ; besides, they responded very sluggishly to the submerged culture techniques particularly. Contrary to this, an early strain of P. chrysogenum (NRRL, 1951), duly isolated from the moldy fruits, was observed to yield much higher yields of penicillin. Consequently, the high-yield strain was duly subjected to careful treatment with a broad-spectrum of time-tested mutagenic agents, for instance: UV-radiations, X-rays, and mechlorethamine (MBA)—a nitrogen mustard. Obviously, these mutagenic agents helped a long way in the appropriate selection of several higher yielding mutants in particular; and, in general, the judicious application of these ensuing mutagenic agents in sequence, along with certain repetitive treatments, ultimately grave rise to the newer strain Q-176, that essentially had the ability of producing maximum yields of penicillin.

Q-176 strain produced > 1000 Units/ml

NRRL-1951 strain produced ~ 200 Units/ml

Production Media:

Though the precise and exact compositions of the penicillin-production media really employed in any industry are more or less impossible to quote and determine, by virtue of the fact that such information(s) are regarded to be the 'trade secrets' or patented by the actual users. Nevertheless, a large segment of these commonly used media invariably comprises of such ingredients as: cornsteep liquor solids, lactose, glucose, calcium carbonate, potassium dihydrogen phosphate [KH2PO4], edible oil, and a penicillin precursor. Jackson (1958) promulgated a very useful and typical medium having essentially the following composition:

Sl. No.

Ingredients Quantity Remarks

1. Fermentable carbohydrates

Corn steep liquor solids 3.5 Organic Carbon Source

Lactose 3.5

Glucose 1

2. Organic nitrogen source q.s. 3. Phenyl acetic acid q.s. Penicillin precursor

4. Potassium dihydrogen

phosphate [KH2PO4]

0.4

5. Calcium carbonate 1

Acts as buffer

6. Edible oil 0.25

7. Organic salts q.s. Maintain salt-balance in medium

Note :

(1) The pH after sterilization is carefully maintained between 5.5 to 6.0.(2) Higher lactose content ranging between 4 to 5% is desired with vigorously increased aeration and agitation environments maintained within the fermentor (i.e., bioreactor).(3) The 'production media' contains both 'lactose' and 'precursor' which are not included in the inoculums media.

Production and Recovery :

A general and basic flow sheet diagram for the large-scale recovery and purification of 'antibiotics' is illustrated in Fig. 3.21. The various steps that are usually followed in a sequential manner are described as under :

1. Once the entire fermentative procedure is accomplished i.e. at harvest, the completed penicillin fermentation culture is subjected to filtration by the help of heavy duty rotary vacuum filter to get rid to the mycelium plus other unwanted solid residues.

2. The pH of the clear filtered fermented broth is carefully brought down between 2 to 2.5 by the addition of a calculated amount of either phosphoric acid [H3P04] or sulphuric acid [H2SO4] so as to convert the resulting penicillin to its anionic form.

3. The resulting fermented broth (pH 2 – 2.5) is extracted immediately by using a Pod bielniak countercurrent solvent extractor, with an appropriate organic solvent e.g., amyl acetate, butyl acetate, or methyl isobutyl ketone.

4. Penicillin, thus obtained, is back extracted into aqueous medium from the corresponding organic solvent by the careful addition of requiste quantum of KOH or Na0H to give rise to the formation of the corresponding potassium or sodium salt of the penicillin.

5. The resulting aqueous solution, containing the respective salt of penicillin, is again acidified and re-extracted with the organic solvent methyl isobutyl ketone.

6. In fact, these shifts taking place between 'aqueous' and 'solvent' medium help in the ultimate process of purification of the penicillin.

7. The resulting solvent extract is finally subjected to a meticulous back-extraction with aqueous NaOH preferably, a number of times till extraction of penicillin is completed; and from this combine of aqueous extractions different established procedures are adopted to afford the penicillin to crystallize out either as sodium or potassium penicillin.

8. The crystalline penicillin thus obtained is washed, dried under vacuum, and the final product must conform to the requirements/specifications laid down by various Official Compendia.

Fig. 3.22 illustrates a typical 'antibiotic' fermentation plant. In actual practice, the culture medium could be conveniently batched as well as sterilized in the fermentor itself. Nevertheless, most of the fermentors are attached to a batching vessel and subsequently to the respective sterilizers as given in the above figure. The various feed vessels duly connected to the final-stage, fermentor are invariably employed to supplement both nutrients and precursors during the on-going fermentative process. Importantly, the seed fermentor and the final-stage fermentor should be operable under stringent aseptic environments. The bioreactors are made of SS, having a capacity ranging between 30 and 300 m3 agitation by 2/3 flat-peddled impellers, aeration done with compressed sterile air injection, generated heat dissipated by employing chilled-water cooling coils (maintained at 26 ± 2°C). Sterilization of the system done with live-steam injection ports adequately.