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1. Penicillin are group of antibiotics containing B-lactam ring which are effective against several gram positive bacteria. MOA: inhibit the bacterial cell wall synthesis by inhibiting transpeptidase enzyme and cause cell death. Natural penicillin (6-amino penicillanic acid) are ineffective against microorganism that produces B-lactamase ( Eg. Staphylococcus aureus) The various penicillin differ primarily in the nature of R-side chain which is attached by an amido linkage to the chemical nucleus of the molecule. INTRODUCTION

2. If penicillin fermentation is carried out without the addition of side chain precursor, the natural penicillin are formed from which only benzyl penicillin can be isolated Desired penicillin can be obtained by adding suitable side chain precursor into the medium. Such penicillin are called as semi-synthetic penicillin. When compared to natural penicillin, semisynthetic penicillin have improved characters viz acid stability, resistance to plasmid or chromosomally coded B-lactamases, expanded antimicrobial effectiveness and are therefore, extensively used in therapy .

3. Different steps of productions are Selection of microorganism Formulation of medium Production process Recovery and purification

4. Step 1: Selection of microorganism Early Pencillium notatum was used. Currently high yielding strains of Penicillium chrysogenum are preferred (but genetically unstable) Genetic Instability increases with the increase in the yield. However, it can be controlled to some extent by following suitable preservation methods. Preservation methods are generally adopted are A spore suspension is stored in a frozen state under liquid nitrogen. A spore suspension can be lyophilized in an appropriate medium. A spore suspension is mixed with a sterile finely divided inert material like soil or sand and desiccated.

5. High yield Strain Development: developed from the wild P. chrysogenum by a process called sequential genetic selection. This process consists of stepwise development of improved mutant by treating the wild strain of P. chrysogenum with a series of mutagenic agents or exposing to ultraviolet radiation either individually or in combination, such as X-rays and chemical mutagens, is called as strain improvement. Strain development is a laborious and time-consuming process. The selected mutant possesses greater capacity for antibiotic production than the wild type.

6. Innoculum Production: high yielding strain of P. chrysogenum is sub-cultured from stock culture Spores from primary source are suspended in water or in a dilute solution of a nontoxic wetting agent such as 1:10000 sodium lauryl sulfate. The spores are then added to flasks or bottles of Wheat bran + nutrient solution and these are incubated for 5 to7 days at 24°C so as to provide heavy sporulation. The entire process is repeated several times in order to have more sporulation. The resulting spores are used directly to inoculate inoculam tanks or stirred fermenters. The incubation temperature is maintained at 24-27°C for 2 days with agitation and aeration in order to facilitate heavy mycelial growth, which may be added to a second or even a third stage fermentation. The resulting inoculum which is employed in a production tank is tested both by microscopic examination and by sub-culturing method.

7. Step 2: Formulation of medium Medium used consists of corn steep liquor (4-5% dry weight) → Abundantformation of mycelium and spores takes place because it contains important amino acids required for mycelial growth. carbon source: Lactose, Glucose, Sucrose, Glycerol and Sorbitol Nitrogen source: ammonium sulphate or ammonium acetate or ammonium nitrate. An addition of yeast extract, soy meal or whey is done for a good supply of nitrogen. Potassium and phosphorus are supplied in the form of potassium dihydrogen phosphate, magnesium; iron and copper are supplied in the form of sulphate (these are present in corn steep liquor)

8. Phenyl acetic acid (or phenoxyacetic acid) is continuously fed which serves as a precursor for penicillin biosynthesis. Further continuous feeding of sugar is advantageous for good yield. 10% of metabolized carbon contributes to penicillin production, while 65% is utilized towards energy supply and 25% for growth of organism.Efficiency of penicillin production can be optimized by adequate supply of carbon source By adding glucose and acetic acid, the yield can be increased by about 25%

9. Step 2: Formulation of medium Regulation of biosynthesis: Availability of aminoadipic acid plays a significant role in regulating the synthesis of penicillin Biosynthesis is inhibited by glucose through catabolite repression. So penicillin was produced by slowly degraded sugar like lactose. The concentration of phosphate and ammonia also influence penicillin synthesis

10. Step 3: Production process Fermenter with 40000-2 lakhs L are employed carried out as fed batch submerged fermentation in stirred tank aerobic process and a continuous supply of O₂ is very essential. Aeration rate 0.5-1 vvm (volume/volume/minute) Ideal pH 6.5 Optimal temperature 25-27°C 20-40% of fermentation content is drawn off and replaced with fresh nutrients Growth phase 40 hours with doubling time of 6-8 hours Production phase can be extended to 120-180 hours

11. Step 4: Recovery and purification of penicillin Separation of Mycelium: Broth containing 1% penicillin is processed for extraction by employing rotatory vacuum filter. This process should be performed carefully in order to avoid contaminating microorganisms which produce penicillinase enzyme, degrading the penicillin. Penicillin is recovered by solvent (amyl acetate, n-butyl acetate or methyl isobutyl ketone) extraction at low temperature (<10°C) and acidic pH (phosphoric or sulphuric acid) by this way chemical and enzymatic degradation can be minimized. Penicillin containing solvent is treated with activated carbon (charcoal) to remove impurities (pyrogens) and pigment

12. It is sterilized to remove bacteria by using Seitz filter. Penicillin can be can recovered by adding salts (NaCl or KCI). K or Na salts of penicillin can be further processed (in dry solvent such as n-butanol or isopropanol) to remove impurities As water is totally removed, penicillin salts can be crystallized and dried under required pressure. It may be packed as powder in sterile vials or prepared in the form of tablets or in the form of syrups for oral usage

13. Penicillin: USES Used therapeutically in the treatment of infectious diseases of humans caused by Gram positive bacteria in which they inhibit the cell wall synthesis leading to the death of bacteria. 2. commercially produced natural penicillin (Penicillin G) are used38% as human medicine 12% as veterinary medicine 43% as starting material for production of semi synthetic penicillin

14. CITRIC ACID: INTRODUCTION Citric acid is a natural constituent (lemon) and common metabolite of plants and animals (intermediate of Krebs (citric acid) cycle) It is most versatile and widely used organic acid in the field of food and pharmaceuticals. Citric acid was isolated from lemon (contain 7%) but today about 99% of world's citric acid produced from microbial fermentation. Now citric acid mainly produced by submerged fermentation using Aspergillus niger or candida species from different source of carbohydrates

15. STEPS OF PRODUCTION Different steps of productions are Selection of microorganism Formulation of medium Production process Recovery and purification

16. Selection of microorganism

17. Step 1. Selection of microorganism mutant strains of Aspergillus nigger have been developed For improved industrial production of citric acid, The strains that can tolerate high sugar concentration and low pH with reduced synthesis of undesirable byproducts (oxalic acid, isocitric acid and gluconic acid) are industrially important.

18. Step 2. SELECTION OF MEDIA Carbon source includes molasses (sugar cane or sugar beet) starch (from potatoes) date syrup, cotton wastes, banana extract, sweet potato pulp etc sugars that are rapidly metabolized are used eg : Glucose, sucrose, maltose → high yield of citric acid production sugar concentration (12-15%): It is believed that a higher sugar concentration induces increased Glucose uptake and consequently enhanced citric acid production At a concentration less than 5% sucrose, citric acid formation negligible citric acid formation increases as the concentration is raised to 10% and then stabilizes

19. Nitrogen source Ammonium salts, nitrates and urea are used as good sources, as long as they do not adversely effect the pH of the medium If molasses are used for nutrient supply, addition of extra Nitrogen source is not required Exogenous addition of ammonium ions stimulate citric acid production

20. Trace metals : Certain trace elements (Fe, Zn, Mn, Cu, Mg, Co) are essential for growth of A niger some of the trace metals particularly Mn2+, Fe3+ and Zn2+ increases the yield of citric acid. Manganese ions promote glycolysis and reduce respiration; both these processes promote citric acid production. Fe concentration of 0.05-0.5 ppm is ideal for optimal citric acid production

21. Step 3. PRODUCTION PROCESS Biosynthesis: Citric acid is a primary metabolic product (of primary metabolism) formed in the tricarboxylic acid (Krebs) cycle. Glucose is the predominant carbon source for citric acid production. involves glycolysis wherein glucose is converted to two molecules of pyruvate. Pyruvate in turn forms acetyl CoA and oxaloacetate which condense to finally give citrate.

22. Step 3. PRODUCTION PROCESS PH The pH of the medium influences the yield of citric acidmaximal when pH is below 2.5. (At this pH the production of oxalic acid and gluconic acid is suppressed, further at low pH, transport of citric acid is much higher) If the pH is above 4, gluconic acid accumulates at the expense of citric acid And when the pH goes beyond 6 oxalic acid accumulates Another advantage with low pH is that the risk of contamination is very minimal, since many organism grow at this pH

23. Dissolved O₂ The yield of citric acid production substantially increases when the Dissolved O₂ tension is higher This can be achieved by strong aeration or by sparging with pure O2. It has been observed that sudden interruptions in O₂ supply cause drastic reduction in citric acid production, without harming the growth of the organism

24. Step 3. PRODUCTION PROCESS PRODUCTION PROCESSES Surface fermentation processes Solid surface fermentation: Solid medium Liquid surface fermentation: Liquid medium Submerged fermentation processes: most preferred Stirred bioreactor Airlift bioreactor

25. Solid surface fermentation Solid substrates such as wheat bran or pulp from sweet potato starch are used as culture media. The pH of the medium is adjusted to 4-5, and then sterilized.inoculum in the form of spores of Aspergillus niger is spread as layers (3-6cm thicknesses) and incubated at 28°c. growth of organism can be accelerated by the addition of alpha amylaseO takes about 80 to 100 hours for maximal production of citric acid. At the end of the process, citric acid can be extracted into hot water and isolated

26. Liquid surface fermentation carried out in aluminium trays filled with sterile nutrient medium containing beet molasses inoculum in the form of spores is sprayed over the medium A sterile air is passed for supplying O₂ as well as cooling temperature is maintained around 30°c during fermentation As the spore germinate a layer of mycelium is formed over the medium, The pH of the nutrient medium falls to less than 2, as the mycelium grows in size and forms a thick layer on the surface of the nutrient solution. The fermentation is stopped after 7-15 days

27. 2. Submerged processes 2 types of bioreactors are in use---- stirred tanks and aerated towers made up of high-quality stainless steel. Citric acid production mainly depend on the structure of mycelium: The mycelium with forked and bulbous hyphea and branches which aggregate into pellets is ideal for citric acid formation. on other hand, no citric acid production occurs if the mycelium is loose and filamantous with limited branches An adequate supply of O2 (20-25%) is required for good production of citric acid The submerged fermenters have the problem of foam formation which occupy about ½ of bioreactor. Antifoam agents and mechanical antifoam devices are used to prevent foaming Nutrient concentration is very important in the industrial production of citric acid It is estimated that under optimal condition in about 250-280 hours

28. Step 4. RECOVERY OF CITRIC ACID start with the filtration of culture broth and washing of mycelium Oxalic acid is an unwanted byproduct and it can be removed by precipitation by adding lime at pH<3 The culture broth is subjected to pH 7.2 and temperature 70-90°Cfor precipitation of citric acid For further purification citric acid is dissolved in sulfuric acid The final steps for citric acid recovery are - treatment with activated charcoal, cation and anion exchangers and crystallization

29. CITRIC ACID: APPLICATION used as flavouring agent (due to its pleasant taste and palatability) in foods and beverages eg : jams, jellies, candles etc used as antioxidant and preserve flavor of foodused as an antifoam agent in chemical industry and for treatment of textiles In pharmaceutical industry as trisodium citrate, It is used as blood preservative used as agent for stabilization of fats and oils In detergent / cleaning industry

30. VITAMIN B12: INTRODUCTION Vitamins are organic compounds that perform specific biological functions for normal maintenance and optimal growth of an organism. These vitamins cannot be synthesized by the higher organisms, including man, and therefore they have to be supplied in small amounts in the diet. Vitamin B12 also known as cyanocobalamin, its fat soluble vitamin belongs to the cobalamin family of compounds, which are composed of a corrinoid ring and an upper and lower ligand. The upper ligand can be adenosine, methyl, hydroxy, or a  cyano  group.

31. Unlike other vitamin, chemical synthesis of vitamin B12 not practical since it involve 20 complicated steps. Fermentation is the only choice Vitamin B12 is synthesized by prokaryotes and inhibits the development of pernicious anemia in animals. Microbial de novo biosynthesis of Vitamin B12 occurs through two alternative routes: the aerobic or anaerobic pathway, in bacteria and archaea, respectively.

32. STEPS OF PRODUCTION Different steps of productions are Selection of microorganism Formulation of medium Production process Recovery and purification

33. Selection of microorganism

34. Step 1. Selection of microorganism Pseudomonas denitrificans , Propionibacterium freudenreichii , P.shermanii and their mutant strains are commonly used A protoplast fusion technique between Protaminobacter rubber and Rhodopseudomonas spheroides resulted in a hybrid strain called Rhodopseudomonas   protamicus .

35. Step 2. Formulation of medium Medium should contain all the nutrient to support adequate growth of microorganism. Medium should be readily available at low cost and are nutritionally safe. carbon source: Glucose, alcohols (methanol, ethanol, isopropanol) and hydrocarbons. Beet molasses medium supplemented with cobalt chloride is also used as source.

36. Step 3. Production process Process is carried out by adding cobalt in 2 phases Anaerobic phase: preliminary phase may take 2-4 days. In this phase 5’- deoxyadenosylcobinamide is predominantly produced. Aerobic phase: in this phase 5,6-dimethylbenzimidazole is produced from riboflavin which incorporated to finally form coenzyme of vitamin B12 namely 5'-deoxyadenosylcobalamin. Bulk production is by submerged bacterial fermentation with beet molasses medium supplemented with cobalt chloride. pH 7.5 Temperature 30°C

37. Step 4. Recovery and Purification Cobalamin produced by fermentation are mostly bound to the cells. They can be solubilized by heat treatment at 80-120°C for about 30 minutes at pH 6.5-8.5 Solid and mycelium are filtered or centrifuged and fermentation broth collected cobalamin can be converted to more stable cyanocobalamins This vitamin B12 is around 80% purity and can be directly used as a feed additive. However, for medical use (particularly for treatment of pernicious anemia ), vitamin B12 should be further purified (95-98% purity) by adsorbing on IRC-50 resin and then eluted with a phenolic compound. Cyanocobalamin present in solvent gets crystallized by evaporation

38. VITAMIN B12: USES Helps in red-blood cells formation there by prevent anemia . Reduce the birth defects in beginning stages of pregnancy. Support bone health and prevent osteoporosis. Reduce the risk of macular degeneration. Improve mood and symptoms of depression. Improve heart health by decreasing homocysteine. Improves the health of hair, skin and nails.

39. GLUTAMIC ACID: INTRODUCTION L-Glutamic acid was the first amino acid to be produced by microorganism, Corynebacterium glutamicum . Chemically glutamic acid is 2-Amino pentanedioec Acid USES widely used in the production of monosodium glutamate which is commonly known as the 'seasoning salt’. Monosodium glutamate is a condiment and flavor enhancing agent and is commonly used in convenient food-stuffs.

40. STEPS OF PRODUCTION Different steps of productions are 1.Selection of microorganism 2. Formulation of medium 3. Production process 4. Recovery and purification

41. SELECTION OF MICROORGANISM

42. Step 1. SELECTION OF MICROORGANISM improved strain of Corynebacterium glutamicum which can give high yield and tolerate high concentration of biotin and lysozyme. A suitable strain of Corynebacterium glutamicum from stock culture is inoculated in sterilized medium in shake flask and incubated at 35°C for 16 hours. inoculum culture is grown in shake flasks and transferred to the first seed tank (1000-2000 L) in size. After suitable growth the inoculum is transferred to the second seed tank (10,000-20,000L) which serves as inoculum for the production tank (50,000-500,000 L)

43. Step 2. FORMULATION OF MEDIUM Carbon sources: such as glucose, sucrose, fructose, maltose, sugar beet molasses and sugarcane molasses and starch hydrolysates from corn or cassava. Nitrogen source: inorganic compounds such as ammonia or ammoniumsulfatePhosphates , Vitamins and other necessary supplements are usually provided with corn steep liquor. Growth factor: Biotin (its concentration influenced by carbon sources)

44. Step 2. FORMULATION OF MEDIUM Glutamic acid is the intracellular component hence, production and extraction is dependent upon cell permeability of acid producing bacteria. increased permeability to the acid can be achieved in the following ways: Ensuring biotin deficiency in the medium Treatment with fatty acid derivatives Ensuring oleic acid deficiency in mutants requiring oleic acid Addition of penicillin during growth of glutamic acid bacteria (prevents cell wall formation)Use of oleic acid auxotrophs Use of glycerol auxotrophs Addition of surfactants

45. Cells treated in one of the first three ways have cell membranes in which the saturated to unsaturated fatty acid ratio is abnormal, therefore the permeability barrier is destroyed and glutamic acid accumulates in the medium. Glutamic acid production is greatest when biotin is sub optimal limiting When biotin is optimal (0.5 mg per g of dry cells) growth is luxuriant and lactic acid is excreted (not glutamic acid) with higher amounts glutamic acid production falls. The prepared culture medium is sterilized in a fermenter by steam.

46. Step 3. PRODUCTION PROCESS When the temperature of the medium cools down to 30°C, the micro-organism is added to the fermenter in a proper inoculum size. Done in production tank (50,000-500,000 L) By batch or fed-batch method: fed-batch preferable The micro-organism is incubated for 36-48 hrs during which time the pH (7-8), Temperature (30-35°C), The oxygen concentration should be optimum for production of acid. High concentration inhibits growth of the organisms low concentration → produce lactic acid and succinic acid

47. Step 4. RECOVERY AND PURIFICATION After fermentation, the cells are filtered using a rotary vacuum filter which can be improved by using filter aids ( kiesselghur which are based on diatomaceous earth) The extraction done by chromatographic (ion exchange) method or the concentration-crystallization method. Crystallization: By the pH of the at the isoelectric point (zero net charge). Lowering the temperature Precipitation with salts like ammonium and calcium salts and with metals like zinc.

48. Ion Exchange Resin: used for the extraction and purification of amino acids The adsorption of amino acids by ion exchange resins is strongly affected by the pH of the solution and by the presence of contaminant ions. There are two types of ion exchange resins; cation exchange resins and anion exchange resins. Cation exchange resins bind positively charged amino acidso anion exchange resins bind negatively charged amino acids Anion exchange resins are generally lower in their exchange capacity and durability than cation exchange resins and are seldom used for industrial separation.

49. GLUTAMIC ACID: USES important in brain metabolism → used in rearing carious neuropathic diseases Monosodium glutamate has been used as a flavour enhancing agent both by thefood industry and the general public. Poly glutamic acid (PGA): naturally occurring anionic polymer that is biodegradable, edible, and non-toxic towards human and environment. industrial applications including thickener, bitterness reliving agent, Cryoprotectant. Curable biological adhesive Heavy metals absorbers. Cosmetics: L-Glutamic Acid is Widely used. a. as hair restorer in Cosmetics. As hair restorer: in treatment of hair loss. b. As wrinkle: in preventing aging

50. GRISEOFULVIN: INTRODUCTION Griseofulvin is an antifungal agent which acts specifically on fungi with chitinous cell wall which is isolated from penicillium species. USES in the treatment of various fungal skin infections. MOA: Although the exact mechanism of action of griseofulvin is not known, it is believed that Chitin biosynthesis is adversely affected. The chemical synthesis is less frequently used due to high cost. The fermentation is carried out by an aerobic submerged process

51. STEPS OF PRODUCTION Different steps of productions are Selection of microorganism Formulation of medium Production process Recovery and purification

52. Step 1. SELECTION OF MICROORGANISMS uses high yielding strain of Penicillium Griseofulvum , Penicilliumpatulum Different other strains of fungi are used for production of griseofulvin such as Penicillium nigricans, Penicillium janczewskit , Penicillium urticae , Penicillium albidum, Khauskia oryzae,Nigrospora musae , N.splaerica , etc. Spore suspensions as well as vegetative cells are suitable to develop inoculum for laboratory scale fermentation.

53. Step 2. FORMULATION OF MEDIA The media for inoculum development, fermentation and culture conditions are quite similar for all antibiotic producing fungal cells. Czapek -Dox agar medium is commonly used for growth and maintaining fungal culture on agar plates. Rich in glucose and sodium nitrate Composition of Czapek -Dox agar medium

54. P. griseofulvum may grown in different media such as sporulation medium (whey powder lactose, whey powder nitrogen, KH2PO4, potassium chloride, corn-steep liquor solids), germination medium ( protopeptone , malted cereal extract, glucose, starch, NaNO3, KH2PO4, MgSO4, FeSO4) and seed stage medium (corn-steep liquor, brown sugar, chalk, corn oil, Hodag MF) Medium composition contains in production of antibiotic is Corn steep liquor, CaCO3, KH2PO4, and KCI. Carbon source (12%) phosphate (0.5%) and chlorine are most commonly used for better yield production.

55. Step 3. PRODUCTION CONTROL inoculated with organism into fermentation medium carried out by an aerobic submerged process with a glucose rich medium. Nitrogen is supplied in form of sodium nitrate. The optimal conditions for fermentation are- Temperature 23-26°C, pH 6.8-7.3, Aeration 0.8-1 vvm and the Period is 7-10 days.

56. Step 4. RECOVERY AND PURIFICATION The broth is heated above 60°C for 20-30 min. After heating, sufficient coagulation of material occurs to produce a valuable improvement in separation characteristics of the broth. The period of heating may be short, 5-10 min at 80°C having been found to provide a satisfactory increase in filtration rate. The wet mycelium is collected from a rotary drum discharge filter Extracted three times with butyl acetate/ cold acetone. The colour of the extract can be improved by the addition of calcium hydroxide

57. GRISEOFULVIN: APPLICATIONS used in the treatment of various fungal skin infections. in the treatment of plant diseases caused by Biotrytis and Alternaria solani . used to treat infections such as ringworm, athlete's foot, jock itch, and fungal infections of the scalp, fingernails, or toenails.