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Production of Penicillin by Fermentation

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Penicillium chrysogenum is used as the commercial strain for the production of penicillin.

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Production of Penicillin by Fermentation

  1. 1. Ubaid Tariq Bhat M.Pharm Biotechnology Delhi Pharmaceutical Science Research University E. Mail: bhatubaid55@gmail.com 1
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  3. 3.  Also known as Penicillium notatum.  It is common in temperate and subtropical regions and can be found on salted food products, but it is mostly found in indoor environments, especially in damp or water-damaged buildings.  It is the source of several β-lactam antibiotics, most significantly penicillin which inhibits the biosynthesis of bacterial cell walls affecting lysis of the cell. 3
  4. 4.  Kingdom: Fungi  Division: Ascomycota  Class: Eurotiomycetes  Order: Eurotiales  Family: Trichocomaceae  Genus: Penicillium  Species: Chrysogenum 4
  5. 5.  Penicillium chrysogenum exhibits typical eukaryotic cell structure; it has a tubulin cytoskeleton which is used for motility. 5 TAM structure of P. Chrysogenum Structure of P. Chrysogenum
  6. 6.  This image displays the typical filamentous hyphae that contain many conidia.  The oblong structures in the image are conidia, the asexual spores of the fungus.  In P. chrysogenum, the conidia are blue to blue-green.  These conidia are the cause of pathogenicity in humans as in the cases of allergy and endophthalmitis.  The conidia originate from complexes known as conidiophores.  The growth of conidiophores begins when a stalk sprouts out of a foot cell.  The stalk swells at the end and forms a vesicle. Sterigmata form from the vesicle which give way to long chains of conidia. 6
  7. 7.  It produces the hydrophobic β-lactam compound penicillin.  Penicillium chrysogenum remains the primary producer of Penicilian G and Penicilian V  P. chrysogenum has been used industrially to produce Penicilian G and Penicilian V and Xanthocillin X, and to produce the enzymes polyamine oxidase, phosphogluconate dehydrogenase, and glucose oxidase.  Penicillium chrysogenum can be used to assist crops to fight off other pathogenic species. 7
  8. 8.  P. chrysogenum is high yielding strain and therefore most widely used as production strain. Inoculum Preparation:  Purpose is to develop a pure inoculum in an adequate amount. To do so various sequential steps are necessary like: 1) A starter culture is needed for inoculation. 2) After getting growth on solid media, one or two growth stages should allowed in shaken flask cultures to create a suspension, which can be transferred to seed tanks for further growth. 3) After about 24-28 hours, the content of the seed tanks is transferred to the primary fermentation tanks. 8
  9. 9. 4) All the bio parameters like temperature, pH, aeration, agitation etc. should be properly maintained. Bio parameters  pH: near 6.5  Temperature: 26°C to 28°C  Aeration: a continuous stream of sterilized air is pumped into it.  Agitation: have baffles which allow constant agitation (200rpm). 9
  10. 10.  Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms.  Generally, it contains a carbon source, nitrogen source, mineral source, precrsors and antifoam agents. Carbon Source  Lactose in a concentration of 6%.  Other carbohydrates like glucose & sucrose.  Complex as well as cheap sources like molasses, or soya meal can also be used which are made up of lactose and glucose sugars. 10
  11. 11. Nitrogen Source  Ammonium salts such as ammonium sulphate, ammonium acetate, ammonium lactate or ammonia gas are used for this reason.  Sometime corn steep liquor may be used. Mineral Source  These elements include phosphorus, sulphur, magnesium, zinc, iron, and copper which generally added in the form of water soluble salts. Precursors  Various types of precursors are added into production medium to produce specific type of penicillin. 11
  12. 12.  For example, if phenyl acetic acid is provided then only penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced.  Phenoxy acetic acid is provided as precursor for penicillin-V production.  When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore it is widely used in the production of penicillin-G. 12
  13. 13. Anti-foam agents  Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation. Recovery  The recovery of penicillin is carried out in three successive stages: 1. Removal of mycelium 2. Counter current solvent extraction of penicillin 3. Treatment of crude extracts 13
  14. 14.  At harvest the fermentation broth is filtered on a rotatory vacuum filter to remove the mycelium and other solids.  Phosphoric or sulfuric acids are added to lower the pH (2 to 2.5) in order to transform the penicillin to the anionic form.  Then the broth is directly extracted in a Podbielniak Counter Current Solvent Extractor with an organic solvent such as methyl isobutyl ketone, amyl acetate or butyl acetate.  Penicillin is then again extracted into water from the organic solvent by adding an adequate amount of potassium or sodium hydroxide to form a salt of the penicillin.  The resulting aqueous solution is again acidified & re- extracted with methyl isobutyl ketone. 14
  15. 15.  This shifts between water and solvent help in purification of the penicillin.  The solvent extract is carefully back extracted with NaOH and from this aqueous solution; various procedures are utilized to cause the penicillin to crystalize as sodium or potassium penicillinate.  The resulting crystalline penicillin salts are then washed and dried.  Sometimes the crude extract of penicillin is passed out from charcoal treatment to eliminate pyrogens; even sterilization can also be done. 15
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