microbial production of antibiotics penicillin and streptomycin

microbial production of antibiotics penicillin and streptomycin

1. Microbial production of Antibiotics (Penicillin and streptomycin) Subhananthini Jeyamurugan , 18py17, M.Sc., microbiology. Ayya nadar Janaki ammal college Sivakasi.

2. Antibiotic production Antibiotics are metabolites having preferential antimicrobial activity. Therefore, they are widely used for curing of human ailments caused by microorganisms. Antibiotic compounds are used either in their natural form or as semi synthetic derivatives; the latter arc usually produced by isolating the antibiotic nucleus and subjecting it to chemical modification. Antibiotics are produced by both fungi and bacteria. Antibiotics are generally produced in stainless steel fermenters (30,000-200,000 1 medium volume) used in the batch or fed-batch mode.

3. Microbial production of antibiotics: Penicillin Streptomycin

4. Penicillin: Penicillin is an antibiotic produced by microorganisms. Generally the penicillin antibiotic is produced by some Actinomycetes and some filamentous fungi. It is used against Gram positive bacteria as well as high dosage can be used against Gram negative bacteria. Penicillin is not harmful to plants, animals or human beings except in some cases of allergies.

5. Structure of penicillin

6. Mode of action The penicillin antibiotic acts on the cell wall synthesis of growing Gram positive bacteria. Penicillin is a β lactamase antibiotic. Beta lactam ring attaches to the DD trans peptidase. It results in inhibition of formation of cross linkage in the cell wall The growth and development of cell is stopped. Results in protoplast. Whereas gram negative cell do not loss its cell wall completely so called as spheroplast. After inhibition of cell wall the protoplast and spheroplast are formed. Due to increase in osmotic pressure the cell undergoes its lysis.

7. Production of penicillin: Penicillin fermentation is carried out anaerobically by submerged aerated fermentation process. Fermentation medium Fermentation organism Inoculum preparation Fermentation process Recovery and Harvest of penicillin.

8. Fermentation medium: Carbon source – Glucose, Lactose and Sucrose. Nitrogen source – Ammonium sulphate, Ammonium acetate, Ammonium lactate. Amino acids – Corn steep liquor. Precursors for penicillin G – phenylacetic acid. Precursors for penicillin X – Hydroxyphenylacetic acid. Precursors for penicillin V – Phenoxyacetic acid.

9. Fermentation organism: Initially   Penicillium notatum  strain was used for fermentation of penicillin. Then   Penicillium chrysogenum  was used. The strain  Penicillium chrysogenum   gave a yield of 200 units/ ml. Further this strain was improved by using strain improvement program and after strain improvement the strain gave a yield of 761 units /ml by using submerged cultured method. The strain is maintained in dormant state by using lyophilization technique, stored in liquid nitrogen in frozen state or in spore form.

10. Inoculum preparation: Pure culture of  Penicillium chrysogenum is prepared  in adequate amount . The primary stock is added in special agar, the special agar should provide sporulation of spores and the sporulating medium is used to prepare this working stock . The sporulated spores are suspended in SLS solution in a proportion of 1:10,000 further this spores are added in a nutrient medium of wheat bran plus nutrient and the flask are incubated for 5 to 7 days at 24 ° C . This medium is used for heavy sporulation . Now this spores are used as a innoculum in fermentation tank.  

11. Fermentation process: Fermentation of penicillin is carried out in trays or by submerged culture method. The 10 % of innoculum is added in the fermentor in aseptically. The temperature of about 25 ° C to 26 ° C is maintained . The sterile air supply is provided continuosly . The fermentation is carried out for about 3 to 5 days. During this fermentation process the samples are withdrawn aseptically and checked for yield of penicillin, pH and contamination. It is monitored for foam formation also if foam is produced it is controlled by antifoaming agent. 

12.  The pH of the fermentation media increases to 7.0 to 7.5 due to deamination and release of ammonia. Now at this point the micro-organism starts product synthesis by utilization of lactose and production of penicillin.  Initially during 20 to 30 hours the fungal spores utilizes carbohydrates and corn steep liquor and fungal spores develop as mycelium and further in the duration of 48 to 96 hours the mycelium starts production of penicillin product. The yield obtained is 3 % to 5 % and 1500 unit per milliliter of fermentation medium.

13. Harvest and Recovery: Initially the mycelium and other solid suspended particles are removed by filtration process. Treated with solvent extraction procedure for separation of penicillin. The penicillin is converted to anionic form by using phosphoric acid and sulphuric acid. Further the broth is extracted by using a organic solvent like methyl isobutyl ketone or amyl acetate. The extracted penicillin in solvent is back extracted in water by use of potassium hydroxide and sodium hydroxide in form of salt. The aqueous penicillin is acidified and re-extracted by methyl isobutyl ketone . Thus extraced penicillin is then evaporated, crystallized and standardised.

14. Streptomycin: Streptomycin is an bactericidal antibiotic drug belonging to the class of aminoglycosides . Used against TB. Derived from actinobacterium Streptomyces griseus Espescially used against gram negative bacteria

15. Chemical structure: Chemically it contains 3 sugars derived from glucose with C, O, N and H elements. Chemical formula : C21H39N7O12

16. Medium: Carbon source: starch, dextrin, glucose, glycerol Nitrogen source: natural agricultural by-products, soybean meal, corn steep liquor, cotton seed flour, casein hydrolate , yeast. Inorganic N salts – ammoniumsulphate and ammonium nitrate Animal oil, vegetable oil and mineral oils. Inoculum : S. griseus spores are maintained in soil stocks or lyophilizedin carrier are inoculated into sporulation medium which bulds up mycelial inoculum .

17. Hockenhul medium: Glucose – 2.5% Extracted soya meal – 4% Distillers dried soluble – 0.5% Sodium chloride – 0.25% pH – 7.3-7.5.

18. Fermentation process: Temperature :25 to 30 degree C pH range : 7-8 Time : 5-7 days High aeration and agitation It involves 3 phases which are as follows..

19. Phase 1 Initial fermentation phase – little production Rapid growth with production of mycelial biomass Proteolytic enzymatic activity releases NH3 from soyameal , raising the pH to 7.5 Characterized by the release of ammonia Carbon nutrients of soya meal are utilized for growth Glucose is slowly utilized with slight production of Streptomycin

20. Phase 2 Little production of mycelia Glucose added to the medium and ammonia released from soya meal are consumed pH remains fairly constant ranging between 7.6 - 8

21. Phase 3 Final phase of fermentation Depletion of carbohydrates from the medium Streptomycin production ceases and bacterial cells begins to lyse Ammonia from lysed cells increase the pH

22. Recovery and Purification: Mycelium is separated from broth byfilteration and streptomycin is recovered Broth is acidified, filtered and neutralized Then it is subjected to column containing cation exchange resin to absorb Streptomycin from the broth and column is washed with water and it is eluted with HCl . Dissolved in ethanol and filtered Acetone is used to precipitate Precipitate is washed with acetone and purified

23. Uses Treatment of diseases: TB, Plaque veterinary medicine against gram negative bacteria Pesticides and fungicides Cell culture Protein purification

24. Thank you