Fermentation process in process chemistry

Fermentation process in process chemistry

1. MICROBIAL PRODUCTION OF ANTIBIOTICS
(PENICILLIN AND STREPTOMYCIN)
Subhananthini Jeyamurugan,
18py17, M.Sc., microbiology.
Ayya nadarJanaki ammalcollege 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 anaerobicallyby
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 –phenylaceticacid.
Precursors for penicillin X –Hydroxyphenylaceticacid.
Precursors for penicillin V –Phenoxyaceticacid.

9. FERMENTATION ORGANISM:
InitiallyPenicilliumnotatumstrain was used for fermentation of penicillin.
ThenPenicilliumchrysogenumwas used.
The strainPenicilliumchrysogenumgave 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 lyophilizationtechnique,
stored in liquid nitrogen in frozen state or in spore form.

10. INOCULUMPREPARATION:
Pure culture ofPenicilliumchrysogenumis preparedin adequate amount.
The primary stock is added in special agar, the special agar should
provide sporulation of spores and the sporulatingmedium is used to
prepare this working stock.
The sporulatedspores 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 innoculumin fermentation tank.

11. FERMENTATION PROCESS:
Fermentation of penicillin is carried out in trays or by submerged
culture method.
The 10 % of innoculumis added in the fermentorin 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 milliliterof 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
ketoneor amyl acetate.
The extracted penicillin in solvent is back extracted in water by use of
potassium hydroxide and sodium hydroxide in form of salt.
Theaqueous penicillin is acidified and re-extracted by methyl isobutyl ketone.
Thus extracedpenicillin 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 actinobacteriumStreptomycesgriseus
Espesciallyused 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 –ammoniumsulphateand
ammonium nitrate
Animal oil, vegetable oil and mineral oils.
Inoculum: S. griseusspores are maintained in soil stocks or lyophilizedincarrier are
inoculated into sporulationmedium which buldsup mycelialinoculum.

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 mycelialbiomass
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 byfilterationand streptomycin is
recovered
Broth is acidified, filtered and neutralized
Then it is subjected to column containing cationexchange 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