fermentaion M.Pharmacy P.chemistry 2nd sem

1. FERMENTATION
Pharmaceutical Process Chemistry

2. FERMENTATION
• The word fermentation comes from the Latin word "FERVER" which
means to "boil".
• Fermentation applies to growth of the microorganisms in a medium under
either condition that is aerobic or anaerobic to produce economically useful
products.
• Fermentation is a metabolic process that produces chemical changes in
organic substrate through the action of the enzymes which are produced
by microorganisms such as yeasts, or bacteria.

3. Types of Fermentation
1. Batch fermentation
2. Continuous fermentation
3. Anaerobic fermentation
4. Aerobic fermentation

4. 1. Batch Fermentation:-
Batch fermentation is a process where the
microorganism is inoculated into a fixed
volume of medium in a closed container.
As the growth takes place the nutrients that
are consumed and the product of growth
keeps getting stored in the fermenter.

6. 2. Continuous fermentation
• In this type of fermentation, the product
are removed continuously along with the
cells and the same is replenished with the
cell growth and addition of culture media.
• This type of fermentation is used for the
production of single cell protein,
antibiotics and organic solvents.

7. 3. Anaerobic fermentation
• Fermentation process carried out in the absence of oxygen is called anaerobic
fermentation.
• There are two types of the anaerobic fermentation for example:-
• FACULTATIVE ANAEROBE is an organism that are able to withstand by
aerobic respiration if oxygen is present, but is capable of switching to fermentation
by anaerobic respiration if oxygen is absent. eg. Lactic acid bacteria. however
certain organs like yeast require an initial aeration to build up high yield before
anaerobic conditions are created.
• OBLIGATE ANAEROBE are the species like clostridium species which cannot
withstand oxygen or remain active only in the absence of the oxygen.

8. 4. Aerobic fermentation
• A Fermentation process carried out in the presence of oxygen is called aerobic
• Oxygen is required for the reproduction and growth of microorganisms
(Yeast/Bacteria etc.)
• Yeast requires oxygen for a number of processes essential for reproduction.

10. PRODUCTION OF PENICILLIN
• Penicillin production is previously achieved by surface process i.e.. Solid state fermentation
and surface liquid fermentation. Now a days commercial production is carried out by fed
batch process
• Inoculum (Organism): Penicillium chrysogenum (improved strain)
1. Inoculum preparation:
• For inoculum preparation, spore from heavily sporulated working stocks are suspended in
water or non-toxic wetting agent's (sodium sulfonate 1: 10000)
• Theses spore are then added to flask containing wheat bran and nutrient solution for heavy
sporulation
• Incubate for 5-7 days at 24 ℃.
• Spore are then transferred to seed tank and incubated for 24-48 hours at 24℃ with aeration
and agitation for sufficient mycelial growth
• These mycelia can be used for production fermenter.

11. 2. Production fermentation:
• Method: fed-batch or batch
• Substrate: glucose, phenoxyacetic acid (fed component used for production of side chain),
Corn steep liquor, Additional nitrogen source i.e., soyameal, yeast extract, Lactic acid,
inorganic ions, growth factors
• Fermenter: stirred tank or air lift tank
• pH: set at 5.5 t0 6.0 which increased up to 7-7.5 (optimum) due to liberation of NH3 gas
and consumption of lactic acid. If pH is 8 or more, CaCO3 or MgCO3 or phosphate buffer
is added
• temperature: 25-27 ℃
• aeration: 0.5-1 vvm (initially more, latter less O2 )
• agitation: 120-150 rpm
• time: 3-5 days
• antifoam: edible oil (0.25%)

12. 3. Product recovery:
• Harvest broth from fermenter tank by filtration (rotary vacuum filtration)
• chill to 5-10 ℃ (because penicillin is highly reactive and destroyed by alkali and
enzyme)
• acidify filtrate to pH 2.0-2.5 with H2SO4 ( to convert penicillin to its anionic form)
• extract penicillin from aqueous filtrate into butyl acetate or amyl acetate (at this very
low pH as soon as possible in centrifugal counter current extractor)
• discard aqueous fraction
• allow the organic solvent to pass through charcoal to remove impurities and extract
penicillin from butyl acetate to 2% aqueous phosphate buffer at pH 7.5
• acidify the aq. Fraction to pH 2-2.5 with mineral acid and re-extract penicillin into
fresh butyl acetate ( it concentrated up to 80-100 times)
• add potassium acetate to the solvent extract in a crystallization tank to crystalize as
potassium salt
• recover crystal in filter centrifuge
• sterilization
• further processing
• packaging

13. An outline of flow chart for penicillin Fermentation

14. Production of Streptomycin
• Spores of S.griseus are inoculated into a medium to establish a culture with high mycelial
biomass for introduction into inoculum tank, using inoculum to initiate the fermentation
process.
• Yield in production vessel responds to high aeration & agitation conditions. Other conditions
involve-
• Temperature range 25-30°C pH range 7-8 Time 5-7 days
• The fermentation process for production of Streptomycin involves 3 phases.
PHASE 1
• Initial fermentation phase and there is little production of streptomycin.
• Rapid growth with production of mycelial biomass.
• Proteolytic enzymatic activity of S.griseus releases NH3 from soya meal, raising the pH to 7.5
• Characterized by release of ammonia.
• Carbon nutrients of soya meal are utilized for growth.
• Glucose is slowly utilized with slight production of Streptomycin

15. PHASE 2
• Little production of mycelia.
• Glucose added to the medium & the NH3 released from soya meal are consumed.
• pH remains fairly constant ranging between 7.6 to 8.
PHASE 3
• Final phase of fermentation.
• Depletion of carbohydrates from medium.
• Streptomycin production ceases & bacterial cells begin to lyse.
• Ammonia from lysed cells increase the pH.
RECOVERY & PURIFICATION
• Mycelium is separated from broth by filtration & streptomycin is recovered.

16. • The streptomycin is dissolved in methanol & filtered.
• Acetone is used in filtrate to precipitate the antibiotic.
• Precipitate is washed with acetone & dried in vacuum.
• Purification is done by dissolving in methanol to form pure S. chloride complex.
Further by, adsorbing it onto activated charcoal & eluting with acid alcohol.

17. Production of Vitamin B12
Production by Streptomyces olivaceus :
A. Preparation Of Inoculum:
• Pure slant culture of S. olivaceus is inoculated in 100-250ml of inoculum medium, contained
in Erlenmeyer flask.
• Seeded flask is incubated on platform of a mechanical shaker to aerate the system.
• This flask culture is then subsequently used to inoculate larger inoculum tanks.
B. Production Medium :
• Consist of carbohydrate, proteinaceous material, and source of cobalt and other salts.
• It is necessary to add cobalt to the medium for maximum yield of cobalamin. Cyanide is
added for conversion of other cobalamins to vitamin B12.
C. Sterilization of the medium :
• Sterilization can be done batch wise of continuously.
• ‣ Batch - medium heated at 250°F for 1 hour.
• › Continuous - 330°F for 13 min by mixing with live steam

18. D. Temperature, pH, Aeration and Agitation :
• Temperature: A temperature of 80°F in production tank is satisfactory during fermentation.
• pH: At starting of process pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis
of mycelium. pH 5 is maintained with H2SO4 and reducing agent Na2SO4.
• Aeration and Agitation Optimum rate of aeration is 0.5 volume air/volume medium/min.
E. Antifoam agent, Prevention of contamination : Antifoam agent: Defoaming agents like soya bean
oil, corn oil, lard oil and silicones can be used.
Prevention of contamination : Essential to maintain sterility, contamination results in reduced yields,
equipment's must be sterile and all transfers are carried out under aseptic conditions.
F. Recovery: During fermentation, most of cobalamin is associated with the mycelium; boiling mixture at
pH 5 liberates the cobalamin quantitatively from mycelium. Broth containing cobalamin is subjected to
further process to obtain crystalline B12.

19. Production of Vitamin B2
Upstream
• The upstream processes include preparation and sterilization of the medium.
• The medium's composition does not allow sterilization of all components mixed together and
using classical batch conditions (121°C, 20 minutes). Therefore, the medium would be
divided into several groups:
I. glucose and sunflower oil,
II. peptone, yeast and malt extracts,
Ⅲ. salts in water
IV. methionine.
• The latter is sterilized by filtration.
• Sulfuric acid does not require sterilization.

20. Fermentation
• In several steps the necessary seed cultures are prepared in different seed fermenters.
• The last seed culture is the start inoculum for the main fermentation.
• The duration of a seed-fermentation is around 50 hours, while the main fermentation
lasts about 500 hours.
• During this time the strain produces 27 g/L riboflavin.
• Fermentation requires aeration accomplished by a gas compressor and a sterile filter.
• Exhaust gases are filtered by a second filter.
• A small fraction of the harvested broth is put into another tank and is used as inoculum
for the next batch.

21. Downstream
• After fermentation the broth is harvested into the harvest tank.
• Part of the product crystallizes in the fermenter and also in the harvesting tank. Crystallization is
completed in the crystallizer by evaporation of some of the water.
• Afterwards the suspension is stored in tank.
• From the decanter three streams are harvested, two liquid phases and the cell/crystal suspension
.To achieve higher purity, a washing step is used with a second separation.
• The last step is drying, either using a spray dryer to obtain a powdered product or applying a
spray granulation to obtain granulate.

22. Production of Lovastatin
• Culturing of A.terreus by Solid State Fermentation (SSF)
Wheat bran (40g) as substrate
Inoculated with spore suspension (107/8ml spores) of A. terreus.
Incubated at 28°C for 7 days
Inoculated substrate was dried at 40°C for 24h
Crushed to powder
Ethyl acetate (150 ml) was added
Filtrate was dried using rotary vacuum

23. Purification of lovastatin
One gram of dried crude lovastatin extract was loaded on to pre-packed silica gel
column
Elution with benzene (100%), And combination of Benzene: Acetonitrile in the
following ratio 95:5, 90:10, 85:15, 80:20 Acetonitrile (100%)
Thin Layer Chromatography (TLC)

24. REFERENCE
• https://www.onlinebiologynotes.com/penicillin-production-commercially-by-
fermentation-biotechnology/
• https://www.slideshare.net/poojabasule1/biopharmaceutics-or-process-chemistry-
fermentation
• https://www.slideshare.net/SoodShipra/streptomycin-production
• https://www.slideshare.net/mahyar_emee/riboflavinvitamin-b2-fermentation-process
• https://www.slideshare.net/vijaysrampur/fermentation-pptx