This one describe about fermentation principles, processes and pharmaceutical applications.

1. FERMENTATION
DR.Senthil Prabhu R
COP,MMC,Madurai

2. DEFINITION
Process of breaking bigger organic particles
into smaller ones with the help of enzymes
released by the microbes

3. TYPES OF FERMENTATION
• Aerobic
• Anaerobic
• 1)True anaerobic
• 2) Facultative anaerobic

4. AEROBIC FERMENTATION
• Need oxygen for growth and metabolism
• Examples Of Fermentative Products:
• Penicillin from Penicillium notatum,
Penicillium chrysogenum
• Citric acid from Aspergillus niger
• Streptomycin from Streptomyces griseus
• Riboflavin from Eremothecium ashybii,
Ashbya gossypii

5. TRUE ANAEROBIC OR
STRINGENT ANAEROBIC
• Do not required oxygen both for growth and
metabolism
• Examples of fermentative products
• Acetone or Butanol from Clostridium
acetobutylicum.
• Lactic acid from Lactobacillus delbruckii,
Lactobacillus leishmanii, Lactobacillus caseii,
Lactobacillus bulgaricus, Streptococcus lactis

6. FACULTATIVE ANAEROBE
• Need oxygen either for growth or for
metabolism
• Example of fermentative product:
• Alcohol from Saccharomyces cerevisiae,
Saccharomyces ellipsoidens, Candida Utilis,
Candida pseudotropicalis
• Latest development is from Zymomonas
mobilis

7. Pretreatment for the media
• Black strap molasses: Initial sugar
concentration is 46%. Determined by
Balling hydrometer. This much high
concentration itself will kill the
organism which gives the
fermentative product. Hence,
pretreat this by reducing the
concentration of sugar to 2 to 8 % by
diluting with water or water plus
stillage

8. Pretreatment of media
• Lactose of Whey can be used after removal of
proteins
• Sulphite of waste liquor after removal of SO2

9. PROCESSES OF FEMENTATION
• Stationary mat culture
• Shake flask fermentation
• Deep aerated submerged fermentation

10. STATIONARY MAT CULTURE
• Medium is stagnant and inoculated with
organism. The bottle is shaken then and there
so that organisms receive oxygen from top
and medium nutrients from down.
• Disadvantages: Yield is less( exception is
Diastase ); while mixing the contents of all the
bottles containing the fermentative product,
even if one bottle is contaminated then all
bottles will get contaminated.

11. SHAKE FLASK METHOD
• The conical flasks of different capacity can be loaded
with two third volume of medium and inoculated
with organism, shaken at the incubated shaker at
specific temperature.
When the organism goes down, it picks up the
nutrients and when it comes up , it picks up the
oxygen. There should be sufficient space for the
contents to get shaken. The shaker speed is also
deciding factor in giving yield.

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13. DEEP AERATED SUBMERGED FERMENTATION
• For large scale production, we can use
• Deep aerated means, supply of air from bottom(submerged).
• Supply of sterile air through bacteria proof filter
• Baffles at the sides to avoid vortexing
• There are ports for pH, dissolved oxygen content, sample
withdrawl, temperature , for adding antifoaming agent,
inoculum

15. DEEP AERATED SUBMERGED FERMENTATION
Agitator occupies two third diameter of fermenter
There are ports at down to recover the product, spent
broth(means which has been utilized by the respective
organism to produce the required product)
Fermenter itself acts as an autoclave by supplying steam from
external boiler.
Capacity may vary from 1 liter to gallons.
Do not continue the fermentation process for longer duration
for a meager increase in yield. Means, suppose if you have
got 90 % yield within 3 days and to get another 10 % yield
don’t extend for another 3 days. It is a waste of time, man
power, electricity. Moreover, the consistency of spent broth
should be easily filterable.

21. ALCOHOL FERMENTATION
• pH of fermentation is 4 to 5. No other organisms
can grow in this pH. To maintain the pH, lactic
acid can be used as buffer.
• Alcohol is itself an antimicrobial
• As the sugar concentration used is high, that itself
won’t allow the growth of microbes. For example,
in case of syrup I.P, we don’t need preservative
since it exhibits high osmotic pressure. No
microbes can grow.

22. ALCOHOL FERMENTATION
Name of the microbe
• Sacharomyces cerevisiae
• Sacharomyces ellipsoidens
• Candida utilis
• Candida pseudotropicalis
Medium used
• Black strap molasses
• Sulphite of waste liquor
• Lactose of Whey

23. • In the first part, the yeast breaks down glucose
to form 2 pyruvate molecules.
• This part is known as glycolysis.
• In the second part, the 2 pyruvate molecules are
converted into 2 carbon dioxide molecules and 2
molecules of ethanol, otherwise known as
alcohol.
• This second part is called fermentation.

26. PENICILLIN FERMENTATION
• Organism: Penicillium chrysogenum,
Penicillium notatum
• Type: Aerobic fermentation
• History: Both organisms are mould type. Able
to grow at pH of 5.5 -6.0. Inventor is Alexander
Flemming in 1929. It was Penicillin F. Narrow
spectrum. Active against gram +ve, Nocardia
and actinomycetes but not against gram –ve
except at higher levels.

27. PENICILLIN DETAILS
• Types: G,V,F,X
• Mechanism: interferes with cell wall synthesis
of invading organisms and is active only
against growing cells.

28. CHEMISTRY OF PENICILLIN
Precursor : C6H5CH2COOH
R group : C6H5CH2
General Name : PENICILLIN G
Chemical name : Benzyl Penicillin

29. CHEMISTRY OF PENICILLIN
Precursor : C6H5OCH2COOH
R group : C6H5OCH2
General Name : PENICILLIN V
Chemical name : PHENOXY METHYL Penicillin

30. CHEMISTRY OF PENICILLIN
Precursor : C6H4OHCH2COOH
R group : C6H5CH2
General Name : PENICILLIN X
Chemical name : Hydroxy methyl Penicillin

31. Other types of Penicillin
R= CH3CH2CH=CHCH2, PENICILLIN F,
2-pentenyl penicillin
R= CH3(CH2)3 CH2, DIHYDRO PENICILLIN F, n-
Penetenyl Penicillin
R= CH3(CH2)5CH2, PENICILLIN K, n-heptyl
Penicillin

32. • Penicillin in its structure itself has 2 amino
acids namely L-Cysteine and D-Valine. Hence,
if you use these 2 amino acids ,then it is
possible to get maximum yield.

33. Problem to be solved
• In Penicillin fermentation, sometimes coliform
group of bacteria like Staphylococcus aureus
contaminates the product and degrades
Penicillin (active form) into inactive Penicilloic
acid(inactive form). Because these organisms
release Penicillinase enzyme to degrade the
Penicillin.
• Solution to the problem: Use
NTG(Nitrosoguanidine) in the medium

34. Penicillin converted into Penicilloic
acid in presence of Penicillinase

35. PROCESSES
• 1. Stationary mat culture
• 2. Deep aerated submerged fermentation
• 1. Stationary mat culture:
• Medium sterilized in shallow layers in bottles,
flasks, trays or pans made of metal or glass
• Add spores as inoculum. Incubate at 24 to 28°C
for 6 to 7 days.
• Harvest Penicillin by pooling all the bottles.

37. DISADVANTAGES OF STATIONARY MAT CULTURE
• O2 penetrates poorly into the mycelial mat
• Organisms at surface do not receive nutrients
properly
• When we pool all the bottles, even if one
bottle is contaminated with penicillinase
enzyme then all will get spoiled.

38. DEEPAERATED SUBMERGED FERMENTATION
• Gives more yield than stationary mat culture
• Agitation: Organism should receive nutrients
and oxygen for their growth. Hence, agitate.
Agitator occupies 2/3 rd diameter of
fermentation tank. Blades attached to central
shaft. Rotated at a speed which delivers 2 to3
KW of power per kilolitre of aerated liquid.

39. AERATION
• Sterile air supplied through SPARGER( small
holed pipe) when the viscosity of the medium
is high. Impeller or agitator itself is sufficient
to pass air through the medium.
• Required for Penicillin fermentation = 0.5 VVM
to 1 VVM(Volume of air per Volume of liquid
per Minute)

40. AIR STERILIZATION
• Oxygen supplied should be free from
contamination.
• To avoid the Penicillinase action, sterilize the
air supplied or add NTG(Nitrosoguanidine).
• Air is sterilized by passing thorugh fibrous
depth filter or polymeric filter(Poly vinyl
alcohol/PTFE).

41. AIR STERILIZATION
• Pore size in these filters are very small. It
should be passed in the following way.
• Prefilter →Compressor→Separator→ Mist
eliminator→ Fibrous air filter→Local
membrane filter→Tank

42. MASTER STOCK STRAIN
• Identity of strain producing Penicillin should
not be lost. Frequent subculturing and
periodic transfer from one media to other
media may lead to contamination. Preserve in
the following way.
• A) Freeze dry the spores
• B) add spores in sterilized soil-sand mixture

43. MEDIA
• JACKSON MEDIUM(1955)
Corn steep liquor 3.5% (nitrogen source)
Lactose 3.5% (carbon source for prodn.)
Glucose 1%(carbon source for growth
CaCO3 1%( Buffer)
KH2PO4 0.4% (buffer)
Soyabean oil 0.25%(antifoaming, carbon
source, increases the yield)
Precursor for particular type of penicillin = 0.05g

44. MEDIUM
• SYLVESTER & COGHILL(1954)
Corn steep liquor 30G (nitrogen source)
Lactose 30G(carbon source for prodn.)
Glucose 5G(carbon source for growth
CaCO3 3G( Buffer)
ZnSO4 0.444G (buffer)
Mg2sO4 0.25g
Sodium Nitrate 3 g
Soyabean oil 0.25%(antifoaming, carbon
source, increases the yield)
Precursor for particular type of penicillin = 0.05g

45. Feed and Feed Management
• Either continuously or intermittently, supply
the feed of medium or precursor. The process
should not stop because of lack of supply.
• To achieve this, have best knowledge on
biosynthesis of Penicillin

46. SUGAR FEED
• Glucose for growth
• For production, Lactose is the carbon source.
• Lactose is degraded into Glucose and
Galactose. But this is a very slow process.
Organism starve for Glucose. During that time,
it releases some product which is nothing but
Penicillin.

47. NITROGENOUS FEED
• Supply continuously or intermittently.
• Small addition of ammonia and urea are
sufficient.
• Glutamine plays main role in penicillin
production.
• When Ammonia level is low, glutamine level
is high and hence Penicillin production is
more.

48. LIPID Nutrients
• Increases growth and yield also.
• Fatty acids like Lard Oil, Soybean oil and fatty
acids of greater than 14 carbon chain lengths
and their esters are especially effective. Some
oils are antifoaming agents.

49. PRECURSOR FEED
• It should not be very high at initial stage.
Otherwise, it is toxic to organism.
• Small addition is a trigger for the early
formation of enzyme.
• Regular feeding of precursor is started some
hours after the growth has been initiated.

50. Media sterilization
• Either in batch or continuously
• Continuous is someway advantageous than
that of batch.
• It helps in heat recovery and turn round time is
lessened.

51. Disadvantages of continuous sterilization
1) protein containing medium leads formation of
a thin film of protein leading to excessive heat
2) property of medium gets affected
3)particles in the slurries which are moving
faster may not be sterilized.
From these angles, batch sterilization is better.

52. INOCULUM DEVELOPMENT
• From master stock, revive the culture on a
good sporulating medium at 25°C for a 4 to 6
days. Surface turns into green
• Prepare spore suspension from the above and
store at 4°C until use. You can use this for 3 to
4 weeks by keeping in cold temperature.
• Inoculate the Germinator containing
germinating medium at 25°C for 2to 3 days.

53. Inoculum devt.(contd.)
• Transfer the biomass into seed tank containing ten
times more medium as that of germinator but without
Lactose and Precursor.
• This avoids extended LAG phase.
• Run this for 15 to 20 hours also at 25°C.
• Check for the absence of contamination and then
transfer 8 to 10% to the production Fermentor
containing Lactose, precursor.
• As the LAG phase is virtually absent, production of
penicillin sets within 20 to 24 hours.
• During the process, in between withdraw the samples
and check for absence of contamination.

54. PROCESS PARAMETERS
• pH: 5.5 to 6.0
• While production starts, the pH changes and
reaches to pH of 8. Penicillin production is less at
this pH of 8. Increase in pH is due to utilization of
corn steep liquor. CSL has Phenylalanine and its
breakdown products are Phenethylamaine and
phenyl acetic acid.
• Amino acids from CSL are deaminated and
ammonia production sets in leading to pH.
• Hence, use buffering agent like CaCO3 or NaOH
or HCl or Mg2CO3

55. Process parameters(contd.)
• During initial stage of 20 to 30 hours, fungal
growth becomes thick and heavy due to
utilization of Glucose and Corn Steep
Liquor(CSL)
• Penicillin yields with time are linear from 48
to 96 hours. Lactose is slowly degraded which
is helpful for Penicillin production.

56. CYCLE TIME
• Both short (120-140 hrs) and extended
cycles(180-240hrs) are known in commercial
practice.
• Long cycle runs utilize the nutrients fully and
time.
• In practice, short cycle runs give more output
at the enhanced cost of the process.

57. HARVEST TIME
• Do it at suitable time
• When production falls below a certain threshold ,
then not to continue the process. Recover it.
• Moreover, the status of mycelium and filtration
characteristics determine the harvest time.
• If recovered earlier, medium unutilized properly.
• If delayed , filtration rate is affected. Hence do the
recovery at correct time.

58. PRODUCT RECOVERY
• Fermented broth is filtered using rotary
vacuum filter.
• Filtrate is converted into acidic(anionic) form
by adding H3PO3 or H2SO4 by bringing pH to
2 to 2.5
• Extract using Podbielnaik counter current
solvent extractor with amyl acetate, methyl
isobutyl ketone, butyl acetate.

59. Product recovery(contd.)
• Back extracted using aqueous solvent like
NaOH or KOH. From this , we get salt of
Penicillin
• Again acidified and re extracted with organic
solvent. These shifts between w ater and
organic solvent help in purification
• Again treated with NaOH or KOH solution to
get salt form

60. PRODUCT RECOVERY
• By azeotropic distillation or Ion exchange
adsorption method, Penicillin is separated.
This salt form of Penicillin is obtained in
crystalline form after filtration.
• Crystalline mass is washed and dried. This is
the final product
• Solvents used can be recovered for reuse.

61. • Important notes
1. Fermenter and its components.
2. Types of fermentation process.
3. Various types of fermentation media.
4. Fermentation products.

62. Thank You