This presentation detail introduction to Fermentation. This Presentation contains, Introduction to Fermentation. Media Formulation. Historical Background. Types of Fermentation. Production of Antibiotics. Production of Vitamins Production of Statins.

1. FERMENTATION
Presented By: Guided By:
Laxman D Bulbule Mr. M. Bhosale Sir
M Pharm II Sem M. Pharm
Department of Pharmaceutical Chemistry.
2020-2021
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Pravara Rural College of Pharmacy, Pravaranagar

2. Table of Contents:
 Introduction to Fermentation.
 Media Formulation.
 Historical Background.
 Types of Fermentation.
 Production of Antibiotics.
 Production of Vitamins
 Production of Statins.
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3. What is the Fermentation?
 Fermentation is the Metabolic Process in which specifically selected
Population of microorganism converts Primary Metabolites into
Secondary Metabolites i.e. conversion of Sugar into the Acids,
Gases & Alcohol.
 The different Microorganism Used in Fermentation are:-
a) Bacteria: Acetobacter lacti, Acetobacter woodi.
b) Algae: Spirulina maxima, Chlorella sorokiniana.
c) Fungi: Aspergillus oryzae, Aspergillus niger, Saccharomyces
cervisae.
d) Actinomycetes: Streptomyces griseus, Streptomyces noursei.
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4.  Fermenters are required to carry out the fermentation.
 Fermenters are the closed vessels with all required Accessories to
provide Aseptic & Strict Controlled Environment to perform
Microbial or Biochemical Reaction, mediated by Microbial Cells.
 The medium on which the Microorganism grows to bring out the
Fermentation, such medium is called as Nutrient Medium.
 Nutrient media generally contains:
i. Water
ii. Carbohydrates
iii. Oil & Fats
iv. Growth Factor
v. Buffers
vi. Oxygen
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5. Media Formulation:
 Water: It acts as a Major Component of Nutrient Media, plays
major & vital role in Fermentation Procedure. Water is mainly used
for vaporization which helps to convert Primary Metabolites into
secondary Metabolites.
 Carbohydrates: Sucrose is used as a Energy source in Nutrient
Medium. They are taken in the form of Powder or Juice.
 Oils & Fats: They act as Anti-Foaming Agent. Vegetable Oils &
Nitrogen substrate are used as Anti foaming Agent.
 Growth Factor: They used for Development of Media. Vitamins,
Amino acids, Fatty acids, Sterol, Biotin are used as Growth
Factor.
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6.  Buffer: To Control the PH Value. Calcium Carbonate, Phosphates,
Sodium Hydroxide, Sulfuric Acid are used as Buffer.
 Oxygen: Helps in Fast Metabolism. High Oxygen Demand(HOD)
may occur due to less supply of oxygen that affect on rate of
Fermentation.
Nutrient Medium & Optimum Condition
Microorganism Fermenter Product
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7. History of Fermentation:
 The Development in Traditional Biotechnology (upto1971) is due to
Fermentation.
 In 1837 Charles Cagniard, T. Schwann & K.T Friedrich
independently published papers concluding as result of microscopic
investigation, that Yeast is Living Organism.
 Schwann boiled Grape Juice to kill the yeast & found that would not
Fermentation occur until new was added.
 The turning point came when Louis Pasteur, during the 1850s &
1860s repeated Schwann’s experiment and showed Fermentation is
initiated by Living Organism.
 In 1857 Pasteur showed Lactic Acid Fermentation is caused by
Living Organism.
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8.  In 1860 Louis Pasteur demonstrated how bacteria cause Souring in
Milk.
 So Louis Pasteur became First Zymurgist.
 In 1877 Pasteur published his famous paper on Fermentation
“Etudes sur la Biere”. Which was translated into English in 1879
as “Studies on Fermentation”. In this he defined fermentation as
“Life Without Air”
 But real successes on Fermentation came in 1897 when German
Chemist Eduard Buechner ground up yeast, extracted a juice from
them, then found to his amazement this “Dead” liquid Ferment a
Sugar Solution, forming Carbon dioxide & Alcohol.
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9.  Buechner’s result are considered to mark the Birth Of
Biochemistry. From that time on, the term Enzyme came to be
applied to all Ferments. It was then understood Fermentation is
cause by Enzyme produced by microorganism.
 In 1907 Buechner won the Nobel Prize in Chemistry for his work.
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10. Types of Fermentation:
 Based on Respiration:
1. Aerobic Fermentation.
2. Anaerobic Fermentation.
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11. Aerobic Fermentation:
 Aerobic Fermentation or Aerobic Glycolysis is a metabolic process
by which cells metabolize Sugar via Fermentation in the Prescence
of Oxygen and occurs through the repression of normal respiratory
metabolism. It is referred as the Crabtree Effect in Yeast.
 Aerobic Fermentation helps to covert nutrient such as Glucose &
Glutamine more efficiently into Biomass by avoiding unnecessary
catabolic oxidation of such nutrient into Carbon dioxide.
 Chemical Reaction of Aerobic Fermentation:
C6H12O6 + 6O2 6CO2 + 6H2O + 36ATP.
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12. Diagram Showing Aerobic Fermentation.
 Glycolysis is the first step in Aerobic fermentation which occurs in
the cytoplasm.
 This process breaks down glucose into two Pyruvate molecule.
 2 ATP & 2 NADH are the yield of this process.
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13.  Now the Pyruvate molecule synthesized in Glycolysis undergoes
Oxidative Decarboxylation to form Acetyl-CoA.
 During this process 2NADH is produced.
 This Acetyl-CoA enters into Citric Acid Cycle or Krebs Cycle.
Krebs cycle occurs in the Mitochondrial Matrix.
 A complete break down of Acetyl-CoA into Carbon dioxide occurs
in Krebs cycle, regenerating the starting compound Oxaloacetate.
 During Krebs cycle 2GTPs, 6NADH & 2 FADH2 are produced.
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14.  The last step in Aerobic Fermentation is Oxidative
Phosphorylation i.e. ETC.
 It occurs in inner membrane of Mitochondria.
 In ETC NADH & FADH2 Produced during Glycolysis, Pyruvate
oxidation, Krebs cycle are passed from one member of the transport
chain to another in series of Redox reaction to generate ATP.
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15. Anaerobic Fermentation:
 Anaerobic fermentation is referred to chemical break down of
organic substrate by microorganism into Ethanol or Lactic Acid in
absence of Oxygen.
 Typically it gives Effervescence & Heat.
 Fermentation occurs in the Cytoplasm of microorganism such as
Yeast, Parasitic worms & Bacteria.
 Initially Glucose enters into glycolysis pathway to produce
pyruvate.
 The net yield of Fermentation is only 2 ATPs.
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16.  Based on pyruvate oxidation pathway, Anaerobic Fermentation
consist of two types:
A. Ethanol Fermentation.
B. Lactic Acid Fermentation.
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17. Ethanol Fermentation:
 Ethanol fermentation mainly occurs in Yeast in absence of Oxygen.
 In this process, removing the CO2 results in the decarboxylation of
Pyruvate into Acetaldehyde.
 Acetaldehyde then converted into Ethanol by using the Hydrogen
atom of the NADH produced during Glycolysis.
 The effervescence occurs due to the release of carbon dioxide gas
into the medium.
 Chemical Reaction:
C6H12O6 2C2H5OH + 2CO2 + 2ATP
Glucose Ethanol
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18. C6H12O6 Glycolysis 2(CH3-CO-CO-OH) Decarboxylase 2(CH3-COH)
Glucose Pyruvic Acid Acetaldehyde
2ADP 2ATP CO2 NADH + H+
2NAD+ 2NADH Dehydrogenase
2Pi 2Pi, 2H2O NAD+
2(CH3-CH2-OH)
Ethanol
Table showing Ethanol Fermentation
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19. Lactic Acid Fermentation:
 Lactic Acid fermentation mainly occurs in Bacteria in the absence
of Oxygen.
 During Lactic Acid Fermentation, the pyruvate coverts into Lactic
Acid by using the Hydrogen atoms of the NADH produced during
Glycolysis.
 Chemical Reaction:
C6H12O6 2C3H6O3 + 2ATP
Glucose Lactate
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20. C6H12O6 Glycolysis 2 (CH3-CO-CO-OH) Dehydrogenase 2(CH3-CHOH-CO-OH)
Glucose Pyruvic Acid Lactic Acid
2 ADP 2 ATP NADH + H+ NAD+
2Pi, 2 NAD+ 2 NADH
2Pi, H2O
Table Showing Lactic Acid Fermentation:
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21. Glucose
2ATP Glycolysis
2 Pyruvate
Oxygen NO Oxygen
Aerobic Anaerobic
Fermentation Fermentation
Krebs cycle Alcohol Lactate
ETC
Chart: Aerobic vs Anaerobic fermentation
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22. Role of Fermentation in Industrial Production:
 Production of Antibiotics: Penicillin & Streptomycin.
 Production of Vitamins: B2 & B12.
 Production of Statins: Lovastatin, Simvastatin.
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23. Production of Penicillin:
 Antibiotics are antimicrobial agents produced naturally by other
microbes.
 The first antibiotic was discovered in1896 by Ernest Duchesne and
in 1928 rediscovered by Alexander Fleming from the filamentous
fungus Penicillium notatum.
 Penicillin was the first important commercial product produced by
an aerobic fermentation.
 Penicillin is active against certain Gram Positive bacteria.
 It is water Soluble
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24. Commercial Production of Penicillin:
 Spores of the Fungi Penicillium chrysogenum is the source of
inoculum.
 This spore are suspended in water or non toxic Lauryl Sulphonate.
 Then added to the flask containing wheat bran and nutrient solution.
 Jackson’s medium is used for production of penicillin i.e. in 1958
Jackson prepared a media for production of Penicillin.
 Aeration or supply of oxygen in fermenter is the limiting factor in
penicillin biosynthesis as well as temperature and PH should be
maintained.
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25. Process:
 The inoculum is inoculated on the medium which contain sugar,
nitrogen source, buffers, minerals & added into Bioreactor or
Fermenter.
 In 6 days 10 times remove 30% of culture & add 30% new medium
into Fermenter.
 Removed 30% culture is filtered with rotating filter. The filtrate is
dissolved in Butylacetate.
 Now the potassium ions are added to precipitate salt of penicillin.
 This ppt is washed, filtered and then dried to get 99.5% pure
penicillin
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26. Production of Streptomycin:
 Streptomycin is Bactericidal Antibiotic belongs to class
Aminoglycosides.
 Derived from Actinobacterium Streptomyces griseus.
 Used in treatment of TB, useful against Gram Negative Bacteria.
 It is used as Pesticide & fungicide.
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27. Commercial Production of Streptomycin:
 Inoculum: Spores of Streptomyces griseus maintained in soil stock.
 The culturing media for Streptomycin consist of starch, Dextrin,
Glucose, Glycerol as a source of Carbon. Soyabean meal, Corn
steep liquor, Cotton seed flour is used as N source.
 Inorganic N salts like Ammonium sulphate & Ammonium nitrates
are also used.
 Animal oil, Vegetable oil, Mineral oils are used.
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28. Process:
 Spores of Streptomyces 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.
 The fermentation process for production of Streptomycin involves 3
phases:
 Phase-1: Initial fermentation phase and there is little production of
streptomycin. Rapid growth of culture.
Proteolytic enzymatic activity of Streptomyces griseus release NH3
soya meal, raising the PH to 7.5.
Carbon nutrients of soya meal are utilized for growth.
Glucose is slowly utilized with slight production of Streptomycin.
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29.  Phase-2: Glucose added to the medium & the NH3 released from
soya meal are consumed.
PH remains fairly constant ranging between 7.6-8.
 Phase-3: Final phase of Fermentation.
Depletion of carbohydrates from media.
Streptomycin production raises & bacterial cell begin to lyse.
Ammonia from lysed cells increase the PH.
The streptomycin is dissolved in methanol & filtered.
Acetone is used in filtrate to precipitate the antibiotic.
Purification is done by dissolving in methanol.
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30. Production of Vit-B2 (Riboflavin):
 Riboflavin was first isolated by Blyth in 1879 from whey. The water
soluble, yellow, fluorescent material was named Lactochrome.
 According to IUPAC rules, Riboflavin is called 7,8 dimethyl-10-(d-
1-ribityl)isoalloxazine, also known as vitamin B2 or Lactoflavin.
 The daily human demand for riboflavin is around 1.7gm &
deficiencies lead to various symptoms such as version of dermatitis.
 The vitamin can not be stored in the body and a constant intake is
required.
 Green plants, most bacteria & moulds can produce their own
Riboflavin.
 At present three organism are used for the industrial production of
riboflavin: Fungi Ashby gossypii, Yeast Candida famata, Bacteria
Bacillus subtills.
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31. Procedure:
 Production process involves 3 processes: 1) upstream, 2)
Fermentation,
3) Downstream.
1) Upstream: The upstream process include preparation and
sterilization of the medium.
The medium composition does not allow sterilization of all
components mixed together. The medium divided into several groups:
I. Glucose & Sunflower oil.
II. Peptone, Yeast & malt extract.
III.Salts in water.
IV.Methionine.
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32. 2) Fermentation: In several steps the necessary seed cultures are
prepared in different seed fermenter.
The last seed culture is the start inoculum for the main fermentation.
The duration of a seed-fermentation is around 50 hours, while main
fermentation last about 500 hours.
Fermentation requires aeration accomplished by a gas compressor and
sterile filter.
A small fraction of the harvested broth is put into another tank and is
used as inoculum for the next batch.
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33. 3) Downstream: After the fermentation broth is harvested into the
harvest tank.
Part of the product crystalizes in the fermenter and also in the harvest
tank. Crystallization is completed in the crystallizer by evaporation of
some of the water.
Afterwards suspension is stored into the tank.
To achieve higher purity a washing is done.
The last step is drying either using spry dryer to obtain powder product
or applying spray granulation to obtain granulate.
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34. Production of Vitamin B12:
 Vitamin B12 also called Cobalamin, is a water soluble vitamin that
has key role in the normal functioning of brain and nervous system
and the formation RBC.
 It is involved in the metabolism of every cell of the human body,
especially affecting DNA synthesis, fatty acid and amino acid
metabolism.
 It is synthesized only by microorganism and not by Animals and
Plants.
 People with B12 deficiency may eventually develop Pernicious
anemia.
 It is the largest and most structurally complicated vit and can be
produced industrially only through Bacterial Fermentation
Synthesis.
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35. Process:
 Vitamin B12cis entirely produced on commercial basis by the
Fermentation.
 Most of the B12 fermentation process use Glucose as a Carbon
source.
 The microorganism that may be employed in the industrial
production process are:
i. Streptomyces griseus.
ii. Streptomyces olivaceus.
iii. Bacillus megaterium.
iv. Bacillus coagulans.
v. Pseudomonas denitrificans.
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36.  Pure slant culture of Streptomyces olivaceus is inoculated in 100-
250 ml of inoculum media.
 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.
 Media consist of carbohydrates, proteinaceous material and source
of cobalt and other salts.
 It is necessary to add cobalt to the medium for max yield of
cobalamin.
 Cyanide is added for conversion of other cobalamins to vit B12.
 Sterilization of the medium is done by heating media at 2500F.
 Temperature of 800F is satisfactory during fermentation.
 PH 5 is maintained with H2SO4 and reducing agent Na2SO4.
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37.  During fermentation most of cobalamin is associated with the boiling
mixture at PH 5 liberates the cobalamin quantitively from mycelium.
 Broth containing cobalamin is subjected to further process to obtain
crystalline B12.
 Filtration of broth is done. Filtered broth is treated with cyanide to bring
conversion of cobalamin to cyanocobalamin.
 Adsorption of cyanocobalamin from the solution is done by passing it
through adsorbing agent packed in a column.
 Cyanocobalamin is then eluted from the adsorbent by the use of n aqueous
solution of organic bases or solutions of Na-cyanide & Na-thiocyanate.
 Extraction is carried out by countercurrent distribution between cresol,
amyl phenol or benzyl alcohol and water or a single extraction into an
organic solvent e.g. phenol is carried out.
 Chromatography on alumina and final crystallization complete the
process.
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38. Production of Lovastatin:
 Lovastatin are natural statins produced as secondary metabolites by
predominantly Aspergillus and Penicillium spp.
 Lovastatin produced by fungus, Aspergillus terreus isolated from topical soil.
Process: Wheat bran 40gm used as substrate.
Inoculated with spore suspension of A. terreus.
Incubated at 280C for 7 days.
Inoculated substrate was dried at 40OC for 24hr.
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39. Crushed into Powder.
Ethyl Acetate is added.
Filtrate was dried using rotary vacuum evaporator.
Purification done by adding 1gm of dried crude lovastatin extract was
loaded on to pre-packed silica gel column.
Elution with benzene(100%)
Purification done by TLC
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40. Production of Simvastatin:
 Simvastatin is obtained a selective enzymatic deacylation of
Lovastatin.
 Many statins ae chemically synthesized but lovastatin is still
required to produce Simvastatin.
 From reducing blood cholesterol levels simvastatin cause pleotropic
effect and has potential to treat various kinds of disorders including
neurodegenerative disease.
 15 Simvastatin is a semi synthetic compound derived from natural
product lovastatin, a fungal polyketide by Aspergillus terreus as a
secondary metabolites.
 The synthesis of simvastatin from lovastatin is multi step process
involving replacement of 2-methylbutyryl side chain 2,2 dimethyl
butyryl group with an additional methyl group at C2 position of the
lovastatin side chain.
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41. Thank
You.
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