Fermentation

•Fermentation is enzymatically controlled transformation of an organic compound.

•The fermentation product may be a metabolite or biomass.

• Metabolites are the intermediate products produced during metabolism, catalyzed by various
enzymes that occur naturally within cells. Eg., antibiotics, and pigments.
• The term metabolites are usually used for small molecules.
• The various functions of metabolites include; fuel, structure, signalling, catalytic activity, defence and
interactions with other organisms.
• The metabolites are produced by plants, humans and microbes.

•Biomass is the term loosely used to define the total amount of carbon and nitrogen
incorporated into all polymeric organic material by biological processes.
108
What are Primary Metabolites
• Primary metabolites are small chemical compounds that are directly involved in
the growth, development, and reproduction of living organisms.
• Therefore, they are key components in the maintenance of normal physiological
functions in the body.
• Thus, primary metabolites are often referred to as central metabolites.
• Primary metabolites are usually formed during the growth phase due to energy
metabolism.
• They are major components of proper growth.
• Ethanol, lactic acid, nucleotides, vitamins, and some amino acids are considered
as primary metabolites.
• In industrial microbiology, ethanol is the most common primary metabolite
produced in large-scale by fermentation.
• Morely, amino acids like L-lysine and L-glutamate are produced in large-scale.
• Citric acid is the other common primary metabolite produced in large-scale.
• It is used as an ingredient in food production.
What are Secondary Metabolites
• Secondary metabolites are small organic compounds produced through the
modification of primary metabolites.
• They are formed near the stationary phase of growth.
• Secondary metabolites do not play a role in growth, development, or
reproduction.
• However, they play a role in ecological functions like defense mechanisms, serve
as antibiotics, and produce pigments.
• Atropine and antibiotics like erythromycin and bacitracin are commercially
important secondary metabolites produced in large-scale.
• Atropine serves as a competitive antagonist for acetylcholine receptors.
• It is derived from various plants which can be used to treat bradycardia.
• Erythromycin is an antibiotic with wide antimicrobial spectrum.
 117
Primary and Secondary Metabolites. Depending on the particular organism, the
desired product may be formed during or after growth.
117
 Organic Acid
• Organic acid is an organic compound that is characterized by weak
acidic properties and does not dissociate completely in the presence of
water.

• An organic acid is an organic compound with acidic properties.

• Given that these acids are organic, a carbon atom must exist in its
structure. One of the most common organic acids is carboxylic acid,
which has the molecular formula RCOOH.

• Organic acids are commonly weak acids.

Organic Acid
Citric Acid
Citric acid is a weak organic acid found in citrus fruits
It comprises as much as 8% of the dry weight of the fruit
Available in three popular varieties
-Monohydrate Citric acid
-Anhydrate citric acid
-Sodium citrate
First isolated in 1784 by the Swedish chemist Carl Wilhelm, who
crystallized it from lemon juice
Structure

 Citric acid's chemical

formula is C6H8O7

 IUPAC nomenclature:
2-Hydroxy-1,2,3-propane
tri carboxylic acid
 Microbial Fermentation
Bacterial species:
Bacillus licheniformis, B. subtilis, Aerobacter,
Pseudomonas, Micrococcus etc.
Yeast species:
Candida sps, Hansenula , Pichia, Debarymyces,
Trichosporan
Nematospora, Saccharomyces, Zygosaccaromyces etc.
Fungal species:
Aspergillus niger, Penicillium janthinellum, Mucour
pyriformis, Ustilina vulgaris, Botrytis

Many microorganisms including filamentus fungi, yeasts and bacteria

could be used to produce citric acid, however, the mutants of A. niger
are generally used for commercial production.
Why Aspergillus niger is used ????

Advantages
Efficient and high yielding strain
Have uniform biochemical characteristics
Easy to cultivate and handle
Widely Available
Utilization of cheap raw materials
Production of less oxalic acid
Major source of citric acid
This organism accounts for over 99% of global citric acid production, or more than 1.4 million tonnes
per annum
Industrial Production of Citric Acid
I. Solid State Fermentation
• SSF involves the growth of microorganisms on solid, normally
organic, material in the absence or near absence of free water.
• The substrates used are often cereal grains, bran, legumes and
lignocellulosic materials, such as straw, wood chippings, etc.

II. Liquid State Fermentation

(A) Surface Process
(B) Submerged Fermentation
I. Solid State Fermentation

Substrates: Wheat bran, Soya grains

or rice etc
pH: The pH has to be adjusted to 4.5
before sterilization by steaming
Temp: It is cooled to 30-35 0C and
inoculated with A. niger
Moisture : 70%
Subsequently, cultured for 5-7 days
The pH drops to 1.8-2.0
The fermented liquor is harvested
and treated for obtaining Citric Acid

128
II. Liquid State Fermentation
This process is of 2 types

(A) Surface process

(B) Submerged Process
(A) Surface process
This is the 1st fermentation process to produce Citric Acid cheaply
Substrate: Cane molasses or beet molasses
Medium pH is always maintained below 3.5
Fermentation carried for 7-10 days
The fermented broth is drained and separated from mycelia by centrifugation
Shallow trays made of stainless steel-316 which are non-corrosive are used
Media is poured to a depth of 1-8 cm (max is 2.5 cm)
Medium is inoculated with A.niger and incubated at 28-35 0C. 129
B. Submerged fermentation
In this process, fermenters made of
cylindrical stainless steel or glass are
employed
The nutrient media after inoculating with
mycelia are subjected to vigorous
controlled aeration and agitation
Aeration is provided at 0.5-1.5 VvM
(volume of air, volume of medium, per
minute)
Process is carried for 3-7 days at 30-35 0C

pH maintained below 3.5

131
Antifoam agents are added to prevent any foam
Mycelia pellets produced during the process can be reused
After the stipulated time, broth is drained from the vessel and the
mycelia are separated by centrifugation or filtration
Citric Acid is further extracted from the broth
Recovery of Citric Acid
Recovery of Citric Acid is difficult due to
 Presence of unconverted sugars
 Presence of other products (oxalic / gluconic acid)
 Presence of trace salts or impurities
Method
 The crude broth containing citric acid is filtered to separate mycelial mat and
liquid

 A slurry of hydrated lime Ca(OH)2(calcium hydroxide) is added & the broth

solution is heated to 80-90 0C to precipitate calcium citrate

133
133
Recovery of Citric Acid
The precipitated calcium citrate is filtered and washed several times with
water
It is then treated with H2SO4 to liberate citric acid, leaving the precipitated
calcium sulphate( CaSO4.2H2O)

The solution is filtered to separate citric acid from CaSO4.2H2O

Citric acid solution is subjected to decolourization by adding activated

charcoal or passing through ion exchange resin columns

The solution is then passed through circulating evaporators or through low

temperature crystallizers where citric acid is crystallized as citrate
monohydrate 134
 Nutrient medium
Evaporation &
crystallization
Aspergillus niger Fermentor

Demineralization Flow Chart for CA Filter

production

Decolorization by
Activated charcoal or
passing through ion
exchange resins Culture filtrate

filter H2SO4 Ca(OH)2

80-90C

Citric acid CaSO42H2O Tank Washed with 135

Ca.citrate 135
water
 Lactic Acid
• Lactic acid (2-hydroxypropanoic acid)
was discovered and isolated in 1780 Lactic acid
by the Swedish chemist Scheele from
sour milk .
• In 1857, however, Pasteur discovered L (+) Lactic acid
that it was not a milk component, but
a fermentation metabolite generated
by certain microorganisms.
• It is the first organic acid produced
D (-) Lactic acid
microbiologically in 1881 by Charles
E. Avery at Littleton, Massachusetts,
USA .
 Lactic acid bacteria
There are two groups of lactic acid bacteria
1. Heterofermentative
2. Homofermentative.

• The heterofermentative (e.g. Luconostoc

mesenteroides) lactic acid bacteria produce
many by-products other than lactic acid and are
not suitable for commercial processes.

• In case of homofermentative bacteria

(Lactobacillus sp.), very little substrate is used for
producing cell mass and other metabolites and
majority of the carbon source is converted to
lactic acid.
•The homofermentative LAB usually metabolize glucose via the Embden-Meyerhof
pathway (i.e. glycolysis).
•Since glycolysis results only in lactic acid as a major end-product of glucose
metabolism, two lactic acid molecules are produced from each molecule of glucose
with a yield of more than 0.90 g/g
C6H12O6 + 2NAD+ + 2 ADP + 2Pi  2C3H6O3 + 2NADH + 2ATP
The Homofermentative lactic acid bacteria are
facultative anaerobes which can be grown in low
oxygen concentration and are used for industrial
production of lactic acid.
Biosynthesis of Lactic Acid
 Industrial Production of Lactic Acid
Substrates: Glucose + Nitrogen Source + Vitamin B (cofactor)

Carbon Sources
Hydrolyzed potato starch, corn, straw, whey, cotton seed hulls,
grapefruit,
Sugar normally 5- 20 %
Molasses is cheap but give low yields of lactic acid and have expensive
purification processes
Nitrogen Sources
• yeast extract
• malt sprouts
• malt combing nuts
• grass extract
• peptones
• beef extract
Microbes: Lactobacillus bulgaricus and L. delbruckii

Other Conditions
pH: 5.5 to 6.5
Temp: ~45-50oC
Time: ~72 hrs depending on strain and substrate
The starchy materials used for lactic acid production include sweet
sorghum, wheat , corn , cassava , potato , rice ,rye , and barley.

These materials have to be hydrolyzed into fermentable sugars before

fermentation, because they consist mainly of α(1,4)- and α(1,6)-linked
glucose.

This hydrolysis can be carried out simultaneously with fermentation .

Amylase-producing L. amylophilus and L. amylovorus are often used for

the direct fermentation of starchy materials into lactic acid.
 Lactic acid is produced in the form of L or D lactic acid or as its racemic mixture

 Organisms that form the L form or D form have two lactate dehydrogenases (LDH),
which differ in their stereospecifity

 Some Lactobacilli produce L form, which on accumulation induces a racemase,

which converts it into D lactic acid until equilibrium is obtained

143
143
 Recovery of lactic acid
• A number of methods can be applied for the separation of lactate salt from
fermented medium which involve extraction by solvents or separation by
ion-exchange, adsorption, vacuum distillation and membrane filtration .
• The medium after completion of fermentation consists of either pure lactic
acid or its salt or the mixture of the two.
• Addition of excess of calcium carbonate to the medium at the end of the
fermentation and pH adjusted to 10, heated and filtered.
• In this procedure all the lactic acid is converted in to calcium lactate, the
bacteria are killed and protein in the medium gets coagulated.
• To the Calcium lactate, sulphuric acid is added to precipitate calcium as
calcium sulphate and removed by filtration.
• Lactic acid is extracted with isopropyl ether directly from the heated and
filtered fermentation broth, by counter-current continuous extraction method
Recovery of lactic acid
Medium
Calcium pH 10;
carbonate Heated & Filtered
Calcium Lactate

Sulphuric
Acid
Calcium
Sulphate

Extracted with
isopropyl ether
145
Lactic
acid
 (B) AMINO ACIDS
 Amino acids are important biochemical constituents required by living
being and they are precursors for polypeptide synthesis

Global amino acid production:700,000 metric tonnes per year

Glutamic acid and Lysine: 250,000 metric tonnes per year

The increase in demand for amino acids is around 10% every year

146
146
 Commercial uses of Amino Acids
Amino acids have extensive industrial applications

About 65% of the AA produced are used in food industry

 As Flavor enhancing agents Ex: Sodium glutamate

 For taste in fruit juices Ex: Sodium aspartate, L-Alanine
 Improving quality of bread during baking Ex: Cysteine
 Antioxidant in fruit juices Ex: Cysteine
 Antioxidant & preservation of powdered milk Ex: Tryptophan & Histidine
 As a low calorie sweetener in soft drinks Ex: Aspartame (L-Aspartyl-L-Phenylalanine & L-
Aspartic acid)

147
Amino acids can be placed into five families based on
biosynthetic origins

1. Glutamate family

2. Pyruvate family

3. Aspartate family

4. Shikimate family

5. Serine family 148

 Methods of Amino Acid Production

Commercial manufacture are of 3 types

1. Extraction of AA from Protein hydrolysates
Ex: Cysteine, Leucine, Asparagine, Tyrosine

2. Chemical Synthesis
Ex: Glycine, Alanine, Methionine, Tryptophan

3. Microbiological Production
Ex: Glutamic acid, Lysine, Aspartic acid, Tryptophan, Alanine, Valine, Tyrosine,
Serine, Histidine, leucine, Isoleucine, Arginine, Methionine, Phenylalanine, Proline, Ornithine
149
149
Microbiological production
It is of 3 types
1. Direct fermentation of amino acids
By using different carbon sources, such as glucose, fructose, molasses,
starch, n-alkanes, ethanol, glycerol etc
2. Converting inexpensive intermediate products
E.g. Conversion of glycine to serine, alanine to aspartate etc.
3. Enzymes or immobilized cells
Batch / continuous process involving enzyme-membrane reactors
A. Use of an enzyme amino acylase from A. oryzae to selectively split a
chemically synthesized amino acid (alanine, methionine, phenylalanine,
tryptophan)
B. Amino acids are produced by reductive deamination of α-ketoacids
using amino acid dehydrogenases (alanine, leucine)
150
150
 GLUTAMIC ACID
Microbial production of Glutamic acid is predominantly achieved on rich carbohydrate media

About 2000 microbes on different media were screened for glutamic acid production

Organisms: (commonly called as glutamic acid bacteria)

Corynebacterium glutamicum, C.callunae, C.herculis,
Brevibacterium flavum, B. lactofermentum, B. divaricatum,
B. thiogenitalis, Microbacterium ammoniaphilum etc.

Under optimal conditions, glutamic acid bacteria convert about 50% of the supplied
carbohydrate into glutamic acid, with little formation of by- products

Temp: ~30oC
151
Hybrid strains were also developed from Brevibacterium & Corynebacterium 151
Cornybacterium glutamicum Brevibacterium
153
 Biosynthesis
 Glucose is broken down into C3 &
C2 fragments by glutamic acid
producing bacteria through the
EMP Pathway and Pentose-
phosphate cycle, and the
fragments are channeled into the
TCA cycle

 The key precursor of glutamic acid

is α-ketoglutarate, which is formed
in the TCA cycle via Citrate &
Isocitrate and then converted into
glutamic acid through the
reductive amination with free NH4+
ions

 The last step is catalyzed by

NADP dependent Glutamate 154
154
dehydrogenase
 Isocitrate NADP Glutamic
acid

Isocitrate dehydrogenase Glutamate

dehydrogenase

Reductive amination
CO2 + NADPH2
α-ketoglutarate
α-ketoglutarate
+ NH4+

Reductive amination involves the conversion of a carbonyl group to an amine

The carbonyl group is most commonly a ketone or an aldehyde

156
156
 Pathway of Glutamic acid production
Glucose

Pyruvic acid

Acetyl CoA

Oxaloacetic acid Citric acid

α-ketoglutaric acid
GLUTAMIC ACID
1 mole of glutamic acid is produced
Amination
from 1 mole of glucose or 3 moles of
acetate 157
Regulatory control:
• Good supply of glucose and efficient conversion of phosphoenol
pyruvate to oxaloacetate
• Phosphoenol pyruvate carboxylase and pyruvate carboxylase,
pyruvate dehydrogenase
• a-ketoglutarate dehydrogenase (low activity by adding penicillin,
surfactants)
• Glutamate dehydrogenase (high activity)

1 mole of glucose should produce 1 mole of glu

In practice, efficiency is 70%
• Glu is synthesized intracellularly
• Carrier mediated processess
• Biotin is essential co factor (for Acetyl CoA carboxylase), deficiency of biotin
affects fatty acid biosynthesis, membrane formation alters, permeability is
affected and intracellular export of glu is altered

FACTORS INFLUENCING PRODUCTION

1. Carbon sources
2. Nitrogen source: ammonia for carbon to glu pH control
3. Growth factors :biotin
4. O2 supply: high conc inhibits growth and low O2 leads to lactic acid
production and succinic acid, Affects Glu production in both cases
Effect of Permeability
Production & excretion of glutamic acid is dependent upon
cell permeability
Increased permeability in glutamic acid bacteria can be
obtained through biotin deficiency
Biotin (>5 µg/L) is generally required in growth for fatty acid synthesis,
which results in oleic acid synthesis
This increases phospholipid concentration, making the cell incapable of
excreting glutamic acid

Therefore, glutamic acid up to 25-35 µg/mg of dry weight accumulates

intra-cellularly resulting in feedback inhibition

160
161
161
Recovery

 Centrifugation is done at the end of the fermentation process to

separate cell material
 Glutamic acid is obtained after
 Acidification , precipitation at the Isoelectric point
 Ion exchange chromatography
 Electro dialysis
 Extraction with organic solvents

162
165
165
• Ethanol is an organic chemical compound.

• It is a simple alcohol with the chemical

formula C2H6O (CH3−CH2−OH or C2H5OH

• Ethanol is a volatile, flammable, colorless

liquid with a characteristic wine-like odor
and pungent taste

• Ethanol is naturally produced by

the fermentation of sugars by yeasts or
via petrochemical processes such
as ethylene hydration.

• It has medical applications as

an antiseptic and disinfectant.
Microorganisms:
Yeast species are Saccharomyces, Torulopsis, Kloekera, Candida
Top fermenter yeast – S. cerevisiae (uniform, ring on wall)
Bottom fermenter yeast – S. carlbergensis

Candida are preferred when whey and waste sulphite liquor are employed in alcohol
fermentation

Bacterial species include Zymomonas mobilis, Thermo anaerobacter

167
Desirable characteristics of strain
(i) It should be fast growing
(ii) It should be an efficient strain (yield more alcohol)
(iii) It should have tolerance to alcohol, as well as to osmotic
pressure (for accumulation)
(iv) It should possess uniform and stable biochemical
properties

168
 C6H12O6
C3H4O3

Biosynthesis
of Ethanol

Alcohol Dehydrogenase
C2H5OH C2H4O 169
Ethanol Production Process

3 steps
1. Preparation of Media
2. Fermentation
3. Distillation

171
 1. Preparation of Media
 A variety of carbohydrate available as waste products of agricultural
industries are used for preparation
The simple hexose sugars will be converted to ethanol by the organism
The choice of raw material is critical as raw material costs make up to
75% of the final selling price
The raw materials are classified into
 Saccharine materials: Molasses, whey , fruit juices
 Starchy materials: potato starch, corn starch, wheat flour, cereals (oats)
 Cellulose materials: sulphite liquor waste, wood etc

172
Molasses
 Molasses, is a viscous substance resulting from
refining sugarcane or sugar beets into sugar
 commonly used substrate for ethanol fermentation
 Molasses is extracted from Sugar beet chips with
hot water and from sugar cane.
Bagasse:-
 is the dry pulpy fibrous material that remains after
crushing sugarcane or sorghum stalks to extract
their juice
 This can be burned as an energy source for
distillation of alcohol

173
Starch

The starch is liquefied by boiling

under pressure and then
hydrolyzed by cellulose degrading
enzymes
When starch grains are used they
are to be soaked at 40-50 0C for
several hours and then liquefied The most imp starch root is derived from the
tropical plant Manihot esculenta
Saccharomyces has no amylase
activity and hence the starch must
be hydrolyzed
174
Cellulose
 Yet to be used in the commercial
production of ethanol

 Needs to be hydrolyzed with

cellulases before usage

175
 2. Fermentation
Batch fermentations are more commonly employed for ethanol
fermentation

Production is carried in fermentors (600 m3) with either starch

hydrolysate or molasses with 3-5% inoculum

The process is run at a temp of 35-380C & pH about 4.0-4.5 for about
24-36 h

With in 12 h 10% (v/v) ethanol is produced with 10-12 g dry cell

weight

80% of the cells can be recycled as inoculum for next round

176
3. Distillation
 Before distillation the cell mass is separated by
centrifugation or filtration or sedimentation

 The separated broth is distilled and ethanol is collected in

the condensing collector

 The concentrate is analyzed and processed further

 The distillation step is most important which require energy

for boiling the broth
177
 Ca2+
Water
Corn meal/molasses Heat (60-80 degree) Cooler (heat recovery)

α-Amylase
Steps in Ethanol Liquefaction
Fermentation

Starch digestion

ETHANOL
Cell material Inoculum

Fermentation (Temp 30-350C)

Distillation Separation pH 4.5-5.5
178
Applications of Ethanol
Ethanol is an active solvent of dyes, lubricants, adhesives, pesticides, detergents, paints,
explosives

Used as an organic solvent for extraction of organic compounds

Used in manufacture of rubber

Manufacture of acetaldehyde

Used as a fuel in internal combustion engines

Used in manufacture of perfumes

179
 PHENYLALANINE
• Amino Acid
• ESSENTIAL
• Neutral and non-polar (due to benzene
ring)
• L-phenylalanine to make proteins and
other important molecules (tyrosine,
dopamine, epinephrine and
norepinephrine).
• It has also been studied as a treatment Phenylalanine (C9H11NO2)
for several medical conditions,
including skin disorders and
depression
• 2 types- L and D (DL also
synthetically)
 PHENYLALANINE (C9H11NO2)
• It is the only form of phenylalanine found in proteins. Major dietary sources
of L-phenylalanine include meat, fish, eggs, cheese, and milk.
• Phenylalanine should be avoided in people with certain inherited disorders
that cause their bodies to build up too much phenylalanine.
• Phenylalanine is the starting compound used in the synthesis of flavonoids
 Antibiotics
• Antibiotics are medicines/ antimicrobial substance that fight bacterial
infections in people and animals.
• It is the most important type of antibacterial agent for fighting bacterial
infections, and antibiotic medications are widely used in
the treatment and prevention of such infections
• They work by killing the bacteria or by making it hard for the bacteria to grow and
multiply.
• Antibiotics can be taken in different ways:
• Orally (by mouth). This could be pills, capsules, or liquids.
• Topically. This might be a cream, spray, or ointment that you put on your skin. It could also
be eye ointment, eye drops, or ear drops.
• Through an injection or intravenously (IV). This is usually for more serious infections.
 Penicillin
• Class of Beta-lactum ring antibiotic
• Secondary metabolite
• Used to treat bacterial infections
• Firstly isolated from mold (accidentally discovered by alexander Fleming in 1928
from filamentous fungi Penicillin notatum-actinga gainst staphylococcus bacteria)
• Examples: ampicillin, amoxicillin, etc.
• Penicillin mostly active against gram positive bacteria
• But, amoxicillin is active against gram negative except pseudomonas aeruginosa
• Classification includes- natural/biosynthetic and synthetic or semi-synthetic
• Natural- harvested from mold through Fermentation (Penicillin V and G)
• Synthetic derivatives- slight modifications (Ampicillin, oxacillin etc)- resistant to stomach
acid
• Penicillin acts on the cellwall and lyse it
• It does not affect the human cells because of the cell wall absence
 Penicillin
• Microbe: Penicillium Chrysogenum
• Aerobic
• Batch/Fed batch
• Carbon: Lactose or starch
• Nitrogen: Yeast extract or soy meal
• pH: ~6.5
• Temp: 25-27oC
• Agitation
• HRT: 40 hrs
• Submerged Fermentation
• Trace eements: magnesium and zinc for mycelium elongation
• Precursor: Phenyl acetic acid (Penicillin G) and Phenoxyacetic acid (Penicillin V)
 • The first step is the condensation of three amino acids—L-α-
aminoadipic acid, L-cysteine, L-valine into a tripeptide

δ-(L-α-aminoadipyl)-L-
• Before condensing into the tripeptide, the amino acid L-valine

Condensation

synthetase (ACVS)
cysteine-D-valine
must undergo epimerization to become D-valine

• The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-

cysteine-D-valine (ACV).
• The condensation reaction and epimerization are both
catalyzed by the enzyme δ-(L-α-aminoadipyl)-L-cysteine-D-

synthase (IPNS)
Tripeptide ACV

isopenicillin N
valine synthetase (ACVS), a nonribosomal peptide
synthetase or NRPS.
• The second step in the biosynthesis of penicillin G is the
oxidative conversion of linear ACV into the bicyclic intermediate

•
isopenicillin N by isopenicillin N synthase (IPNS)

• Isopenicillin N is a very weak intermediate, because it does not

show strong antibiotic activity

• The final step is a transamidation by isopenicillin N N-

acyltransferase, in which the α-aminoadipyl side-chain of
isopenicillin N is removed and exchanged for a phenylacetyl
side-chain.
 Penicillin-Downstreaming
• Recovered by filtering the mycelia
• Extracted in organic solvent (Amyl or butyl acetate) in which it dissolves
• Potassium salts are added and penicillin is precipitated
• pH-2 to 3
 Vitamin
• Organic molecule that is essential micronutrient required by organism in minute
quantities for proper functioning of body (normal growth and development along
with metabolism)
• May not be synthesized in body (higher organisms) hence to be obtained from
diet
• Classified as fat soluble and water soluble
 Vitamin B12
• Vitamin B12 or Cobalamin
• Complex biochemical structure (4
Corrin rings, Cyano group and
adenosine ligand form the six
coordination sites)
• Water soluble
• Cofactor in DNA synthesis
• Important for functioning of nervous
system and maturation of RBC in bone
marrows
• Deficiency causes pernicious anaemia

• Can be obtained as byproduct of

Streptomyces fermentation in
production of some antibiotics
(Streptomyces, chloramphenicol, etc.)
 Vitamin B12-Fermentation
• Microbe: Pseudomonas denitrificans, Streptomyces griseus, Bacillus megaterium,
Lactobacillus, Mycobacterium, Cornebacterium, Salmonella, and Propiobacterium etc.,
• Medium: Glucose, beet molasses, Betain, nitrogen source, Cobalt (*), and 5,6 dimethyl
benzimidazole
• pH 7.5
• Temperature 30oC
• 2 phases: Aerobic (Co at the last) or Anaerobic
(Co from the start)
• Submerged fermentation
• Fedbatch and continuous modes
• Time- 3 to 5 days
• Diet: fish, almonds, milk, red meat, eggs, chicken,
cheese etc.,

• Cyanide is required for conversion of

other cobalamins to B12
 Vitamin B12- Recovery
• Most of the cobalamin is associated with mycelium
• 6 steps
• Heating: Bound vitamin solubilized by heating at 80-120oC for 30 min @ pH 5-6
• Filtration: remove the mycelium (centrifugation or filtration)
• Treatment with Cyanide: Filtrate with cyanide to convert the cobalamin to cyanocobalamin
• Adsorption of cobalamin: the adsorbing agent of column (charcoal, ion exchange resins etc)
is used to adsorb the cobalamin from the solution
• Elution: uses aqueous solutions like HCl or water etc
• Precipitation: can be precipitated as zinc or copper cyanide complex