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Fermentative production of vitamins and amino acids
2ND MSC BIOTECHNOLOGY
Vitamins are essential micronutrients required
in trace quantities that cannot be synthesized
They are essential for metabolism for all living
Apart from their nutritional –physiological roles
as growth factor ,vitamins are increasingly
being introduced as food/feed additives ,as
medical therapeutic agents .
Today many processed foods, feeds
pharmaceuticals, cosmetics and chemical
contain extraneously added vitamins or vitamin
Presently few of the vitamins are chemically
synthesized or via extraction processes
With growing consumer consciousness led to
substituting with biotechnological processes.
FAT SOLUBLE VITAMINS
Most abundant among fat soluble vitamins and has
the highest antioxidant activity in vivo.
In nature, only photosynthetic organisms are
capable of producing α-tocopherol.
In humans, ∞-tocopherol is believed to play a
major role in prevention of light induced
pathologies of the skin, eyes and degenerative
disorders such as atherosclerosis, cardiovascular
diseases and cancer.
Industrial application of ∞-tocopherol includes its
use in preservation of food, in cosmetics and
Currently ∞-tocopherol is obtained by chemical
synthesis and by extraction from vegetable oils
Extraction from oil is not efficient ,as these typically
contains low levels of ∞-tocopherol.
Several strains of freshwater microalgae Euglena
gracilis Z and marine microalgae Dunaliella
∞-tocopherol in concentrations higher than
production of high amounts of vitamin E has
been successfully demonstrated by E. gracilis Z.
using two-step culture.
In the first step of the batch culture, E. gracilis Z.
was photo- heterotrophically cultivated in modified
Oda and modified Hunter media at high light
When the cells reached late exponential phase,
they were separated, washed and resuspended in
the same volume of Cramer and Mayers (CM)
medium for the second step of cultivation.
The two-step cultures using high cell densities
gave high productivity of antioxidant vitamin
Fermentative Production of
Tani and Taguchi have reported that as much
as 182 mg/L MK was produced using
detergent supplement culture and a mutant of
Lactic acid bacteria are reported to produce
MK with the yield of 29–123 g/L MK-7, MK-8,
MK-9 and MK-10.
In fermented soybeans, Bacillus subtilis
produces menaquinones, the major component
being MK-7 and the minor one being MK-6.
Sumi studied production of MKs by the
fermentation of okara with seven different
The highest production rate of 36.6 mg/g
was seen in the Chinese natto strain
followed by (in mg/g of okara-natto wet
mass): 14.2 in Naruse, 11.9 in Asahi, 6.8 in
Takahashi, 1.9 in Miyagino (natto bacilli for
food production), and 5.2 in Nitto and 1.9 in
Meguro (natto bacilli for medicine) after
incubation for 4 days at 37 °C.
The water-soluble vitamin K was isolated as
a dark yellow powder by DEAE Sepharose
chromatography and membrane filter
VITAMIN B 12
Cyanocobalamin, by definition vitamin
B12, is the industrially produced stable
cobalamin form which is not found in
Vitamin B12 is obtained exclusively by
Important dietary component, requirement
Cyanocobalamin consist of a cobinamide
linked to a nucleotide.
Cobinamide – cobalt linked to cyanide grp,
surrounded by 4 reduced pyrrole ring.
Nucleotide – 5 , 6 – dimethyl
INDUSTRIAL PRODUCTION OF
VIT. B12 Streptomyces griseus , S. olivaceus , Bacillus
B. coagulans , Pseudomonas denitrificans ,
Propionibacterium freudenreichii , P.
a mixed fermentation of a Proteus spp and a
Manufactured by submerged fermentation
Aeration and agitation of medium essential
Fermentation process completed in 3 to 5 days
VIT.B12 PRODUCTION USING
Streptomyces olivaceus NRRL B-1125
PREPARATION OF INOCULUM
Pure slant culture of Streptomyces olivaceus
NRRL B-1125 is inoculated and grown in 100
to 250 ml of inoculum medium.
Seeded flask are kept on shaker for incubation
Flask cultures are used to inoculate large
amount of inoculum media arranged in series
of tank .
2 or 3 successive transfers are made to obtain
required amount of inoculum cultures.
Inoculum of production tank must be 5% of the
volume of production medium
Consist of carbohydrate ,proteinaceous material ,
and source of cobalt and other salts .
Sterilization of medium batch wise or continuously .
Batch – medium heated at 250°F for 1 hr
Continuous – 330°F for 13 min by mixing with live
COMPONENTS AMOUNT (%)
Distillers solubles 4.0
Dextrose 0.5 to 1
COCl2.6H2O 1.5 to 10 p.p.m.
TEMPERATURE , PH ,
AERATION AND AGITATION
Temperature : 80°F
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
0.5 vol air/vol medium/min. Excess aeration
ANTIFOAM AGENT ,
CONTAMINATION Antifoam agent : soya bean oil , corn oil,
lard oil and silicones (sterilized before
Prevention of contamination : essential to
maintain sterility ,
contamination results in reduced yields ,
equipments must be sterile and all
transfers are carried out under aseptic
Yield : yield of cobalamin are usually in the
range of 1 to 2 mg. per litre in the
DOWNSTREAM PROCESS OF
DISSOLVED VITAMIN B
CREAM CONCENTRATION OF CELL
Eg :alcohol such as
Dissolved vitamin B12
Pure vitamin B12
Crystallisation from organic
Riboflavin, or vitamin B2, is used for human
nutri- tion and therapy and as an animal feed
Its deficiency in humans is correlated with loss
of hair, inflammation of skin, vision deterioration,
and growth failure.
This vitamin has also been found to be
successful in treatment of migraine and malaria .
Riboflavin has been produced commercially by
chemical synthesis, by fermentation and by a
combination of fermentation and chemical
MICRO –ORGANISMS IN
INDUSTRIAL PRODUCTION OF
Although bacteria (Clostridium sp.) and yeasts
(Candida sp.) are good producers, two closely
related ascomycete fungi, Eremothecium
ashbyii and Ashbya gossypii, are considered
the best riboflavin producers.
Ashbya gossypii produces 40 000 times more
vitamin than it needs for its own growth.
OTHER MICRO -ORGANISMS
PRODUCING VIT. B 2
Gene amplification and substitution of wild type
promoters and the regulatory regions with strong
constitutive promoter from Bacillus subtilis have
resulted in increased riboflavin production .
Lactococcus lactis MG 1363 strain using both direct
mutagenesis and metabolic engineering for
simultaneous overproduction of both folate and
Improved strains for the production of riboflavin
were constructed through metabolic engineering
using recombinant DNA techniques in
PREPARATION OF INOCULUM
Starts from slants or spores dried on sand.
After 1 or 2 stages, further propagation is
carried on 1 or 2 tank inoculum stages
Fermentor 10,000 to 1,00,000 gals range.
Production medium designed according to type of
Ashbya gossypii : sources - palm oil ,corn steep
molasses , whey, collagen , soya oil , glycine.
Stahmann et al. reported riboflavin yields in excess of
15 g/L of culture broth in a sterile aerobic submerged
fermentation of Ashbya gossypii with a nutrient medium
containing molasses or plant oil as
major carbon source.
Ertrk et al. studied fermentative production of riboflavin by
Ashbya gossypii in a medium containing whey.
The quantities of riboflavin produced by Ashbya gossypii in whey
with different supplements.
Supplement Quantity of riboflavin
Glycine + peptone 120
Yeast extract 68.4
Soyabean oil 17.5
For Eremothecium ashbyii - still slops from alcohol industry with skim milk ,
soya bean meal or casein(protein source),maltose/ sucrose/glucose (carbohydrate
low cost organic wastes as flavinogenic factors and the various concentrations at
which they induced flavinogenecity resulting in higher yields of riboflavin
Organic wastes like beef extract, hog casings, blood meal or fish meal supported
the production of riboflavin from Eremothecium ashbyii NRRL 1363.
Recent studies with wild type of E. ashbyii have yielded 3.3 g/L of riboflavin
using molasses and peanut seed cake as carbon and nitrogen source, respectively
pH : 6 to 7.5
Temperature : 26 to 28 °C
Fermentation : submerged aerated fermentation
Fermentation time : 96 to 120 hrs
Aeration & agitation required.
Yield : 3 to 6 g or more / litre
RIBOFLAVIN Riboflavin is recovered from the broth by
centrifu- gation after inactivation of the
microorganisms by heat.
Pasteurization of the broth ensures that no
viable cells of the production organism are
present in the final product.
After heating, the cell mass is separated
from fermentation broth by centrifugation.
Differential centrifugation leads to separation
of cells and riboflavin crystals because of
differences in size and sedimentation
Riboflavin is then recovered from cell-free
broth by using evaporation and vacuum
L-ascorbic acid finds its use mainly in food
industry, being a vitamin as well as an
Majority of commercially manufactured L-
ascorbic acid is synthesized via Reichstein
process using D-glucose as a starting
Approximately 50 % of synthetic ascorbic acid
is used in vitamins supplements and
Because of its antioxidant properties and its
potential to stimulate collagen production, it is
also widely used as an additive to cosmetics.
Yeast Based Fermentative
Saccharomyces cerevisiae and
Zygosaccharomyces sp. produce L-ascorbic
acid intracellularly when incubated with L-
Over-expression of the D-arabinose
dehydrogenase and D-arabinono-1,4-lactone
oxidase in Saccharomyces cerevisiae
enhances this ability significantly.
FERMENTATION BY ALGAE
Skatrud and Huss described a method that
involved initial growth of Chlorella pyrenoidosa
ATCC53170 in a fermentor with a carbon source
that is sufficient for the cells to grow to an
intermediate density. At the depleted stage,
additional carbon source was added sequentially
or continuously to maintain the carbon source
concentration below a predetermined level until
the addition is terminated. This resulted in the
production of 1.45 g/L of L-ascorbic acid.
Euglena gracilis Z. is one of the few
microorganisms which simultaneously produce
antioxidant vitamins such as carotene (71
mg/L), vitamin C (86.5 mg/L) and vitamin E (30.1
BIOTIN (VITAMIN H)
Biotin (vitamin H) is one of the most fascinating
cofactors involved in central pathways in pro- and
eukaryotic cell metabolism.
While humans and animals require several hundred
micrograms of biotin per day, most microbes, plants
and fungi appear to be able to synthesize the cofactor
Biotin is added to many food, feed and cosmetic
Majority of the biotin sold is synthesized chemically.
The chemical synthesis is linked with a high
environmental burden, much effort has been put into
the development of biotin-overproducing microbes
Biosynthesis of Biotin
The conversion of
dethiobiotin to biotin
has not been
Ogata et al. screened microorganisms and
demonstrated that the bacterium B.
sphaericus can excrete significant quantities
of biotin synthetic pathway intermediates from
precursor, Pimelic acid.
Microbial production of l-glutamic acid has been
extensively studied by a large number of research
The most popular Coryneform species include
C.glutamicum, Corynebacterium, Brevibacterium
flavum, Brevibacterium lactofermentum,
Brevibacterium divarticum, Brevibacterium
ammoniagenes, Brevibacterium thio- genetalis,
Brevibacterium saccharoliticum, and
Brevibacterium roseum .
Other glutamic acid-producing organisms
include Escherichia coli, Bacillus
megaterium, Bacillus circulans, Bacillus
cereus, and Sarcina lutea.
Industrially, glutamic acid is usually
manufactured by batch/fed-batch
submerged fermentation processes using
genetically modified strains of
Corynebacterium or Brevibacterium.
The seed medium composition can be used:
glucose (8%), NH4Cl (0.5%), corn steep liquor
(0.3%), K2HPO4 (0.5%), KH2PO4 (0.5%),
MgSO4·7H2O (0.03%), CaCO3 (1.0%), and
deionized water to make 100%.
The pH of the medium -7.2
The inoculated flasks are grown in an orbital
shaker incubator maintained at 30°C and 230
rpm for 15 h.
The entire contents of the one flask is then
transferred to a 2.0 L capacity fermenter with
500 mL of sterile nutrient medium containing
molasses (20%), KH2PO4 (0.5%), KH2PO4
(0.5%), MgSO4·7H2O (0.3%), urea (0.8%),
CaCO3 (1.0%), and deionized water to make
In most cases, the optimum pH of the medium
was recorded as 7.0.
The fermentation is usually initiated with
continuous agitation and aeration for 48 h at
AND THERAPEUTIC ROLE
The greatest application of glutamic acid and
its salt is in the food industry as a flavor
To aid in peptic ulcer healing
One of the leading roles of glutamic acid in
pharmaceuticals is that of a neurotransmitter.
The blockage of NMDA receptors can greatly
affect the memory and overall mental
performance of an individual.
Glutamic acid and aspartic acid have the
capability to combine with NMDA receptors
thus increasing cation conductance,
depolarizing the cell membrane, and
deblocking the NMDA receptors.
l-Lysine is one of the leading and most
exploited amino acid among the
essential amino acids list.
l-lysine can be synthesized from α-
aminoadipic acid by yeast and
Neurospora mold, or from
diaminopimelic acid (DAP) by E. coli
Organism: mutant strain of C.glutamicum
In commercial-scale starches, molasses and
glucose are mostly used as the carbon
Care must be taken to create a balance
between carbon and nitrogen sources such
as corn steep liquor, soybean cake acid
hydrolysate, yeast extract, peptone, and the
Inorganic salts such as KH2PO4, K2HPO4,
MnSO4·7H2O, (NH4)2 SO4.
In most cases, the optimum pH of the
medium has been 7.2 and
temperature at 30°C.
The seed stage cultivation requires
around 24 h, whereas the
fermentation stage is complete by
approximately 96 h.
After this, harvesting is done and the
product l-lysine is recovered using
some suitable and economical method
AND THERAPEUTIC ROLE
It is an important additive to animal feed for
optimizing the growth of pigs and chickens.
In the food industry, l-lysine is used in a
number of dietary or nutritional supplements
that are popularly used by athletes, weight
lifters, bodybuilders, and even some
individuals to boost their energy level and
protect their muscles from deterioration.
l-lysine is also recommended for the
treatment of some viral infections, for
example, herpes simplex, cold sores,
shingles, and human papillomavirus
infections such as genital warts and genital
No scientific reports were available
relating to the microbial direct
production of tryptophan. During this
period, more attention was given by
researchers looking into the possibility
of tryptophan production
With the introduction of efficient
strains of Corynebacterium and E.
coli, now tryptophan is largely
produced by fermentation
Genetically modified strain of C. glutamicum
that is capable of producing tryptophan.
Fermentation medium may be prepared from
molasses (30%), corn-steep liquor (0.7%),
KH2PO4 (0.05%), K2HPO4 (0.15%),
MgSO4·7H2O (0.025%), (NH4)2SO4 (1.5%),
and calcium carbonate (1%).
In addition vitamin B1, biotin, l-phenylalanine,
pH adjusted to 6.8
1.0 mL of 20% silicon RD in deionized water
is added as antifoam.
The fermenters can be harvested after
Product recovery is usually done using
ultracentrifugation at around 10,000
rpm, followed by treatment with cation
exchange resin and decolorization
with activated carbon.
After further centrifugation, the mixture
can be subjected to drying under a
AND THERAPEUTIC ROLE
Tryptophan has a wide range of
applications in the feed and
As an essential amino acid with a
unique indole side chain, which
indicates its use as a precursor for a
number of neurotransmitters in the
Its application in the chemical
synthesis of some antidepressant