Gisha G P
• Vitamin B12
• Vitamin B2
• Vitamin C
• proVitamin A
2Industrial production of vitamins
• Vitamins are defined as essential micronutrients that are required
in trace quantity and are very important compounds in diet
• Synthesized by prototropic microorganisms
• Microbes excrete vitamins in excess of their metabolic needs under
highly specific and artificial condition
3Industrial production of vitamins
(Vitamin B 12)
Industrial production of vitamins 4
• Cyanocobalamin, by definition vitamin B12, is the industrially
produced stable cobalamin form which is not found in nature.
• Vitamin B12 is obtained exclusively by fermentation process.
• Important dietary component, requirement 0.001 mg/day.
• 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 benziminazole
5Industrial production of vitamins
Micro –organisms in industrial
production of vit. b12
• Streptomyces griseus , S. olivaceus , Bacillus megaterium ,
B. coagulans , Pseudomonas denitrificans ,
Propionibacterium freudenreichii , P. shermanii and
a mixed fermentation of a Proteus spp and a Pseudomonas sp.
7Industrial production of vitamins
• Manufactured by submerged fermentation
• Aeration and agitation of medium essential
• Fermentation process completed in 3 to 5 days
8Industrial production of vitamins
Vit.B12 production using
Streptomyces olivaceus NRRL B-1125
Industrial production of vitamins 9
Preparation of inoculum
Industrial production of vitamins 10
• 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 of production tank must be 5% of the volume of
• Consist of carbohydrate ,proteinaceous material , and source of cobalt
and other salts .
• Sterilization of medium batchwise or continuously .
• Batch – medium heated at 250°F for 1 hr
• Continuous – 330°F for 13 min by mixing with live steam.
12Industrial production of vitamins
Components Amount ( %)
Distillers solubles 4.0
Dextrose 0.5 to 1
COCl2.6H2O 1.5 to 10 p.p.m.
Temperature , pH , aeration and
• 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 aeration is
0.5 vol air/vol medium/min. Excess aeration cause foaming.
13Industrial production of vitamins
Antifoam agent , prevention of
• Antifoam agent : soya bean oil , corn oil,
lard oil and silicones (sterilized before adding) .
• Prevention of contamination : essential to maintain sterility ,
contamination results in reduced yields , equipments must be
sterile and all transfers are carried out under aseptic conditions .
14Industrial production of vitamins
• Yield of cobalamin are usually in the range of 1 to 2
mg. per litre in the fermented broth
15Industrial production of vitamins
• Cobalamin associated with mycelium- boiling mixture at pH 5
liberates the cobalamin quantitatively from mycelium.
• Broth containing cobalamin is subjected to further work up
depending on type of product to be produced
16Industrial production of vitamins
• Filtration - to remove mycelium.
• Filtered broth treated with cyanide – (cobalamin to
• Adsorption chromatography , ion exchange chromatography –
adsorbents : activated charcoal , bentonite , fuller’s earth .
Industrial production of vitamins 17
Bentonite Fuller’s earth
• Elution : water, water-acetone and solution of sodium cyanide or
sodium thiocyanate .
• further extraction – countercurrent distribution b/w cresol,
amyl phenol or benzyl alcohol and water or single extraction into
organic solvent (phenol)
Industrial production of vitamins 18
• To aqueous concentrates , dissolve a Zn salt in a slight acidic
solution & then rise the pH to bring about precipitation of
ZnOH(impurities are removed) .
• Chromatography on alumina & crystallization from
methanol-acetone , ethanol-acetone, or acetone-water.
Industrial production of vitamins 19
• To use as feed supplement , final fermented broth is evaporated
• Final broth contain 3% solids – in vacuo evaporation (15 to 20 %
• Syrup – drum dried or spray dried.(contain 10 to 30 mg.lb. of
Industrial production of vitamins 20
Vit.B12 production using
Industrial production of vitamins 21
• Reported to produce the highest yield s of Vit. B12 20 mg. per
• Production media : glucose , corn- steep , betaine , & cobalt
Betaine -0.5 %
Cobalt – 5µg./ml (excess cause reduced cobalamin formatn)
• pH -7.5
• Temperature – 30°C
Industrial production of vitamins 22
• 2 cycles : anaerobic 70 hrs ; aerobic 50 hrs.
• Anaerobic cycle – formation of cobinamide (O2 inhibits the
• Necessary to add 0.1% of 5, 6 – dimethyl benziminazole ( cannot
readily synthesize ).
• Aerobic cycle – nucleotide links with previously formed
• Continuous culture – 2 tanks in series , retention period 60 hrs ,
yield 15µg./ml is obtained .
Industrial production of vitamins 23
Vit.B12 production using
other micro - organisms
Industrial production of vitamins 24
Industrial production of vitamins 25
Species Medium Aeration Temp.
B. megaterium Molases , mineral
Aerobic 30 18 0.45
P. shermanii Glucose , corn-
steep, ammonia ,
cobalt pH 7.0
days) , aerobic
30 150 23
B. coagulants Citric acid ,
corn – steep ,
Aerobic 55 18 6.0
Oxalic acid , betaine
, cobalt , mineral
Aerobic ___ ___ 10
(Vitamin B 2)
Industrial production of vitamins 27
• Another important vitamin.
• Require 0.07 – 0.1 mg./ 0.75 kg of body wt. in humans.
• Recently, fermentation route has been widely used as it produces
the vitamin in a single step, resulting in substantial cost savings.
Industrial production of vitamins 28
Micro –organisms in industrial
production of vit. b2
• 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.
Industrial production of vitamins 29
Industrial production of vitamins 30
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 phage SPO 1 have resulted in
increased riboflavin production . The B. subtilis riboflavin-producing strain developed by
Perkins et al.
• Sybesma et al. developed Lactococcus lactis MG 1363 strain using both direct
mutagenesis and metabolic engineering for simultaneous overproduction of both folate
• Improved strains for the production of riboflavin were constructed through metabolic
engineering using recombinant DNA techniques in Corynebacterium ammoniagenes
Industrial production of vitamins 31
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
Industrial production of vitamins 32
• Fermentor 10,000 to 1,00,000 gals range.
• Production medium designed according to type of micro- organism.
• Ashbya gossypii : sources - palm oil ,corn steep liquor, glucose,
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.
Industrial production of vitamins 33
• 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
Industrial production of vitamins 34
Supplement Quantity of riboflavin (mg./L)
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 source).
• low cost organic wastes as flavinogenic factors and the various concentrations at which
they induced flavinogenecity resulting in higher yields of riboflavin (Kalingan and Liao,
• 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 (Kalingan, A.
E. and M. R. V. Krishnan ,1997)
Industrial production of vitamins 35
• 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
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• 2 steps (for animal feed supplements)
i. Evaporation of the whole broth in multiple effect evaporators
ii. Drying by drum-drier / spray drier
Industrial production of vitamins 37
• For drug & fine food use
i. Heating of fermentation broth at 120°C for 1 hr.
iii. Precipitation by adding dithionite (hydrosulfite).
iv. Other purification steps – crystallization.
Industrial production of vitamins 38
Ascorbic acid (Vit c)
Industrial production of vitamins 40
• L-ascorbic acid is an important metabolite for most living
• Strong reducing agent and plays a part in cellular oxidation –
• Daily intake : 60mg for persons aged 15 & above
50mg for age group b/w 11 to 14
45mg for children under 11
Industrial production of vitamins 41
• L-ascorbic acid finds its use mainly in food industry, being a
vitamin as well as an antioxidant.
• Approximately 50 % of synthetic ascorbic acid is used in vitamin
supplements and pharmaceutical preparations.
Industrial production of vitamins 42
• At present the majority of commercially manufactured L-ascorbic
acid is synthesized via Reichstein process using D-glucose as a
• Reichstein process involves six chemical steps and one
fermentation step for oxidation of D-sorbitol to L-sorbose .
• Bacterium used for this bioconversion is Acetobacter suboxydans.
• Very high level of aeration is required.
Industrial production of vitamins 43
Industrial production of vitamins 45
• With higher sorbitol concentration fermentation is slower in the beginning.
• Production medium is inoculated with culture of Acetobacter suboxydans (3 %
of total medium volume)
• Complete fermentation cycle takes 25 – 30 hrs.
D- Sorbitol 200 -250 g.
Corn –steep liquor 5 ml.
Tap water 1 ltr.
Antifoam as required
Fermentor (for vit.c
• Vertical fermentors , ( 5,000 to 10,000 gal. capacity)
• Designed for contain large volumes of air .
• Fermentors are equipped with a turbo mixer type agitator &
baffles along the side walls to ensure maximal turbulence of the
• Air inlet underneath the agitator.
Industrial production of vitamins 46
• In commercial operations , high velocity jets & 30 lb. head
pressure , used for maximal rate of fermentation , air to solution
ratio is 1:3
• Require provision for cooling the fermentor along with medium ,
cooling achieved by circulating cold water either through coils or
Industrial production of vitamins 47
• Analytical yields are about 95% – 98%.
• Recovery yields are about 85%.
Industrial production of vitamins 48
Other microbes involved in
ascorbic acid production
• Glucanobacter oxydans , Acetobacter melanogenus , Pseudomonas
albosesamae , Bacterium gluconicum , Brevibacterium ketosoreductum
• Saccharomyces cerevisiae and Zygosaccharomyces bailii accumulate L-
ascorbic acid intracellularly when incubated with L-galactose.
• Skatrud and Huss described the method for the efficient production of
L-ascorbic acid in algae ,Chlorella pyrenoidosa ATCC 53170
Industrial production of vitamins 49
Industrial production of vitamins 50
• β-carotene (provitamin A) , occurs naturally as a component of
• β-carotene after ingestion, is converted into vitamin A in the
Industrial production of vitamins 51
Micro –organisms in
• Members of Choanephoraceae family of Phycomycetes.
• Extensive studies on Phycomyces blakesleeanus ,
Choanephora cucurbitarum , and Blakeslea trispora .
• Production of β-carotene by wild-type Phycomyces blakesleeanus can be
stimulated by light, chemicals, regulatory mutations, and sexual interaction
between mycelia of opposite sex.
Industrial production of vitamins 52
Industrial production of vitamins 53
Blakeslea trispora - phase contrast image
• Barnett , Lilly , and Krause (1956) discovered increased yields of
β- carotene results with Choanephora cucurbitarum if both plus and minus
mating types are grown together .
• Similar phenomenon with Blakeslea trispora (Hesseltine and Anderson ,
Industrial production of vitamins 54
• Includes β – ionone, non ionic detergent , vegetable oil , fats , fatty acids ,
waxes, or white grease.
• β – ionone is regarded as a steering factor activates enzyme
responsible for β- carotene formation.
• Inoculum of each mating type is grown separately , & apprx. 5%
of each is added to fermentation medium.
• β – ionone is added 48 hrs after inoculation.
Industrial production of vitamins 55
Industrial production of vitamins 56
Coriolus versicolor in submerged culture
Food-grade pigments from Streptomyces sp. isolated from the
marine sponge Callyspongia diffusa
Industrial production of vitamins 57
Carotenogenic genes from the carotenoid-producing yeast
Xanthophyllomyces dendrorhous were introduced and overexpressed in S.
India based biotech companies in
• The manufacturing set up of Fermenta Biotech Limited located at Kullu in Himachal
Pradesh and Dahej in Gujarat are modern facilities that cater to worldwide supplies of
various grades of Vitamin D3 products and other specialty APIs.
• Jubilant Life Sciences Limited, headquarters in Noida, New Delhi , an integrated
pharmaceutical and life sciences company, is the largest Custom Research and
Manufacturing Services (CRAMS) player and a leading Drug Discovery and
Development Solution (DDDS) provider out of India. Globally No. 2 in Niacin &
Niacinamide / Vitamin B3-Nutrition Ingredients
Industrial production of vitamins 58
• Biotechnological Production of Vitamins , S.A. SURVASE et al.: Production of Vitamins, Food
Technol. Biotechnol. 44 (3) 381–396 (2006)
• Microbial Production of Riboflavin Using Riboflavin Overproducers, Ashbya gossypii, Bacillus
subtilis, and Candida famate: An Overview ,Seong Han Lim1, Jong Soo Choi2, and Enoch Y.
Park1* , Biotechnol. Bioprocess Eng. 2001, 6: 75-88
• Carotene-Super producing Strains of Phycomyces , F. J. Murillo et.al, Applied and Environmental
Microbiology, Nov. 1978, p. 639-642
• High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive
Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous
• Industrial Microbiology by A H Patel
• Industrial Microbiology by Casida
59Industrial production of vitamins