1. Fermentative Production of Vitamins:
Indian and Global Scenario
Irfan Ahmad
M. Tech. Pharm. Tech. (Biotech.)
National Institute of Pharmaceutical Education and Research, S.A.S. Nagar

2. CONTENTS
1. Introduction to Vitamins.
2. Industrial Production of Vitamins.
3. Vitamins Market Scenario.
4. Fermentative Production of Water Soluble Vitamins.
i. Vitamin B₁₂
ii. Vitamin B₂
iii. Vitamin C
iv. Vitamin H
5. Fermentative Production of Fat Soluble Vitamins.
i. Vitamin E
ii. Vitamin K
6. Summary.

3. Vitamins
Vitamins are defined as essential micronutrients that are
required in trace quantity and cannot be synthesized by
mammals but are essential for metabolism of all living
organisms
VITAMINS
Fat Soluble Vitamins Water Soluble Vitamins
Vitamin A Vitamins B
Vitamin D Vitamin C
Vitamin E Vitamin H
Vitamin K Vitamin P, etc.

4. Role of Vitamins
1 Nutritional and Physiologic Role
.
2 Food/Feed Additives
3 Therapeutic Agents
4 Health Aids
5 Technical Aids

5. Industrial Production Of Vitamins
Extraction Chemical Fermentation

6. Industrial Production of Vitamins
Production Method World Production (tonne)
Biotech. Chem. Extr. 1980 1990 2000 2010
Thiamine (B1) + 1700 4200 3700
Riboflavin (B2) + 2000 2400 4400
Niacin + + 8500 22000 2800
Pantothenic acid + + 5000 7000
Pyridoxine (B6) + 1600 2550 3800
Biotin + + 2.7 2.5 88 112
Folic acid + 100 400 534
Vitamin B12 + 2 10 25 175
Vitamin C + + 40000 60000 10700
Vitamin A + + 16800 2500 2700
Vitamin D + + 5000
Tocopherol (E) + + + 6800 22000 30000
Vitamin K + 500
Survase SA, Bajaj IB, Singhal RS: Biotechnological production of vitamins

7. Microbial Production of Vitamins
Vitamin Enzyme (Microorganism)
Vitamin C 2,5-diketo-D-gluconic acid reductase (Corynebacterium sp.).
ucu
Biotin Fermentation (Serratia marcescens); Multiple enzyme system
(Bacillus sphaericus).
Riboflavin Fermentation (Eremothecium ashbyii, Ashbya gossypii,
Bacillus sp.).
Fermentation (Propionibacterium shermanii, Pseudomonas
Vitamin B12
denitrificans)
Vitamin E Freshwater microalgae, Euglena gracilis.
Shimizu S: Vitamins and related compounds: microbial production

8. Fermentative Production of Vitamins
Advantages Disadvantages
i. Efficient,
i. Complex,
ii. Less Energy Intensive,
iii. Carried out at ambient ii. Require Careful Monitoring,
temperature and pressure, iii. Limited Operations Region,
iv. Produce Renewable iv. Susceptible to Substrate and
byproducts (e.g., Biomass), Product Inhibition,
v. Low Cost of Waste Disposal,
v. High Cost of Downstream
vi. Highly enantioselective and Processing,
regioselective,
vii. Don’t suffer from consumer vi. High Risk of Contamination,
consciousness with regard to vii. Tendency to Elicit Allergic
safety. Manifestations.

9. Vitamin Market Scenario
• Europe represents the largest regional market; the US
constitutes the single largest market globally.
• Asia-Pacific is likely to emerge as the fastest growing market,
with a CAGR of about 4.0% over the analysis period.
• Vitamin E represents the largest segment, owing to the
extensive use of these vitamins in cosmetics, pharmaceuticals,
and food end-use applications
• The cosmetics industry, though relatively small in terms of
the percentage share, is emerging as a key end-user with a
CAGR of 4.6% over the analysis period.
• The global vitamin market was $2.3 billion in 2000.
However, the vitamin market is expected to reach US$3.2
Billion by 2017.

10. Top Five Sources of US Vitamin Imports
China Belgium Other Germany India Netherland
The vitamin industry was once
dominated by a core group of
3% 3%
producers in the developed economies 7%
but is now far more open and 13%
competitive as an increasing number
of manufacturers in emerging
57%
economies such as China and India
17%
weigh in.

11. Hoffmann La Roche
(Switzerland), BASF
(Germany), ADM
(USA), Hubei Guangji
(China), Merck
(Germany) and Takeda
(Japan) are some of the
global leaders in vitamin
production.

12. Sales
Single vitamin
In 2011, the Indian vitamin segment 0.3%
grew 7.5 per cent in volumes (against
Single
0.1 decline in the year 2010) and 10.3 Mineral
per cent in value (against 5.4 per cent 9%
Tonics
growth in 2010).By the end of 12%
2014, the vitamins and minerals
category will be worth INR
21,174.6m ($486.7m), with an
expected CAGR of 3% between 2009
and 2014.
Multivitamins
79%
Five of the top 10 players are foreign
multinational firms. Among Indian
companies, the largest vitamin
producers are Wockhardt, Raptakos
Brett, Piramal Healthcare and Alkem.

13. WATER SOLUBLE VITAMINS
Vitamin B₁₂
Vitamin B₂
Vitamin C
Vitamin H

14. Vitamin B₁₂( Cyanocobalamin)
 Cyanocobalamin is the industrially produced
stable cobalamin form which is not found in
nature.
 It is obtained exclusively by fermentation
process.
 Merck began production of vitamin B₁₂ by
Pseudomonas denitrificans in 1952 and have
improved the efficiency of culture more than 30-
fold relative to the performance of the original
soil isolates by genetic manipulations and
microbial screening.
 Mutagenic treatments have resulted in improved
activity.
Sumi: Microbial Production of Vitamin B12

15. Vitamin B₁₂

16. Biosynthesis of Vitamin B₁₂

17. Downstream Process of Vitamin B₁₂

18. Vitamin B2 (Riboflavin)
• Riboflavin has been produced commercially by chemical
synthesis, by fermentation and by a combination of
fermentation and chemical synthesis.
• Recently, fermentation route has been widely used as it
produces the vitamin in a single step, resulting in substantial
cost savings. In contrast, chemical processes are
multistage, and incur a lot of cost.
• Most of the producers like
BASF, Roche, ADM/Aventis, Hubei Guangji prefer
fermentative production of riboflavin over chemical process.

19. Vitamin 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 40000 times more vitamin than it needs for
its own growth.
• Recently, Nippon Roche, Japan, has developed and commercialized a
single step fermentative riboflavin production using a recombinant
Bacillus subtilis strain, which effectively produces riboflavin directly
from glucose in fed-batch operation.
• Improved strains for the production of riboflavin were constructed
through metabolic engineering using recombinant DNA techniques in
Corynebacterium ammoniagenes.
• Sybesma et al. developed Lactococcus lactis strain using both direct
mutagenesis and metabolic engineering for simultaneous overproduction
of both folate and riboflavin.

20. Vitamin C (Ascorbic Acid)
• L-ascorbic acid finds its use mainly in food industry, being a
vitamin as well as an antioxidant.
• Majority of commercially manufactured L-ascorbic acid is
synthesized via Reichstein process using D-glucose as a
starting material
• Approximately 50 % of synthetic ascorbic acid is used in
vitamins supplements and pharmaceutical preparations.
• Because of its antioxidant properties and its potential to
stimulate collagen production, it is also widely used as an
additive to cosmetics.
• The current global market of L-ascorbic acid is in excess of
US$ 585 million with an annual growth rate of 3 %.

21. Vitamin C (Ascorbic Acid)
.
D-Glu
D-Sorbitol
Reichestein Process
L-Sorbose
L-
Ascorbic
Diacetone-L- 2-Keto-L- acid
Sorbose Gluconic
acid methyl
ester

22. Bacterial Fermentation of Vitamin C
2-Keto-D-gluconic acid
Sorbitol Pathway
Pathway

23. Yeast Based Fermentation Process
• Saccharomyces cerevisiae and Zygosaccharomyces
bailiiaccumulate produce L-ascorbic acid intracellularly
when incubated with L-galactose.
• Over-expression of the D-arabinose dehydrogenase and D-
arabinono-1,4-lactone oxidase in Saccharomyces cerevisiae
enhances this ability significantly.

24. Algae Based Fermentation Process
• 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 mg/L).

25. Vitamin H (Biotin)
 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 themselves.
 Biotin is added to many food, feed and cosmetic
products, creating a world market of 10–30 t/year.
 Majority of the biotin sold is synthesized chemically via
Goldberg and Sternbach syntheses.
 The chemical synthesis is linked with a high environmental
burden, much effort has been put into the development of biotin-
overproducing microbes

26. Biosynthesis of Biotin
bioF gene
bioA gene
bioD gene
The conversion of
bioB gene
dethiobiotin to biotin
has not been resolved.

27. Fermentative Production of Biotin
• 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.
• Brown et al. studied the production of biotin by recombinant
strains of E. coli. The strain used was E. coli C268 having a
bio A genotype and plasmid pTG3410 with two expression
cassettes .
• Biotin production under limiting growth conditions by
Agrobacterium/Rhizobium HK4 transformed with a modified
Escherichia coli bio operon.

28. FAT SOLUBLE VITAMINS
Vitamin E
Vitamin K

29. Vitamin E
 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 sunscreens.

30. Vitamin E
Extraction
The synthetic form is a
mixture of eight stereoisomers
Extraction from oils is not
collectively known as all-rac-
efficient, as these typically
alpha-tocopherol.
contain low levels of Vitamin E
∞-tocopherol when administered in equal
amounts, the bioavailability of
natural with respect to
synthetic ∞-tocopherol is 2:1.
Chemical Synthesis
Chemical Synthesis

31. Vitamin E
Fermentative Production of Vitamin K₂
 Microalgae Euglena gracilis Z and marine microalgae
Dunaliella tertiolecta produce ∞-tocopherol in
concentrations higher than conventional foods.
 Carballo-Cardenas et al. studied ∞-tocopherol production
with Dunaliella tertiolecta and Tetraselmis
suecica, demonstrating that nutrient composition can be used
as a tool to improve α-tocopherol productivity.

32. Vitamin K₂
Vitamin K₁
.
Vitamin K Vitamin K₂

33. Vitamin K
Fermentative Production of Vitamin K₂
 Tani and Taguchi have reported that as much as 182 mg/L
MK was produced using detergent supplement culture and a
mutant of Flavabacterium.
 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 natto bacilli.

34. Summary
 Fermentative productions of vitamins have many
advantages compared to conventional chemical
synthesis processes.
 Different methods like media
optimization, mutation and screening, genetic
engineering and biocatalyst conversion have been
used for improvement of the production of vitamins.