Riboflavin, also known as vitamin B2, is produced industrially through three main processes - chemical synthesis, biotransformation, and direct fermentation. Direct fermentation, which accounts for 30% of production, involves fungi and bacteria that synthesize riboflavin including Ashbya gossypii, Candida famata, and genetically engineered Bacillus subtilis. The industrial standard strain is A. gossypii, which is cultured for 7 days at 28°C to produce 10-15 g/L of riboflavin using glucose, corn steep liquor, and other nutrients as carbon and energy sources. After fermentation, riboflavin is released from the cells, purified

1. RIBOFLAVIN FRMENTATION

2. Occurrence and Economic
Significance
 Riboflavin is also called lactoflavin or
Vitamin B2.
 First isolated from whey by Kuhn, Gyorgy
and Wagner-Jauregg in 1933.
 Structure was confirmed by Kuhn and
Karrer in 1935 by synthesis.
 Present in milk as free riboflavin but in
other food (liver, heart, kidney, eggs) as a
part of flavoproteins, which contain the
prosthetic group FMN or FAD.
 The daily human demand for riboflavin is
around 1.7 mg, and deficiencies lead to
various symptoms such as, e.g., versions
of dermatitis.

3.  Deficiency in rats causes stunted growth, dermatitis
and eye damage.
 Ariboflavinosis-a kind of dermatitis is a disease is a
disease in humans caused by its deficiency.
 In USA riboflavin, thiamine and nicotinic acid frequently
added to flour to make vitamin enriched bread.
 There are 3 main production processes:
1. Chemical synthesis: primarily for pharmaceutical
use. (20%)
2. Biotransformation: here glucose is converted to D-
ribose and subsequently to riboflavin by Bacillus
pumilus. (50%)
3. Direct fermentation: more than 2000 tons per
annum on world wide basis. (30%)

4. Strains Used
 Synthesized by many microbes including bacteria, yeast
and fungi.
 Two Ascomycete fungi are industrially important.
1. Eremothecium ashbyii (yield: 2 g/l, initially used)
2. Ashbya gossypii (yield: 10-15 g/l, used since 1946)
 Despite this yield there is a tough competition between
all 3 processes.

5.  At present, three organisms are used for the
industrial production of riboflavin by fermentation:
 The filamentous fungus Ashbya gossypii (BASF,
Germany)
 The yeast Candida famata (ADM, USA)
 A genetically engineered strain of Bacillus subtilis
(DSM, Germany)

6. Structure
 It is an alloxazine derivative
consists of pteridine ring
condensed to a benzene ring.
 The side chain consists of aC5-
polyhydroxy group – a
derivative of ribitol.
 The IUPAC name of riboflavin
is [6,7- dimethyl-9-(d-1’- ribityl)
isoalloxazine].
 The isoalloxazine ring acts as a
reversible redox system.

7. Biosynthesis
 The biosynthetic pathway is derived from experiments
done on yeast and A. gossypii.
 In E. ashbyii and A. gossypii fermentation is not affected
by iron but in clostridia and yeast it is inhibited by very
low conc. of iron. In clostridia 1ppm iron causes 75%
inhibition.
 The intermediates of synthesis are as follows:
1. GTP: Guanosine triphosphate
2. PRP: Phosphoribosyl amino pyrimidine
3. ADRAP: Amino-Dioxy Phosphoribitylamio-Pyrimidine
4. Diaminouracil
5. MERL: Methyldihydroxyethyl ribityllumazine
6. DMRL: Dimethyl ribityllumazine
7. Riboflavin

9. Production process
 Production is carried out with Ashbya gossypii NRRL-
1056 strain.
 A careful sterilization of the culture medium is critical
for high yields, as inoculum size is small (0.75 to 2% of
a 24-48 hour old actively growing culture).
 Originally the fermentation used a medium with
glucose and corn steep liquor; sucrose and maltose
were other suitable carbon sources.
 Lipids were also used as an energy sources and yields
markedly found to increase.
 Riboflavin production (containing corn steep liquor
2.25%, commercial peptone 3.5%, soybean oil 4.5%)
has been further stimulated by the addition of different
peptones, glycine, distillers soluble or yeast extract.

10.  By simultaneous feeding of glucose and inositol the rate
of formation of riboflavin can be further increased.
 The fermentation takes 7 days with an aeration rate of
0.3vvm at 28 ˚C.
 For foam control, silicone antifoam is applied at first and
soybean oil, is added later (also metabolized).
 Riboflavin is present both in solution and bound to the
mycelium in the fermentation broth.
 The bound vitamin is released from the cells by heat
treatment (1hour at 120 ˚C) and the mycelium is
separated and discarded.
 The riboflavin is then further purified.

11. Production using other
organisms
 Production of riboflavin with and aliphatic
hydrocarbon as carbon source has been reported
using Pichia guilliermoendii.
 Using Pichia miso, 51mg/l riboflavin was obtained
on a medium with n-hexadecane, corn steep liquor
and urea.
 Production using Hansenula polymorpha is
reported using methanol.
 Crystalline high purity riboflavin is obtained from
Saccharomyces fermentation with acetate as sole
carbon source.