Cyanocobalamin is produced through bacterial fermentation. Bacteria such as Pseudomonas denitrificans and Propionibacterium shermanii are cultured in sterilized medium to produce precursors to cyanocobalamin. The precursors are then treated with potassium cyanide and heat to convert them into cyanocobalamin. Cyanocobalamin production involves anaerobic fermentation to produce precursors, aerobic fermentation to produce more precursors, and chemical treatment to convert the precursors into the final cyanocobalamin product.

1. Cyanocobalamin Production
Mrs. Praveen Garg
VITS College, Satna

2. Introduction
• Cyanocobalamin is a manufactured form with a cyano
(cyanide) group bound to cobalt.
• Bacterial fermentation creates derivatives of B12 which are
converted to cyanocobalamin by addition of potassium
cyanide in the presence of sodium nitrite and heat.
• It is also known as Vitamin B12.
• Vitamin B12, also known as cobalamin, is a water-soluble
vitamin involved in the metabolism of every cell of the human
body.
• "Cobalamin" actually refers to a group of compounds
(corrinoids) with near-identical structure.
• Cobalamins are characterized by a porphyrin-
like corrin nucleus that contains a single cobalt atom bound to
a benzimidazolyl nucleotide and a variable residue (R) group.

3. Vitamin B12

4. • It is a cofactor in DNA synthesis, and in both fatty
acid and amino acid metabolism.
• It is important in the normal functioning of the nervous
system via its role in the synthesis of myelin, and in the
maturation of developing red blood cells in the bone marrow.
• Vitamin B12 is the largest and most structurally complex
vitamin.
• The vitamin exists in four near-identical chemical forms
(vitamers): cyanocobalamin, hydroxocobalamin, adenosylcobal
amin and methylcobalamin.
Function of Vitamin B12

6. M.Or. Used for Production of Vitamin B12
• Large scale industrial production of vitamin B12 occurs via
fermentation by M.Or.
• Pseudomonas denitrificans,
• Propionibacterium shermanii or Sinorhizobium meliloti
• Cyanocobalamin is commercially prepared by bacterial
Submerged fermentation process.
• Fermentation by a variety of microorganisms yields a mixture
of methylcobalamin, hydroxocobalamin and adenosylcobalamin.
• These compounds are converted to cyanocobalamin by addition
of potassium cyanide in the presence of sodium nitrite and heat.

7. Cobalamin biosynthetic pathway
De novo pathway:
• Cobalamin can be synthesized de novo in prokaryotes through
two alternative routes according to the timing of cobalt
insertion and the molecular oxygen requirement.
• These pathways are the aerobic pathway, which has been
studied in P. denitrificans, and the anaerobic pathway, which
has been studied in S. typhimurium, Bacillus megaterium,
and Propionibacterium shermanii.
• Both routes differ in terms of cobalt chelation and oxygen
requirements (the aerobic pathway requires oxygen to promote
ring-contraction, while the anaerobic pathway does not require
oxygen in this step)

8. Biosynthetic pathway

9. Steps involved in Cyanocobalamine production
 Formation of medium
 Sterilization of medium
 Preparation of Starter culture
 Anaerobic fermentation
 Aerobic fermentation
 Recovery of cyanocobalamine

10. 1. Formation of Medium
 Carbon Source: Soyabean meal/glucose
Beet molasses
Corn steep glucose
 Nitrogen Source: Ammonium phosphate
Ammonium Hydroxide
 Perpared medium is sterilized by autoclaving.
 The sterilized medium is then used for fermentation.
2. Sterilization

11. Preparation of Starter culture
 Different m.or. used for fermentation of cyanocobalamine.
• Pseudomonas denitrificans,
• Propionibacterium shermanii
• S. typhimurium
• Bacillus megaterium
 For preparation of starter culture, nutrients and the inoculum
are added to sterile fermenter.
 Anti-foaming agent may be added.
 Once the desired product is present in the fermenter the
contents are drained off and the product is extracted.

12. Anaerobic fermentation
• Batch and Continuous both fermentation process effective in
anaerobic condition.
• Sterilized medium is filled in stirred tank fermenter. 1% of
inoculum (starter culture) is added in to the fermenter.
• Anaerobic condition is maintained to enhance the production
of dimethyl benzimidazole cobalamine (DBC) by P. shermanii.

13. • In this process, sterile air is pumped in to the fermenter.
• The culture is stirred well for proper aeration.
• Aerobic fermentation is performed for 3-4 days.
• During this process some amount of DBC and pseudovitamin
B12 are produced.
• DBC and pseudovitamin B12 are precursor of
cyanocobalamine.
Aerobic fermentation

14. Recovery of Cyanocobalamine
• The cyanocobalamine exist in the form of natural substances
such as DBC and pseudovitamin B12 Inside the microbial
cells.
• The culture broth contained 10-23mg vitB12/liter.
• It is harvested and centrifuged at high speed to get a
concentrated mass of cells.
• The cell mass is treated with a dilute acid and heat stock at 10-
300C.
• During the treatment precursors of pseudovitamin B12 are
released free.
• Then it is treated with cyanide solution to split the DBC and
pseudovitamin b12.
• As a result cyanocobalamine is released free in the solution.

15. THANK YOU