This document discusses vitamin B12, including its structure, functions, deficiency, production, and uses. It provides details on the microbial production of cyanocobalamin (vitamin B12) through fermentation using Propionibacterium shermanii. The process involves anaerobic fermentation to produce precursors, aerobic fermentation, cell harvesting, acid treatment to release precursors, addition of cyanide to produce cyanocobalamin, and downstream purification using chromatography and crystallization. Vitamin B12 is important for normal functioning of the brain/nervous system, blood formation, and cellular metabolism. Deficiency can cause anemia and fatigue.

1. Dr.K.Gajalakshmi
Assistant professor,
PSGR krishnammal college for Women,
Coimbatore

3. • vitamin B12, vitamin B12 or vitamin B-12, also
called cobalamin, is a water-soluble vitamin with a
key role in the normal functioning of the brain and
nervous system, and for the formation of blood. It is
one of the eight B vitamins.
• It is normally involved in the metabolism of every
cell of the human body, especially affecting DNA
synthesis and regulation, but also fatty acid synthesis
and energy production.
• Vitamin B12 is important for the way the body
works, and people who don't have enough of it may
feel tired or have a lack of energy.

4. • Vitamin B12 helps in the production of healthy
red blood cells that carry oxygen around the
body.
• Not having enough vitamin B12 is called
vitamin B12 deficiency anaemia.
• This condition makes the body produce larger
than normal red blood cells, described as
megaloblastic or macrocytic, which don't do
their job as well.
• Once diagnosed, vitamin B12 deficiency can
usually be treated successfully with B12
injections and sometimes with B12 tablets.

5. Structure of Vitamin B12

6. • Species from the following genera are known to synthesize
B12: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes,
Azotobacter, Bacillus, Clostridium, Corynebacterium,
Flavobacterium, Lactobacillus, Micromonospora,
Mycobacterium, Nocardia, Propionibacterium,
Protaminobacter, Proteus, Pseudomonas, Rhizobium,
Salmonella, Serratia, Streptomyces, Streptococcus and
Xanthomonas.
• Industrial production of B12 is through fermentation of
selected microorganisms. Streptomyces griseus, a bacterium
once thought to be a yeast, was the commercial source of
vitamin B12 for many years.[48][49] The species Pseudomonas
denitrificans and Propionibacterium shermanii are more
commonly used today

7. •
•
•
•
Methylcobalamin (shown) is a form of vitamin
.
B12. Physically it resembles the other forms of
vitamin B12, occurring as dark red crystals that
freely form cherry-colored transparent solutions
in water
Minot and Murphy -reported -liver juice, in
1926.
Rickets and Smith -isolated from liver cells of
animals in 1942.
Rikes - Microbial source of Cyancobalamine
demonstrated in 1948. – Streptomyces griesus

9. Steps involved in Microbial Cyanocobalamine production
Step-6

10. For Media preparation
•
•
•
•
Carbon source- as
1.Corn steep glucose
2. Beet molasses
3.Soyabean meal/Glucose
• Nitrogen source-as
• 1.Ammonium phosphate
• 2. Ammonium hydroxide

11. Step-1 Formulation of medium
Medium+Cobalt salt
Medium+ Cobalt salt
Sterilization
Starter culture of
Propioni bacterium shermanii
o
Inoculati
n
Anaerobic fermentation for 3
days
Aerobic fermentation for 4
days
Centrifugation
Harvested Broth
Cell Harvest
Acid treatment and heating
Released Pseudo-vitamin B12
Bakers coenzyme

12. Released Pseudo-vitamin B12
Bakers coenzyme
Addition of cyanide solution
Cyanocobalamine in liquid
Adsorption
chromatography
Adsorption on IRC-50 resin
Elution with a phenolic
compound
Cyanocobalamine in solvent
Evaporation
Cyanocobalamine crystals

13. 2. Sterilization
• Prepared medium is sterilized by autoclaving
• The sterilized medium is then used for fermentation.

14. 3.Making Starter culture
•
•
The following microbes were suitable for Industrial fermentation of
cyanocobalamine.
1.Bacillus megaterium
2. Streptomyces olivaceous
•
3.Propionibacterium shermanii
•
4. Pseudomonas denitrificans
5.Rhodopseudomonas palustris

15. Inoculum
Wild strains
Mutant strains of--- Inoculum
Improved strain- produce 50,000
times more vitamin B12 than wild
strain .This is the great boon for
biotechnologists to produce vit B12.

16. Batch fermentation
• Most fermentations are batch processes
• Nutrients and the inoculum are added to the sterile fermenter and left
to get on with it!
• Anti-foaming agent may be added.
• Once the desired amount of product is present in the fermenter the
contents are drained off and the product is extracted.
• After emptying, the tank is cleaned & prepared for a new batch.

17. Continuous fermentation
• Some products are made by a continuous
culture system.
• Sterile medium is added to the fermentation
with a balancing withdrawal of broth for
product extraction.

18. • UPSTREAM PROCESSING
• Upstream processing encompasses any
technology that leads to the synthesis of a
product. Upstream includes the exploration,
development and production.
• DOWNSTREAM PROCESSING
The extraction and purification of a
biotechnological product from fermentation is
referred to as downstream processing

19. Aerobic Fermentation
• It is a Batch fermentation
• Continuous fermentation also found to be effective.
• Sterilized medium is filled in stirred tank fermenter
1% of inoculum (Starter culture) is added in to the
fermenter.
• Anaerobic condition is maintained to encourage the
production of 5,6-dimethyl benzimidazole
cobalamide (DBC) by P.shermanii

20. Aerobic Fermentation
• Anaerobic Fermentation is over sterile air is pumped
in to the fermenter.
• The culture is stirred well for proper aeration
• Aerobic fermentation is performed for 4 days.
• During this process some amount of DBC and pseudo
vitaminB12 (adeninyl cobalamine) are produced.
• DBC and pseudovitaminB12 (adeninyl cobalamine)
are immediate precursors of cyanocobalamine

21. Recovery of Cyanocobalamine
• Inside the microbial cells the cyanocobalamine exits in the
form of natural substances such as DBC and
pseudovitaminB12 .
• The cultured broth contains 10-23mg vitB12 per 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
10-30o C.
• During this treatment precursors of pseudovitaminB12 are
released free.

22. • Then it is treated with Cyanide solution to split the DBC and
pseudovitamin B12 .
• As a result cyanocobalamine (Vit B12) is released free in the
solution.
• Cyanocobalamine in the liquid is seperated by using an
adsorption column chromatography with IRC-50 resin. The
adsorbed cyanocobalamine is then eluted out of the column
using phenolic solvent.
• The solvent fraction is evaporated by exposing it to
atmospheric air.
• As result crystals of cyanocobalamine is let in the vessel. It is
stored for future use.

23. USES
• It is a food preservative
• It is a co-factor
• It is a protective medicine