This document discusses the microbial production of vitamin B12 through fermentation. It notes that vitamin B12 is exclusively synthesized by microorganisms and outlines several microorganisms used in fermentation processes, including Propionibacterium freudenreichii and Pseudomonas denitrificans. The document describes the two-stage batch process using P. freudenreichii and the one-stage continuous process using P. denitrificans, which is most productive. It provides details on medium composition and recovery methods to produce vitamin B12 at commercial scales.

1. MICROBIAL PRODUCTION OF
VITAMIN B12 (CYANOCOBALAMIN)

2. INTRODUCTION
 Vit B12 is a vitamin that is synthesized in nature
exclusively by microorganisms.
 Vitamin B12 is synthesized by microorganisms in the
large intestinal tract cannot be assimilated.
 So it should be given by outer source such as food.
 Activated sludge from sewage treatment contains 4-
10mg Vit B12 per kg but isolation from these sources
is expensive.
 Vit B12 was first obtained commercially as a by-
product of streptomycin fermentation with yield of
1mg/liter of broth.

3.  As demand of Vit B12 increased, fermentation
processes were developed with high-yielding strains.
 The current annual production (World) is over
15000kg.
 Vitamin B12 production is based on media containing
carbohydrate.
 Most Vit B12 fermentation processes use glucose as
carbon source.
 Common microorganism which are used for producing
Vitamin B12 by fermentation are as follows.

4. Microorganism Production, mg/liter
 Streptomyces olivaceus 3.3
 Micromonospora sp 11.5
 Propionibacterium freudenreichii 19.5
 Propionibacterium shermanii 23.0
 Pseudomonas denitrificans 60.0
 (Note -Propionibacterium and Pseudomonas are the
commercially used genera)

5. PROCESS BASED ON
PROPIONIBACTERIUM FREUDENREICHII
 Propionibacterium fredenreichii as well as other mutant
strains are used in a two-stage process with added
cobalt (10-100mg/L).
 In the preliminary anaerobic phase (2-4 days)
5’deoxyadenosyl cobinamide is mainly produced.
 In the 2nd aerobic phase (3-4 days) the biosynthesis of
5,6- dimethyl benzimidazole takes place so that
5’deoxyadenosyl cobalamine (known as coenzyme
B12) can be produced.
 As an alternative to this two-stage batch process in a
fermenter both stages can also be operated
continuously in two tanks.

6.  During the recovery process, the cobalamins which
are almost completely bound to cell are brought into
solution by heat treatment (10-30 min at 80- 120°C,
pH~ 6.5-.5).
 They are then converted chemically into more stable
cyanocobalamin.
 The raw product with about 80% purity is used as feed
additive.
 Additional purification is done (95- 98% purity) for
medicinal use.

7. PROCESS BASED ON PSEUDOMONAS
DENITRIFICANS
 Pseudomonas denitrificans has bee found to be the
most productive species among Vit B12 producing
microorganisms.
 In this one-stage process, the vitamin is produced
during the entire fermentation.
 Cobalt and 5,6-dimethyl benzimidazole must be added
as supplements.
 Sugar beet molasses is used as low cost carbon
source, which also contains betaine (which is
assumed to cause activation of biosynthesis or an
increase in membrane permeability).

8. MEDIUM USED
Medium A Amount, g/liter
 Sugar beet molasses 60
 Yeast extract 1
 N-Z amine
(enzymatic casein hydrolysate) 1
 (NH4)2HPO4 2
 MgSO47H2O 1
 MnSO4.H2O 0.2
 ZnSO4.7H2O 0.02
 Na2MoO4.2H2O 0.005
 Agar 25
 Tap water To make 1 liter
 pH 7.4
 Medium B is same as Medium A but without agar.

9. Medium C Amount, g/liter
 Sugar beet molasses 100
 Yeast extract 2
 (NH4)2HPO4 5
 MgSO47H2O 3
 MnSO4.H2O 0.2
 Co(NO3)2.6H2O 0.188
 5,6-dimethyl benzimidazole 0.025
 ZnSO4.7H2O 0.02
 Na2MoO4.2H2O 0.005
 Agar 25
 Tap water To make 1 liter
 pH 7.4