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Production of Vitamin B12 Vitamins Microorganisms can be used for the commercial production of certain vitamins, such as thiamine,

, riboflavin, folic acid, pantothenic acid, pyridoxal, vitamin B12 and Biotin.

However, on a worldwide basis only the production of vitamin B12, riboflavin and ascorbic acid have major economic significance.

Vitamin B12 Vitamin B12 (Cyanocobalamin) is a vitamin that is synthesized in nature exclusively by microorganisms; because it is required by animals it is present in every animal tissue in very low concentrations (eg., 1 PPM in the liver).

The vitamin B12 needs of animals are covered by food intake or by absorption of vitamin B12 produced by intestinal microorganisms. The concentrations of vitamin B12 which are present in animal tissues are too low for use in commercial production. Vitamin B12 was firs obtained commercially as a byproduct of streptomycete fermentations for the production of the antibiotics streptomycin, chloramphenicol or neomycin with a yield of about 1 mg/l. The most important manufacturers of vitamin B12 are: Farmitalia S.p.A (Italy); Glaxo Lab., Ltd (England); MercK & Co., Inc. (US); Rhone Poulenc S.A. (France); Roussel UCLAF (France); G. Richter Pharmaceutical Co and Chinoin (both Hungary). The current annual world production of vitamin B12 is estimated at about 12,000Kg.

Vitamin B12 Production Most of the B12 fermentation processes use glucose as a carbon source.

Several producing strains are known, listed here with their yield of B12: Bacillus megaterium (0.45mg / l) Butyribacterium rettgeri (5 mg / l) Streptomyces olivaceus (3.3 mg / l) Micromonospora sp (11.5 mg / l) Klebsiella pneumoniae (0.2 mg / l) Higher yields have been obtained from Propionibacterium freudenreichii (19 mg / l), Propionibacterium shermanii (30 40 mg / l) and (in a process using sugar cane molasses) Pseudomonas denitrificans (60 mg / l). A hybrid strain called Rhodopseudomonas protamicus, made by the protoplast fusion technique between Protaminobacter ruber and Rhodopseudomonas spheroides, produces 135 mg / l vitamin B12 using glucose as carbon source without the addition of 5, 6 dimethylbenzimidazole

Processes with Propionibacteria

Propionibacterium freudenreichii ATCC 6207 and P. shermanii ATCC 13673, as well as other variants and mutants, are used in two-stage process with added cobalt (10 100 mg / l).

In a preliminary anaerobic phase (2-4 days), 5-deoxyadenosylcobinamide is mainly produced. In a second, aerobic phase (3-4 days) the biosynthesis of 5, 6 dimethylbenzimidazole takes place, so that 5deoxyadenosylcobalamin (Coenzyme B12) can be produced. As an alternative to the two-stage batch process in a fermenter, both stages can also be operated continuously in two tanks operated in cascade fashion. During the recovery process, the cobalamins, which are almost completely bound to the cell, are brought into solution by heat treatment (10-30 min at 80-120C, pH 6.5-8.5). They are then converted chemically into the more stable cyanocobalamins. The raw product (80% purity) is used as a feed additive. Additional purification stages yield a medically usable preparation (95-98% purity). The total yield is 75% of the theoretical value.

Processes with Pseudomonas

Pseudomonas denitrificans has been found to the most productive species among the different vitamin B12 producing pseudomonads. In this one-stage process, vitamin B12 is produced during the entire fermentation. Cobalt and 5,6-dimethylbenzimidazole must be added as supplements. It has also been found that additions of the compound betaine result in increased yield; sugar beet molasses is used as a low-cost betaine source. After 12 years of strain development, the yields from this process have been increased from 0.6 mg / l to 60 mg / l.

Laboratory scale production process for Vitamin B12 using Pseudomonas denitrificans

Media composition Medium A (g / l): Sugar beet molasses 60; Yeast extract 1; N-Z-Amine 1; (NH4)2HPO4 2; MgSO4. 7H2O 1; MnSO4. H2O 0.2; ZnSO4. 7H2O 0.02; Na2MoO4. 2H2O 0.005; Agar 25; pH 7.4. Medium B (g / l): Medium A without agar. Medium C (g / l): Sugar beet molasses 100; Yeast extract 2; (NH4)2HPO4 5; MgSO4. 7H2O 3; MnSO4. H2O 0.2; Co(NO2). 6H2O 0.188; 5, 6 dimethylbenzimidazole 0.25; ZnSO4. 7H2O 0.02; Na2MoO4. 2H2O 0.005; pH 7.4