Microbial production of vitamin B12
Vitamins are growth factors required in very small amounts but are very essential part of nutrition of humans, animals and even microbes. Chemically, vitamins are organic compounds. They also function as coenzymes or building blocks for coenzymes of several enzymes. Vitamin B12 is one of the important vitamins of B complex (B1, B2, B3, B6 and folic acid) group. It is also known as cobalamin or cobamide. Structurally, it is very complex compound or rather a group of closely related cobamides. The structure contains cobalt porphyrin nucleus attached to ribose and phosphate. Variations in structure may occur because of different purine, Benzimidazole or other chemical groups attached to cobalt atom. Being water soluble, it needs to be supplied regularly through food such as liver extract and meat. It is essential in the formation of red blood cells and its deficiency causes pernicious anemia. It functions as coenzyme in various cobalamin derivatives which are involved in molecular rearrangement reactions. Microbial synthesis is the only source of commercial production of vitamin B12. There is no other source or synthetic method known that produce B12 vitamin. Vitamin B12 is produced during normal microbial metabolism. In 1948, Rickes and colleagues, first time recovered B12 in active crystalline form from actinomycete Streptomyces griseus. Before this discovery, vitamin B12 was in use as antipernicious anemia factor for the treatment of anemic patients. Many different types of bacteria, fungi and actinomycetes synthesize vitamin B12 but intestinal and rumen bacteria are most prevalent in its production in large amount. Two types of vitamin B12 re produced by microorganisms; one that promote growth only of microbes and other type which is required for normal growth in animals and humans. Commercially, vitamin B12 is synthesized by microbial fermentation. Hundreds of fermentation processes and their modifications have been employed on commercial scale. Some of them are described here.
1. Direct fermentation: In this process, Streptomyces olivaceus is grown in nutrient medium containing glucose as carbon source. Cobalt chloride, CoCl2 (less than 10ppm) is added as precursor at the beginning or intermittently during the production. Higher concentration of CoCl2 is generally toxic. Fermentation is carried out at 27˚C with aeration for about 5 days. At the end of incubation, growth is harvested before mycelial autolysis and destruction of vitamin. The vitamin is recovered from broth by filtration. A part of mycelium containing vitamin B12 is dried and used as cattle and poultry feed. Filtrate and remaining mycelia are acidified or treated with alcohol to recover the vitamin. Heat treatment is sometimes required for complete extraction of vitamin from mycelia; this vitamin is heat labile so heat is applied with caution and prior study. During acidification, sodium sulfite is added to stabilize the vitamin. Acidified broth is filtered to remove mycelial growth and filtrate is evaporated to dryness under vacuum. It can be further purified by treatment with acetone and ion exchange resins. Ultrapurified crystalline form of vitamin is used for medical use. Direct fermentation employing Bacillus megaterium, Propionibacterium freudenreichii, Propionibacterium shermanii and Pseudomonas has also been used for commercial production of vitamin B12. These methods are similar with respect to recovery and extraction but different regarding yield, growth conditions and fermentation medium as per the requirement of fermentative culture.
2. Indirect production: Vitamin B12 is also obtained as byproduct of various antibiotic fermentation processes such as Grisein, Streptomycin and Aureomycin production. It is also formed during acetone-butanol fermentation. It is reported to be present in high concentration in sewage sludge.
3. Genetic engineering: Genetic engineering techniques has allowed us to change the byproduct status of this vitamin towards principle product, especially in case of Streptomyces fermentation. The strains of Streptomyces griseus have been developed via rigorous strain improvement programmes involving mutations and recombination techniques to yield more vitamin B12 as compared to antibiotic production. Genetic approaches like genome shuffling have also been applied in Propionibacterium shermanii to improve yields of vitamin B12.
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