Microbial Production of Vitamins (Vitamin B)

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Microbial production of Vitamins (Vitamin B12)
One of the most interesting and fascinating molecules in the world of science and
medicine is vitamin B12 (cobalamin), which was originally discovered as the anti-pernicious
anemia factor in the early 1920s, when two American physicians, Minot and Murphy,
demonstrated it to cure pernicious anemia, a disorder first described in 1835, with a diet that
included raw liver. In humans, the vitamin is required in trace amounts (approximately 1
mg/day) to assist the action of only two enzymes, methionine synthase and (R)-methylmalonyl-
CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of
bacterial species. The term vitamin B12 is widely used to describe compounds of the cobalamin
group. Natural forms are adenosylcobalamin, methylcobalamin and hydroxocobala-min.
Cyanocobalamin, by definition vitamin B12, is the industrially produced stable cobalamin form
which is not found in nature.
Vitamin B12 is obtained exclusively by fermentation process. It is produced by a number
of pharmaceutical companies to meet annual demands worldwide. Merck began production of
vitamin B12 by Pseudomonas denitrificans in 1952 and have improved the efficiency of culture
more than 30-fold relative to the performance of the original soil isolates by genetic
manipulations and microbial screening. At first, vitamin B12 for human therapy and as a food or
feed supplement was obtained as a byproduct of Streptomyces antibiotic (neomycin,
chlortetracycline) fermentation. Good strains were also isolated from manure and sewage sludge.
Mutagenic treatments have resulted in improved activity, but in all cases cobalt ions and 5,6-
dimethylbenzimidazole (5,6-DMBI) have to be added in addition to the precursors such as
glycine, threonine, and aminopropanol. During the past two to three decades, several
microorganisms have been employed for the efficient production of vitamin B12. The list of
various microorganisms producing vitamin B12 and the respective yields are shown in Table 1.
Table1. Species of microbial producers and microbiological processes recommended for the
production of vitamin B12
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Vitamin B12 biosynthesis is restricted to microorganisms. Most of the steps in the

biosynthesis of vitamin B12 have been characterized in Pseudomonas denitrificans, Salmonella
typhimurium and Propionibacterium freudenreichii. Some authors have reported about the
requirement of more than 30 genes for the entire de novo biosynthesis of cobalamin, which
amounts to about 1 % of a typical bacterial genome. Two different biosynthetic routes for
vitamin B12 exist in nature:
• aerobic, or more precisely an oxygen-dependent pathway that is found in organisms like P.
denitrificans, and
• anaerobic, oxygen-independent pathway investigated in organisms like P. shermanii,
Salmonella typhimurium and Bacillus megaterium.
The major problem in vitamin B12 production using Propionibacterium is the growth
inhibition of the cell due to the accumulation of inhibitory metabolites such as propionic acid and
acetic acid.
Fig. 1. Downstream processing of vitamin B 12

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Recovery of vitamin B12

The study of separation and purification processes of the fermentation products is most
important for their commercial success. Recovery and purification of high value bioproducts
from their crude sources involve various steps (extraction, membrane filtration, and sorption),
which lead to low overall yields. Conventional chromatographic and chemical processes are
capital and energy consuming due to a number of post- and pretreatment steps involved in a
processing scheme. A rapid and selective mode of recovery of the target molecule from the crude
feedstock can prove highly advantageous in improving the product yields and thus reducing the
overall cost of downstream processing. Adsorptive separations are often used in the downstream
processing using various interactions, e.g. ionic, hydrophobic, affinity, etc. for the recovery of
biomolecules, namely antibiotics and vitamins.
The steps in the downstream processing for the recovery of vitamin B12 are summarized
in Fig. 1. The biomass is separated by centrifugation to obtain a cell mass concentrate, which is
then dried. Alternatively, the entire contents of the fermentor can be concentrated or spray dried.
Cell lysis by heating the centrifuged cell mass in an aqueous solution, or by other methods to get
corrinoids, can be used. Corrinoids are converted to vitamin B12 or cyanocobalamin by the
addition of potassium cyanide, usually in the presence of sodium nitrite and heat. The vitamin
solution is clarified subsequently by filtration, treatment with zinc chloride, and then precipitated
out by the addition of tannic acid or cresol to give the product of 80 % purity, which is suitable
for use as animal food additive. For greater purity, which is required for pharmaceutical use, the
clarified solution is extracted with organic solvents, such as carbon tetrachloride, and then with
water and butanol, followed again by organic solvents. In addition, adsorption processes such as
on ion exchangers, aluminium oxide, or activated carbon can be used. Pure vitamin B12 can be
obtained by crystallization after the addition of organic solvents, such as phenol and water.

Microbial Production of Vitamins (Vitamin B)

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1

Microbial production of Vitamins (Vitamin B12)

Vitamin B12 biosynthesis is restricted to microorganisms. Most of the steps in the

Fig. 1. Downstream processing of vitamin B 12

Recovery of vitamin B12

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