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Background  Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. The synthesis of vitamin B12 is limited to a few of bacteria and archaea. Therefore, industrial microbial fermentation is used to meet annual demands worldwide of vitamin B12 and as an alternative method to the chemical synthesis which requires at least 60 steps that is uneconomical.  Bacillus megaterium  is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Therefore, a two-step optimization strategy was established to produce high yield of vitamin B12 by  B. megaterium  through the provision of the production requirements and the suitable conditions for the biosynthesis of vitamin B12.    Results  We achieved the optimum conditions for the fermentation process of  B. megaterium  to produce high yield of vitamin B12 in a practical way based on statistical design and analysis which allowed vitamin B12 production to increase up to 759-fold (204.46 μg/l) as compared with control without parameters (0.26 μg/L). High performance liquid chromatography coupled to variable wavelength detector and mass spectrometry has been used to identify vitamin B12 forms and confirm the results.    Conclusions  We developed the fermentation process of  B. megaterium  to enhance the production of vitamin B12 by providing the required supplements for the synthesis of vitamin B12 (CoCl 2 , δ-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and dividing the fermentation process into three stages. In addition, the optimum incubation times of the three fermentation stages were investigated and performed with reducing number of experimental and evaluated multiple parameters and their interactions by using statistical experimental design and analysis. All of these strategies has proven successful in enhancing the production of vitamin B12 up to 204.46 μg/l and demonstrated that  B. megaterium  could be a good candidate for the industrial production of vitamin B12.

Development of a two-step cultivation strategy for the production of vitamin B12 by Bacillus megaterium