Enhanced vitamin B₁₂ production by isolated Bacillus strains with the application of response surface methodology

Vitamin B₁₂ is a crucial B-group vitamin, first isolated from the liver due to its role in combating pernicious anemia. It is distinguished by its unique and complex structure, which makes its chemical synthesis challenging and expensive. Consequently, vitamin B₁₂ is alternatively obtained through microbial fermentations. Molasses, an affordable and safe agro-industrial waste, can be used as a carbon source for vitamin B₁₂ production, offering a cost-effective alternative to expensive sugars in the production medium.

A total of 87 yeast, actinomycete, and bacterial isolates were screened for vitamin B₁₂ production, with 15 isolates showing high productivity. Bacillus isolates were selected for further analysis using MALDI-TOF and molecular identification. These isolates were identified as four strains of Bacillus subtilis (MZ08, JT10, BY11, and JT17), one strains of Bacillus sp. (CB09), and one strain of Peribacillus acanthi (MZ01). Genetic circuits associated with vitamin B₁₂ production were demonstrated in a closely related strain of Peribacillus acanthi MZ01 strain. Three strains (MZ01, MZ08, and JT17) were selected for further evaluation of vitamin B₁₂ productivity under different sugar types (glucose, sucrose, fructose, lactose, and galactose) and varying inoculum sizes. The inoculum size significantly impacted vitamin B₁₂ production, with an increase from 5 to 10% enhancing yields. The ability of the strains to produce vitamin B₁₂ varied depending on the type of sugar used. Peribacillus acanthi MZ01 strain showed the highest productivity and subsequently, selected for optimizing vitamin B₁₂ production conditions using response surface methodology. Furthermore, the optimized conditions were then applied to molasses-based medium to achieve high vitamin B₁₂ yields by MZ01 strain.

In this study, Peribacillus acanthi was characterized for the first time as a vitamin B₁₂ producer, demonstrating high productivity among various tested strains. The optimization of production conditions using response surface methodology, further enhanced vitamin B₁₂ yields, showcasing the strain's efficiency in microbial fermentations. This research also highlights the potential of using molasses as a cost-effective alternative carbon source, significantly reducing production costs.