**Background:** Vitamin B12 (cobalamin) is an essential nutrient for humans, serving as a cofactor for methylmalonyl-CoA mutase and methionine synthase, which are involved in DNA synthesis and branched-chain amino acid metabolism. Humans cannot synthesize vitamin B12, and its chemical synthesis is highly complex, making microbial biosynthesis the primary commercial production route. However, many purported B12-producing organisms, such as Spirulina, actually produce pseudovitamin B12, an analogue with adenine instead of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand, which is biologically inactive in humans. Propionibacterium freudenreichii is used industrially because it nearly exclusively produces active vitamin B12, attributed to the BluB/CobT2 fusion enzyme that synthesizes and incorporates DMBI. This study aimed to identify new bacterial species capable of producing active vitamin B12 by screening for bluB/cobT2 homologues and using a sensitive LC-MS/MS method for confirmation.
**Methods:** An LC-MS/MS method was developed using a triple quadrupole LCMS-8045 (Shimadzu) with a Luna Omega 3 µm PS C18 column. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a 7-minute gradient program at 0.4 mL/min and 40°C. MS analysis was performed in positive ion mode using electrospray ionization with multiple reaction monitoring (MRM) for the double-charged cyanocobalamin ion [M+2H]2+ at m/z 678.40 and pseudocobalamin ion at m/z 672.75. Method validation assessed linearity (R²=0.9997 over 20-2000 nM), LOD, LOQ, selectivity, accuracy (recovery 94.4%-103.4%), within-day repeatability (RSD <4%), and intermediate precision (RSD <4%). For strain identification, the BluB/CobT2 protein sequence from P. freudenreichii (GenBank: CBL56167.1) was used for BLASTp searches excluding Propionibacterium. Sequences with >50% identity were selected, and six non-pathogenic (risk group 1) strains were obtained from DSMZ: Terrabacter sp. DSM102553, Terrabacter sp. DSM102554, Calidifontibacter indicus DSM22967, Raineyella antarctica DSM100494, Yimella lutea DSM19828, and Blastococcus sp. DSM44272. Strains were cultivated in recommended media at 30°C with aeration. For cobalamin extraction, 25 mL cultures were harvested after 5 days, resuspended in acetate buffer (pH 4.5) with 0.1% KCN, heated at 98°C for 30 min, and purified using C18 columns.
**Key Results:** Among the six candidate strains, three produced active vitamin B12: Terrabacter sp. DSM102553, Yimella lutea DSM19828, and Calidifontibacter indicus DSM22967. The active form was identified by characteristic fragment ions at m/z 146.95 [DMBI+H]+ and m/z 359.10 [DMBI+sugar+PO3+H]+, matching the cyanocobalamin standard. Y. lutea also produced low amounts of pseudovitamin B12 (adenine-containing analogue). Terrabacter sp. DSM102553 showed the highest growth, reaching a maximum scattered light signal of 42 a.u. after 25 h, compared to approximately 25 a.u. after 80 h for Y. lutea. C. indicus showed a lag phase of approximately 20 h followed by exponential growth. Terrabacter sp. DSM102553 was selected for further optimization. In M9 minimal medium, the strain achieved approximately 1.5 times higher maximal cell density compared to standard PP medium, and the highest vitamin B12 yield of 2.65 µg per g dry cell weight (DCW), which was two-fold higher than in PP medium and five-fold higher than in 2xPP medium. Biotin and thiamin supplementation did not affect growth.
**Clinical Implications:** This study identifies new bacterial strains capable of producing active vitamin B12 under aerobic conditions, eliminating the need for the two-step aerobic/anaerobic cultivation required for P. freudenreichii. While the yields (2.65 µg/g DCW) are lower than the 6-15 µg per g wet cell mass reported for P. freudenreichii, the ability to use minimal medium and aerobic conditions offers a simpler, more time-effective production process. The LC-MS/MS method provides rapid (7-minute analysis), sensitive quantification with clear discrimination between active and inactive B12 forms, which is critical for quality control in supplement manufacturing. The identification strategy based on bluB/cobT2 fusion genes could accelerate discovery of additional B12-producing strains for biotechnological applications, potentially addressing vitamin B12 deficiency risks in vegetarian and vegan populations.