Co-Cultivation of Rumen Bacteria Enhances VitB12 (Cobalamin) Biosynthesis under Optimized Substrate Conditions

Abstract

Introduction: Vitamin B12 (vitB12) micronutrient deficiency remains a significant global health issue, driving the search for new microbial sources capable of producing this essential nutrient. While industrial strains have been well studied, there is limited understanding of the genetic and metabolic diversity among culturable ruminal bacteria that may contribute to vitB12 synthesis. Given that most bacteria possess only partial sets of vitB12 biosynthesis genes, we hypothesized that ruminal bacteria rely on synergistic interactions to collectively establish a complete and functional vitB12 biosynthetic pathway.

Objectives: This study presents results on isolating, culturing, and genetically characterising the ruminal isolates while evaluating the impact of substrate supplementation on vitB12 production.

Methods:  VitB12-producing bacteria isolates were isolated using Brain Heart Infusion Agar. These isolates were then phenotypically characterized, including biochemical and physical tests, while genotypic characterization was performed based on the 16S rRNA gene sequence. Followed by characterization of the vitB12 genetic elements in sequenced genomes from the NCBI public repository. We targeted the cob, hem, and cbi gene cluster and pdu operons. We then evaluated the presence of cobA, cobI, and cobM genes in our isolates. The isolates were screened for VitB12, using the quantitative analysis of vitamins in culture supernatants, done by using HPLC equipped with a UV detector, a gold-standard method for accurately measuring vitB12 and its derivatives. We enriched culture media using betaine and molasses substrates to improve the yield of VitB12 production.

Results: A total of 65 colonies were identified from 18 treatments in each of the three ruminal samples. From the 65 colonies, 25 were randomly selected and sub-cultured for morphological and biochemical characterization, of which 10 were from aerobic and 15 from anaerobic culture conditions. Our ruminal isolates showed multiple morphologies, suggesting the presence of different strains. Using homology comparisons, our isolates clustered with Arthrobacter spp., Bacillus spp., and S. ruminantium with similarities ranging from 95.1% to 100%.  Our ruminal isolates expressed CobA and CobM, but not CobI. While most genes tend to be clustered in unique operons, there exists a diversity of genomic organization across different species. Overall, the ruminal isolates can produce vitB12 when co-cultured than when cultured individually. Individually, AnR2-C4 and AnR3-C4 did not produce a detectable amount of vitB12, but AnR2-C3 produced a quantifiable amount, averaging 0.082 mg/L. The combination of AR2-C3 and AnR2-C4 yielded a higher amount (3.116 mg/L) than AR2-C3 alone. Combination of the three, AR2-C3 + AnR2-C4 + AnR3-C4, yielded the highest quantity (4.481 mg/L).  Supplementation of culture media with molasses and betaine enhanced the vitB12 yield.

Conclusions: This study demonstrates that ruminant-derived bacteria, particularly strains resembling Ruminococcus and Lactobacillus reuteri, are promising sources for vitB12 production. Using culturing, genomic analysis, and media enrichment, it showed how these bacteria work synergistically to support a functional B12 biosynthesis pathway. Genomic comparisons revealed species-specific adaptations in cob and cbi genes arrangements, reflecting their response to environmental and metabolic conditions. The limitation of a single isolate having an incomplete set of vitB12 genes is offset by the natural co-existence of multiple strains in a single ecosystem. Additionally, supplementing culture media with molasses and betaine may further boost vitB12 yields.