In Vitro Investigation of the Effects of Bacillus subtilis-810B and Bacillus licheniformis-809A on the Rumen Fermentation and Microbiota

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. <i>Bacillus</i> species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of <i>B. licheniformis</i> and <i>B. subtilis</i> in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of <i>B. licheniformis</i> and <i>B. subtilis</i> strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that <i>B. licheniformis</i> released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of <i>B. licheniformis</i> and <i>B. subtilis</i> survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of <i>B. licheniformis</i> and <i>B. subtilis</i> on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of <i>B. licheniformis</i> and <i>B. subtilis</i>, less CO₂ was produced while dry matter degradation and CH₄ production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants.