Rumen-derived Prevotella and Megasphaera elsdenii mitigate methane production through functional modulation of rumen microbial metabolism

Background Enteric methane (CH 4 ) production represents a major energy loss in ruminant systems and contributes substantially to agricultural greenhouse gas emissions. Increasing ruminal propionate production has been proposed as a strategy to redirect metabolic hydrogen away from methanogenesis, although the underlying microbial mechanisms remain incompletely understood. Results Four rumen-derived Prevotella strains and one Megasphaera elsdenii strain were isolated, genomically characterized, and evaluated using an in vitro rumen fermentation model. Distinct strain-specific responses were observed. Compared with the control, Prevotella strains RH14 and RH35 significantly reduced CH 4 accumulation at 48 h ( P < 0.05), coinciding with lower total gas and carbon dioxide (CO 2 ) production, whereas RH3, RH27, and RH19 showed CH 4 production comparable to the control. Volatile fatty acid (VFA) profiles showed comparatively smaller differences among treatments, although RH14 maintained relatively greater total VFA and propionate concentrations at later incubation stages. Metagenomic analysis indicated that methane mitigation was associated with reduced relative abundance of methanogenesis-related pathways, particularly hydrogenotrophic methanogenesis ( P < 0.05), whereas archaeal community composition remained largely unchanged. However, metagenomic data reflect gene abundance rather than activity and do not directly indicate functional regulation. Conclusions These findings demonstrate strain-specific effects of rumen-derived bacteria on rumen fermentation and methane production. In particular, Prevotella strains RH14 and RH35 showed potential to mitigate methane formation through functional modulation of microbial metabolism, partially displacing rather than completely eliminating methanogens. These results provide a functional basis for the future development of rumen microbial interventions aimed at improving rumen fermentation efficiency and mitigating enteric methane emissions.