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

Background: Enteric methane 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 an effective strategy to redirect metabolic hydrogen away from methanogenesis. However, the functional mechanisms by which specific rumen bacteria regulate methane production remain incompletely understood Results: In this study, four rumen-derived Prevotella strains and one Megasphaera elsdenii strain were isolated, genomically characterized, and evaluated using an in vitro rumen fermentation model. Supplementation with these strains significantly reduced methane yield while increasing total gas production and volatile fatty acid concentrations, particularly propionate. Shotgun metagenomic analysis revealed that methane mitigation was not associated with major alterations in archaeal abundance, but rather with a pronounced functional suppression of methanogenic pathways. Specifically, the dominant hydrogenotrophic (CO₂-reduction) methanogenesis module was significantly downregulated in all supplemented treatments. Concurrently, pathways and enzymes involved in carbohydrate fermentation and propionate synthesis were enriched, indicating a redirection of metabolic hydrogen toward alternative microbial sinks. Conclusions: These findings demonstrate that rumen-derived Prevotella and Megasphaera elsdenii can reduce methane production primarily through functional regulation of microbial metabolism rather than displacement of methanogens. This study provides mechanistic insights into microbially mediated hydrogen redistribution in the rumen and offers a functional basis for developing future probiotic strategies aimed at improving fermentation efficiency and mitigating enteric methane emissions.