Rumen-derived Pichia membranifaciens modulates the rumen microbiome and metabolome and mitigates methane emissions in dairy cows

Methane emissions from ruminants represent a significant environmental challenge and dietary energy loss. While yeasts are potential rumen modulators, specific methane-mitigating species remain poorly characterized. Here, we screened 73 rumen-derived strains in vitro, identifying Pichia membranifaciens M12 as the most effective candidate, reducing methane output by 17.1%. Subsequently, a randomized block trial with 36 dairy cows compared a control group with P . membranifaciens M12 supplementation at 2.5 and 5 × 10 11 CFU/cow/day. Methane yield per unit of dry matter intake significantly decreased in the high-dose group (18.7%, P = 0.003), without compromising lactation performance and animal health. Multi-omics analyses revealed that M12 suppressed hydrogenotrophic methanogens (e.g., Methanobrevibacter ) and hydrogen-producing bacteria (e.g., Ruminococcus and Fibrobacter ), while enriching specific eukaryotic taxa like Orpinomyces and Entodinium . Metabolomic profiling indicated a significant dose-dependent accumulation of metabolites. Metagenomic function analysis demonstrated the decreased abundance of key methanogenesis genes (e.g., mcrABCDG ) and increased abundance of hydrogenase ( hyaABC ), lactate-forming ( ghrB ), and propionate-forming ( mcmA1 and lcdB ), suggesting a redirection of reducing equivalents from methanogenesis toward propionate synthesis, alongside enhanced butyrate production. These findings demonstrate that P . membranifaciens M12 mitigates methane emissions via coordinated ecological and metabolic modulation, highlighting its potential as a sustainable strategy for low-carbon ruminant production.