Abstract
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 × 1011 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.