Metagenome-based microbial metabolic strategies to mitigate ruminal methane emissions using Komagataeibacter-based...

Global warming increasingly threatens organisms in equatorial regions, where temperatures often exceed physiological limits. Rumen methanogens are a major biological source of anthropogenic methane, a potent greenhouse gas. Therefore, ruminal methane mitigation strategies that preserve animal productivity are urgently needed. Our In vitro analysis of Holstein steer rumen fluid-integrating gas production, volatile fatty acid (VFA) profiles, and metagenomic data-demonstrated that kombucha, a fermented beverage, effectively reduces methane emissions by modulating ruminal fermentation. Rumen fluid was incubated for 60 h under three treatments (control, 3-NOP, and kombucha). During the initial 30 h, kombucha reduced methane by 15.07 % compared to the control but was 17.54 % higher than 3-NOP. In the subsequent 30 h, kombucha achieved sustained reductions of 34.72 % versus the control and 26.28 % versus 3-NOP, highlighting its uniquely sustained methane-reducing effect. A metagenomics-guided screening and in vitro validation identified Komagataeibacter intermedius SLAM-NK6B as a key strain underlying the methane-reducing effect of kombucha. The genome of SLAM-NK6B encodes biosynthetic gene clusters for cellulose, malate, citrate, and methanobactin-metabolites that can modulate the rumen microbiota. SLAM-NK6B supplementation reduced methanogen abundance by 53.32 % and increased hydrogen pressure, shifting microbial metabolism. Excluding acetate, VFA production increased significantly, with propionate levels elevated by 15.39-43.81 %. Metagenomic data further indicated activation of alternative hydrogen sink pathways, including citrate-to-propionate and malate-to-propionate conversions. This study proposes a novel microbial metabolic strategy for methane mitigation, enabling both methane reduction and enhanced fermentation efficiency. Such metabolic guidance of the rumen microbiome offers a sustainable approach to low-emission ruminant production.