28Farmed ruminants are the largest source of anthropogenic methane emissions 29 globally. The methanogenic archaea responsible for these emissions use molecular 30 hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, 31 as their primary energy source. In this work, we used comparative genomic, 32 metatranscriptomic, and co-culture-based approaches to gain a system-wide 33 understanding of the organisms and pathways responsible for ruminal H2 metabolism. 34 Two thirds of sequenced rumen bacterial and archaeal genomes encode enzymes 35 that catalyze H2 production or consumption, including 26 distinct hydrogenase 36 subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are 37 differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases 38 from carbohydrate-fermenting Clostridia (e.g. Ruminococcus) accounted for half of all 39 hydrogenase transcripts. Various H2 uptake pathways were also expressed, including 40 methanogenesis (Methanobrevibacter), fumarate reduction and nitrate ammonification 41 (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis predominated 42 in high methane yield sheep, alternative uptake pathways were significantly 43 upregulated in low methane yield sheep. Complementing these findings, we observed 44 significant differential expression and activity of the hydrogenases of the 45 hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic 46 fumarate reducer Wolinella succinogenes in co-culture compared to pure culture. We 47 conclude that H2 metabolism is a more complex and widespread trait among rumen 48 microorganisms than previously recognized. There is evidence that alternative 49 hydrogenotrophs, including acetogens and selenomonads, can prosper in the rumen 50 and effectively compete with methanogens for H2 in low methane yield ruminants. 51Strategies to increase flux through alternative H2 uptake pathways, including animal 52 selection, dietary supplementation, and methanogenesis inhibitors, may lead to 53 sustained methane mitigation. 54 55 56Methane production by livestock accounts for over 5% of global greenhouse gas 57 emissions annually 1 . These emissions mostly originate from the activity of 58 methanogens within ruminants, which generate methane as an obligate end-product 59 of their energy metabolism 2 . Several lineages of methanogenic archaea are core 60 members of the microbiome of the ruminant foregut 3-5 . Of these, hydrogenotrophic 61 methanogens are dominant in terms of both methane emissions and community 62 composition 6,7 , with global surveys indicating that Methanobrevibacter gottschalkii 63 and Methanobrevibacter ruminantium comprise 74% of the rumen methanogen 64 community 5 . These organisms use molecular hydrogen (H2) to reduce carbon dioxide 65 (CO2) to methane through the Wolfe cycle of methanogenesis 8,9 . Rumen 66 methanogens have also been identified that use formate, acetate, methyl compounds, 67 and ethanol as substrates, but usually do so ...

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