Transcriptomic and metabolic analyses of interspecies hydrogen and electron transfer during syntrophic growth of Ruminococcus albus 7 with four different hydrogen-utilizing Methanobrevibacter strains

Methane production by ruminants, a major anthropogenic source of global greenhouse gas emissions, is driven by ruminal methanogens that utilize hydrogen (H₂), an end-product of microbial fermentation. Ruminococcus albus , a dominant ruminal cellulolytic bacterium, produces H₂ during carbohydrate fermentation, providing electrons for CO₂ reduction and methane production by methanogenic archaea. However, current understanding of how hydrogen-producing bacteria and hydrogen-utilizing methanogens interact during interspecies hydrogen transfer remains limited. This study therefore investigated the transcriptomic and metabolic responses associated with syntrophic growth between R. albus 7 and four hydrogen-utilizing Methanobrevibacter ( Mbb .) strains. Transcriptomic and metabolic profiles were compared between pure and co-cultures of R. albus 7 with Mbb. smithii PS, Mbb. ruminantium M1, Mbb. boviskoreani AbM4, or Mbb. sp. D5. Relative to pure cultures, hydrogen metabolism in R. albus 7 showed upregulation of the hydABC genes and downregulation of hydA2 and alcohol dehydrogenase genes. These transcriptional changes resulted in reduced ethanol and increased acetate production, reflecting a redirection of carbon flux toward acetate to enhance ATP generation in co-culture. Transcriptomic analysis of Methanobrevibacter strains revealed upregulation of genes involved in hydrogenotrophic methanogenesis, including formate dehydrogenases and coenzyme F 420 hydrogenases, during co-culture with R. albus 7. Genes related to biofilm formation in R. albus 7 and adhesin genes in Methanobrevibacter strains were upregulated, indicating potentially enhanced microbial aggregation that facilitates hydrogen transfer. R. albus 7 selectively upregulated sulfur and methionine metabolism genes in co-cultures with Mbb. boviskoreani AbM4 and Mbb. sp. D5, coinciding with increased expression of O-acetylhomoserine sulfhydrylase genes in these methanogens, suggesting coordinated sulfur amino acid metabolism. Collectively, these responses revealed partner-specific differences in hydrogen metabolism, energy conservation, aggregation, and nutrient exchange. Together, these findings show that syntrophic interactions between R. albus 7 and Methanobrevibacter extend beyond interspecies hydrogen transfer to include microbial aggregation and partner-specific metabolic interdependencies. The strain-specific regulatory and metabolic responses observed in this study broaden our understanding of microbial interactions contributing to methane production in the rumen.