Partial Treponema spp. as Candidate Probiotics for Rumen Methane Mitigation Revealed by a Module-Based Activity Index

Wei Wang¹Xiaoyun Chen¹Donghui Fang¹Linxiang Li²AG Yueda¹Jia Gan¹Xiaodong Deng¹Xiaoqin Ma¹Ying Chen¹Yi Shi¹Fang He¹Changfeng Wu²Zhixin Yi²Maozhong Fu¹Jun Yi^1*

• ¹Sichuan Academy of Grassland Science, Chengdu Institute of Biology, Chinese Academy of Sciences (CAS), Chengdu, China
• ²Bazhong Academy of Agriculture and Forestry Sciences, Bazhong, China

Methane emissions from ruminants, primarily driven by methanogenic archaea, constitute a major source of anthropogenic greenhouse gases. Current mitigation strategies rely heavily on metagenomic (MG) abundance of methanogens as a proxy for methanogenic potential, despite emerging evidence of functional decoupling between genomic potential and in-situ activity. Here, we interrogate this discrepancy by integrating paired metagenomic and meta-transcriptomic (MT) datasets. Our analysis reveals systematic transcriptional suppression of dominant archaeal genera (MethanobrevibacterMethanobrevibacte and Methanocaldococcus). Paradoxically, expression of core methanogenesis pathways (M00356, M00567357, M00356 and M00563) was significantly elevated in MT versus MG (p < 0.0001). To quantify functional activity, we developed the Methanogenesis Pathway Expression Activity Index (MPEAI), synthesizing expression of four KEGG modules governing hydrogenotrophic, methylotrophic, acetoclastic, and cofactor-synthesis pathways. Random Forest modeling applied to MPEAI identified multiple Treponema species as robust negative correlationes. These findings provide new evidence that redefine rumen methanogenesis as ainvolves bacterial-archaeal synergy synergistic trait, and identify with Treponema species emerging as potential promising probiotic targets.