Biochar modulates methanotrophic metabolism to enhance methane oxidation and mitigate greenhouse gas emissions.

Methanotrophs represent the major biological sink for methane, yet the direct effects of biochar on methanotrophic metabolism remain unclear. This study investigated how biochars produced at different pyrolysis temperatures influence methane oxidation activity, metabolite production, and metabolic pathways in a methanotrophic pure culture and validated the effects in wetland soil microcosms. Biochar amendment enhanced methane consumption, with 400 °C biochar showing the strongest effect and increasing the methane oxidation rate by 25%, likely due to its greater retention of redox-active surface functional groups. Moreover, 400 °C biochar reduced formate accumulation and promoted biomass synthesis, suggesting improved downstream processing and assimilation of methane-derived carbon. Transcriptomic analysis showed upregulation of rsxG/rnfG and rnfD by 4.73- and 3.99-fold, respectively, together with increased multiheme c-type cytochrome expression (2.15-fold), suggesting improved redox regulation during rapid methane oxidation. Biochar was most effective at 0.2 g L-1, reducing inhibition caused by carbon dioxide accumulation. In wetland soil slurries, 400 °C biochar enhanced methane mitigation and reduced carbon dioxide production, accompanied by increased relative abundances of methanotroph-related and putative carbon-fixing bacterial taxa. These findings provide new mechanistic insights into biochar-mediated regulation of microbial methane oxidation and highlight its potential as a promising amendment for mitigating methane emissions in wetland ecosystems.