Pyrogenic carbon-stimulated nitrate-dependent anaerobic methane oxidation: insights into redox activity and conductivity in anaerobic methanotrophic archaea metabolism and microbial dynamics

Pyrogenic carbon (PC) plays a critical role in regulating greenhouse gas emissions by influencing methanogenesis and methane oxidation in aquatic environments. However, its impact on nitrate-dependent anaerobic oxidation of methane (AOM), associated methane emissions, and the underlying mechanisms remain poorly understood. Here, we demonstrated that in nitrate-dependent AOM consortia amended with HNO₃-treated biochar and graphite (representing redox-active and conductive forms of PC, respectively), AOM rates were significantly elevated by 2.7- and 4.4-fold, respectively, compared to unamended biotic controls. This enhancement was accompanied by a pronounced proliferation of anaerobic methanotrophic archaea, specifically "Candidatus Methanoperedens nitroreducens", along with elevated metabolic activity driven by enhanced electron transport and energy conservation, as indicated by significantly increased electron transfer system activity, total adenine nucleotide levels, and concentrations of key redox carrier F₄₂₀. Metagenomic analysis revealed that PC addition reshaped microbial interactions. Notably, graphite facilitated the potential establishment of direct interspecies electron transfer between "Ca. M. nitroreducens" and coexisting denitrifying populations (Bacteroidota sp. and Ignavibacteriaceae sp.), while also fostering the formation of new interspecies networks that enabled division of labor within the denitrification pathway. These findings not only advance the mechanistic understanding of PC-facilitated methane mitigation in aquatic ecosystems but also suggest strategies for engineering AOM-based systems to optimize methane removal and nitrogen cycling in environmental applications.