New insights into nitrous oxide-driven anaerobic methane oxidation mediated by Methylococcales and Gemmatimonadales

The simultaneous biogeochemical transformation of methane (CH₄) and nitrous oxide (N₂O) in anoxic environments is a recently proposed pathway for decreasing carbon emission in wetlands. However, the mechanisms underlying this coupled process have yet to be elucidated. Here, two systems with CH₄ to N₂O molar ratios of 1:1 (R1) and 1:2 (R2) were established. Isotopic tracing showed that the production rate of ¹³CO₂ in R2 consistently exceeded than that in R1, with a maximum N₂O-driven AOM rate of 2.29 μmol∙g^-1_dw∙d^-1, demonstrating the superior performance of R2. Microbial community analysis revealed that Methylococcales and Rhizobiales were the dominant methanotrophs, whereas Gemmatimonadales and Sphingobacteriales represented the primary denitrifiers involved in N₂O reduction. Metagenomic binning further indicated that Methylococcales and Gemmatimonadales harbor complementary genomic potentials for CH₄ oxidation and N₂O reduction, supporting a synergistic interaction driving N₂O-dependent AOM. This cooperation appears to rely on electron transfer between the methanotrophic and denitrifiers. Collectively, these findings provide mechanistic evidence for N₂O-driven AOM, advancing the understanding of coupled carbon-nitrogen transformations and offering new insights into microbial strategies for mitigating greenhouse gas emissions in wetland systems.