Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH₄) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH₄ oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and ¹³CH₄-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH₄ oxidation in supporting denitrification. Our results reveal positive relationships between CH₄ oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH₄ and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, ¹³CH₄-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating ¹³C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of ¹³C-metagenome-assembled genomes and ¹³C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH₄ oxidation, for the coupling of CH₄ oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH₄ oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.