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

Abstract Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH 4 ) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH 4 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 13 CH 4 -DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH 4 oxidation in supporting denitrification. Our results reveal positive relationships between CH 4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH 4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13 CH 4 -DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13 C, which are mostly belonged to Rubrivivax , Magnetospirillum , and Bradyrhizobium . Combined analyses of 13 C-metagenome-assembled genomes and 13 C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH 4 oxidation, for the coupling of CH 4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH 4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.