Nitrous oxide act as an alternative electron acceptor for microbial methane oxidation in oxygen-deficient microcosms

• Functional groups of AMNR were enriched from 4 paddies. • Conventional methanotrophs catalyze AMNR in paddy soils along a latitude gradient. • High N 2 O reduction rates are strongly associated with CH 4 oxidation rates. • Classic aerobic methanotrophs reduce N 2 O, aiding greenhouse gas mitigation. Submerged paddy is a hotspot of nitrous oxide (N 2 O) and methane (CH 4 ) emission, which is typically considered electron donor and acceptor for microbes, respectively. Theoretical calculations suggested the thermodynamic feasibility of anaerobic CH 4 oxidation coupled with N 2 O reduction (AMNR), and anaerobic methane oxidation and denitrification are typically coupled by certain anaerobic microbes, such as Ca . Methylomirabilis sinica from the NC10 phylum. However, the conventional aerobic methanotrophs underlying this novel greenhouse gas sink remain largely unclear. Four typical soil sample from different latitudes in China were used as inoculum. Enrichment reactors were constructed with continuous CH 4 and N 2 O supply for 400 days to cultivate aerobic methanotrophs capable of N 2 O reduction. This study revealed that conventional methanotrophs, such as species from the Methylocystis and Methylobacterium genera, are the key taxa catalyzing the AMNR process. Consistently high N 2 O reduction rate (5.37–6.24 μmol·g −1 -dry soil·d −1 ) was observed in strong association with CO 2 formation, that was nearly matched with the expected stoichiometry (4:1). The N 2 O reduction process occurred in two distinct phases: a rapid reduction phase concurrent with CH 4 oxidation, followed by a slower reduction phase. N 2 O was directly reduced by conventional aerobic methanotrophs harboring the nosZ gene, such as Methylocystis , or by denitrifiers using the fermentative intermediates produced by methanotrophs as electron donors. This suggests that conventional methanotrophs, which typically perform aerobic methane oxidation, could also have denitrification potential, possibly facilitated by the presence of the nosZ gene. Although methanotrophs and denitrifiers are usually considered distinct groups, these results indicate that the AMNR process could allow for the simultaneous oxidation of CH 4 and reduction of N 2 O in paddy soils, thus enhancing the potential for greenhouse gas mitigation.