Nitrous oxide (N2O) is a potent greenhouse gas, with agricultural activities representing its major source. However, the emission mechanism of nitrous oxide efficient by agricultural activities has not yet been fully studied. This study employs metagenomic analysis to elucidate the microbial community structure and functional potential associated with N2O emissions in river and ditch systems of the Yangtze River Delta. The N2O dissolved concentration in the rivers (0.08 ± 0.03 μmol N·L−1) was significantly lower than that in the ditches (0.21 ± 0.14 μ mol N·L−1) (P < 0.05). According to eight wind-based models, agricultural ditches emissions were 3.53–4.70 times higher than those of the rivers. All models significantly overestimated fluxes (P < 0.05), revealing a systematic overestimation of EF values when using the Intergovernmental Panel on Climate Change (IPCC) methodology. Particulate organic carbon supported microbial activity by providing energy and adhesion sites, while electrical conductivity (EC) served as an indicator of ion inputs from surrounding land use, serving as a critical abiotic driver of EF values in the ditches. The co-occurrence network showed that denitrification genes (norB, nirS, nosZ) formed a tightly clustered subnetwork exclusively in the ditches, indicating broader nitrification niches and stronger functional coupling among denitrifiers in these systems. Metagenomic evidence revealed that EF value correlated significantly with denitrification genes, notably napAB, nirK, norBC and nirK/nosZ (P < 0.05), underscoring denitrification as the primary biotic driver of N2O production. These findings demonstrate the value of metagenomic approaches in revealing microbial mechanisms behind N2O emissions and support the development of more accurate, EF estimates for greenhouse gas inventories in agricultural landscapes.
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