Nitrifier denitrification contributes substantially to nitrous oxide production in calcareous farmland soils

Intensively managed agricultural soils are a major source of nitrous oxide (N₂O) emissions, yet the N₂O production pathways in calcareous soils remain unclear. Using sterilization assays, 15 N 18 O tracing, and qPCR analysis, we quantified the contributions of abiotic and microbial processes to N₂O production in calcareous farmland soils under long-term plastic film mulching and varying nitrogen (N) fertilization regimes. The results showed that abiotic contributions to N₂O production were negligible (<4%), but these contributions were higher in the no-N control than in the N-fertilized soils. Among microbial pathways, nitrifier denitrification (ND) dominated total N 2 O production (13.9–93.2%), followed by nitrifier nitrification (NN) and nitrification-coupled denitrification (NCD), with heterotrophic denitrification (HD) being minor (<13.5%). Long-term film mulching significantly reduced N₂O productions, accompanied by decreased potential nitrification rate (PNR), denitrification enzyme activity (DEA), and functional gene abundances (AOB amoA , nirK , and ( nirK + nirS )/ nosZ ratio), but maintained the dominant N₂O production pathways.Under film mulching, N fertilization significantly increased the contributions of ND and NCD while reducing those of NN and HD relative to the no N control, though no significant differences were observed among the N-fertilization regimes (moderate N rate-F 225 , high N rate-F 380 , moderate N plus manure-F 225+M ). N₂O fluxes strongly correlated ( p < 0.01) with NN, ND, NCD, soil TN, SOC, DON, NO₃ − , and microbial parameters (AOB amoA , nosZ , PNR, DEA). These findings indicate the predominance of ND in the studied calcareous farmland soils and reveal how agricultural practices regulate N₂O production at both process and molecular levels, providing insights for targeted mitigation strategies. • Nitrifier denitrification dominated N 2 O production in alkaline agricultural soils • N fertilization significantly altered dominant N 2 O production pathways. • Long-term film mulching reduced soil nitrification-denitrification potential and AOB amoA/nirK gene abundance, lowering N₂O emissions. • Long-term film mulching maintained the stability of dominant N₂O production pathways.