The Microbial Mechanisms Underlying Soil N2O Emission Mitigation via Organic Fertilizer Substitution in Maize Cropping Systems

In mitigating agricultural nitrous oxide (N2O) emissions, partially substituting chemical nitrogen (N) fertilizers with organic amendments is widely advocated for; however, the effectiveness of this approach varies, and the optimal substitution ratio remains unclear. This study employed a long-term field experiment in a maize cropping system, integrating in situ N2O flux monitoring, soil analysis, and metagenomic sequencing to investigate the effects of substituting synthetic N fertilizer with sheep manure at different ratios (one-sixth, one-third, and full substitution) on cumulative N2O emissions and underlying microbial mechanisms. The results showed that substituting one-third of chemical N with sheep manure (OF2) most effectively reduced cumulative N2O emissions, by 18.64% (2021) and 47.00% (2022), compared to conventional chemical fertilization (NPK). This treatment optimized key soil properties, significantly lowering soil nitrate (NO3−-N) and ammonium (NH4+-N) concentrations while increasing soil organic matter. Metagenomic analysis revealed that the mitigation was primarily driven by a coordinated shift in the nitrogen-cycling microbial functional gene network: OF2 significantly enhanced the abundance of N2O reductase genes (nosZ clades I and II) and increased the nosZ/(nirK + nirS) gene ratio, thereby strengthening the genetic potential for complete denitrification (N2O → N2). Partial least squares path modeling confirmed that soil properties indirectly reduced N2O emissions, primarily by regulating the microbial community and functional gene abundances. This study provides mechanistic evidence that partial organic substitution (with one-third sheep manure) mitigates N2O emissions by optimizing the soil habitat and reprogramming the functional gene network of nitrogen-cycling microbes, with a key emphasis on enhancing the N2O reduction pathway.