Revealing microbial pathways of N₂O production in agricultural ditches

Agricultural ditches are recognized as hotspots of nitrous oxide (N₂O) emissions, yet the contributions of different microbial pathways under varying environments remain largely unknown. In this study, we quantified N₂O production from four microbial pathways (nitrifier nitrification, nitrifier denitrification, nitrification-coupled denitrification, and heterotrophic denitrification) across a gradient of ditch types (from large to small: main, branch, collector, and field ditches). Using a dual isotope tracing technique (¹⁵N-¹⁸O), we distinguished pathway-specific N₂O production under different oxygen and organic carbon (C) conditions. Nitrifier denitrification dominated N₂O production in the three largest ditch types (contributing 68.7-83.6 %), while heterotrophic denitrification accounted for 85.4 % of the total N₂O production in the field ditch. As organic C increased, heterotrophic denitrification-derived N₂O production increased significantly, whereas the contribution of nitrifier denitrification of N₂O production decreased significantly. As oxygen concentration decreased, all pathways-derived N₂O rates increased, while the contribution of nitrifier denitrification remained unchanged. Key nitrifiers, including Nitrososphaeraceae sp. TA-21, Nitrospira sp. Clade C and comammox Nitrospira kreftii, regulated N₂O production across NH₄⁺-derived pathways, with their influence modulated by environmental context. These findings improve our understanding of N₂O production in agricultural ditches, providing insights into developing process-based models and mitigation strategies.