Soil fertility in organic agriculture relies on microbial cycling of nutrient inputs from legume cover crops and animal manure. However, large quantities of labile carbon (C) and nitrogen (N) in these amendments may promote the production and emission of nitrous oxide (N2O) from soils. Better ecological understanding of the N2O emission controls may lead to new management strategies to reduce these emissions. We measured soil N2O emission for two growing seasons in four corn–soybean–winter grain rotations with tillage, cover crop, and manure management variations typical of organic agriculture in temperate and humid North America. To identify N2O production pathways and mitigation opportunities, we supplemented N2O flux measurements with determinations of N2O isotopomer composition and microbiological genomic DNA abundances in microplots where we manipulated cover crop and manure additions. The N input from legume-rich cover crops and manure prior to corn planting made the corn phase the main source of N2O emissions, averaging 9.8 kg/ha of N2O-N and representing 80% of the 3-yr rotations’ total emissions. Nitrous oxide emissions increased sharply when legume cover crop and manure inputs exceeded 1.8 and 4 Mg/ha (dry matter), respectively. Removing the legume aboveground biomass before corn planting to prevent co-location of fresh biomass and manure decreased N2O emissions by 60% during the corn phase. The co-occurrence of peak N2O emission and high carbon dioxide emission suggests that oxygen (O2) consumption likely caused hypoxia and bacterial denitrification. This interpretation is supported by the N2O site preference values trending towards denitrification during peak emissions with limited N2O reduction, as revealed by the N2O δ15N and δ18O and the decrease in clade I nosZ gene abundance following incorporation of cover crops and manure. Thus, accelerated microbial O2 consumption seems to be a critical control of N2O emissions in systems with large additions of decomposable C and N substrates. Because many agricultural systems rely on combined fertility inputs from legumes and manures, our research suggests that controlling the rate and timing of organic input additions, as well as preventing the co-location of legume cover crops and manure, could mitigate N2O emissions.
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