Manure and bio-manure substitutions of chemical fertilizers mitigate long-term nitrous oxide emissions from vegetable production systems

Substituting chemical fertilizers with manure or bio-manure (manure inoculated with microbes) offers a promising strategy to mitigate nitrous oxide (N 2 O) emissions, yet the underlying mechanisms remained unclear. Through a five-year field study employing isotopocule (δ 15 N SP N2O and δ 18 O N2O/H2O ) mapping, we elucidated that a 50% substitution with manure or bio-manure significantly decreased cumulative N 2 O emissions by suppressing production of nitrification- and nitrifier-denitrification- and heterotrophic denitrification (HD)-derived N 2 O, and promoted N 2 O reduction to N 2 in denitrification. In contrast, 20% substitutions failed to achieve sustained mitigation. Bio-manure initially exhibited stronger N 2 O mitigation than manure, but this mitigation effect disappeared over time due to a N 2 O production–consumption offsetting effect in HD. Specifically, relative to manure, bio-manure stimulated HD-driven N 2 O production in association with increasing abundances of HD-related genes ( nirK , nirS , fungi- nirK ) and potential denitrification rates. Bio-manure often maintained a higher capacity for N 2 O reduction, evidenced by lower ( nirK + nirS+ Fungi- nirK )/( nosZ ) ratios and unreduced N 2 O fraction. Critically, substitutions enhanced crop yields, with manure increasing yields by 8–21% and bio-manure by 19–30% compared to conventional fertilizer. Consequently, our study revealed that 50% bio-manure substitution is an effective strategy for mitigating N 2 O emissions and enhancing yields from vegetable production systems, providing actionable insights for sustainable resource management and climate change mitigation. • N 2 O source partitioning under bio-manure (BM) and manure (M) substitutions • BM promoted the reduction of N 2 O to N 2 compared with M • Either 50% M or 50% BM reduces N 2 O emissions based on a 5-year field trial • Production-consumption balance in denitrification governs N 2 O flux in BM vs M • The mitigation advantage of BM vs M diminishes under long-term application