Biennial biochar application mitigates N₂O emissions in saline–alkali soils by modulating microbial communities and metabolic pathways

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In the context of global climate change, agricultural soil nitrous oxide (N₂O) emissions have attracted increasing attention, and biochar is considered a potential mitigation strategy. However, biochar effects on N₂O emissions vary with application method, and the underlying mechanisms remain incompletely understood. We conducted long-term field experiments in saline–alkali soils to test biochar application rates (4, 8, and 12 t ha⁻¹) and frequencies (annual, biennial, single application) on soil N₂O emissions, microbial communities, and untargeted metabolomics. Biennial application reduced cumulative N₂O emissions by 21.21–24.44 % relative to the control, with mitigation strengthening at higher rates. Mechanistically, biennial biochar reduced N₂O emissions by restructuring microbial communities and metabolic pathways: (1) it decreased denitrifying taxa ( Myxococcota ) and increased nitrogen-fixing microorganisms ( Vicinamibacteria and Blastocatellia ), directly weakening denitrification capacity; (2) at 4 and 8 t ha⁻¹ , it downregulated glycerophospholipid metabolites (1-oleoylglycerophosphoinositol; phosphatidylinositol lyso 18:0) important for activities of membrane-bound denitrifying enzymes, thereby inhibiting denitrification; (3) at 12 t ha⁻¹ , it upregulated plant secondary metabolites (abscisic acid) and amino acids (L-isoleucine), which enhanced plant nitrogen uptake and lowered soil nitrate (NO₃⁻) availability, thereby suppressing denitrification-derived N₂O production. Mitigation was constrained by application frequency: annual biochar addition did not significantly reduce N₂O emissions, likely because frequent inputs disrupted microbial adaptation, and a single application showed limited effectiveness because suppression was time-limited. Overall, biennial application of 8–12 t ha⁻¹ biochar mitigated N₂O emissions by regulating microbial communities and metabolic processes, providing evidence to guide biochar use in agricultural production to improve soil quality while reducing greenhouse gas emissions. • Biennial biochar use cuts nitrous oxide emissions in saline–alkali farmland soils. • Higher biochar rates boost emission cuts by shifting microbial community balance. • Biochar lowers key metabolites that drive denitrification in agricultural soils.