Divergent legacy effects of biochar on nitrous oxide emissions in acidic soils driven by altered microbial N pathways

Acidic soils are global hotspots of nitrous oxide (N 2 O) emissions, and biochar has been proposed as a promising mitigation strategy. However, most current evidence comes from short-term studies, and the legacy effects and underlying mechanisms remain poorly understood. Here, we collected acidic soil samples from three sites with and without biochar application, representing short-term (3 and 5 years) and long-term (9 years) legacy effects. Using microcosm incubations, isotope-based source partitioning, and microbial analyses, we evaluated N 2 O dynamics and their microbial drivers. The short-term legacy effects of biochar significantly reduced N 2 O emissions by inhibiting gross N 2 O production and enhancing N 2 O reduction. This was primarily attributed to reduced nitrification-derived N 2 O, increased nos Z gene abundance, and enrichment of taxa carrying the nos Z gene, such as Rhodanobacter and Gemmatimonas . In contrast, long-term legacy effects markedly increased N 2 O emissions because biochar suppressed N 2 O reduction more strongly than its production. This was linked to reduced nos Z abundance, increased fungal denitrification, and depletion of dissolved organic carbon and denitrifying bacteria. Together, these findings reveal that the legacy effects of biochar on N 2 O emissions diverge over time, driven by changes in microbial nitrogen cycling pathways. These results underscore the importance of incorporating temporal and microbial perspectives when evaluating the long-term climate impacts of biochar and developing sustainable soil management strategies.