Long-term biochar amendment increased dissimilatory nitrate reduction to ammonium concomitantly suppressing denitrification and anaerobic ammonium oxidation in deep alkaline paddy soil

Dissimilatory nitrate reduction to ammonium (DNRA) conserves soil nitrogen (N), whereas denitrification and anaerobic ammonium oxidation (anammox) can result in soil N loss. Despite biochar’s potential to modulate N cycling pathways, its effects on dissimilatory nitrate reduction in alkaline paddy soils remain poorly characterized. Using 15 N tracer pairing techniques in soil slurries, we quantified denitrification, anammox, and DNRA rates in soils to 80 cm depth that had received surface annual application of 0.05 t/ha of rice straw biochar. We then partitioned nitrate reduction pathways and identified associated microbial communities. Denitrification showed the greatest variability in rates, proportional contribution and community composition, indicating its dominant role in partitioning nitrate reduction pathways in both surface and deep layers. Random forest and structural equation modeling identified the edaphic factors of soil pH, SOC/NO 3 ⁻ ratio, and Fe 2+ concentration as key drivers. In surface layers, biochar amendment optimized these parameters, stimulating both denitrification and DNRA rates. In deep layers, however, biochar progressively elevated pH above optimal thresholds and lowered SOC/NO 3 ⁻ ratio, further suppressing napA gene abundance and denitrification rates while increasing DNRA rates and proportions. Our findings demonstrate that long-term biochar amendment promotes DNRA and suppresses denitrification and anammox in deep alkaline paddy soil. These results provide mechanistic evidence for biochar’s role in conserving soil N through depth-dependent modulation of microbial N cycling pathways in alkaline paddy systems.