Mixotrophic denitrification (combining heterotrophic denitrification and autotrophic denitrification) based bioretention system is a promising sustainable technology for nitrate nitrogen removal from stormwater runoff due to the ease of start-up and long operational cycle. However, the initial release of organic matter and ammonia nitrogen from most biomass carbon sources and the ineffectiveness for real-time treatment during rewetting period limit its engineering application. In this study, a novel heterotrophic-autotrophic sequential bioretention system was constructed by sequential packing waste reeds and sulfur in the upper and lower layers of the submerged zone. The removal of nitrate nitrogen, total nitrogen, ammonia nitrogen, and chemical oxygen demand maintained at 92.45%-99.54%, 84.80%-95.60%, 57.27%-82.86%, and 67.79%-85.82%, respectively, under different influent nitrate nitrogen loads of 20 mg/L, 14 mg/L, and 8 mg/L, respectively. The system solved the massive leaching of ammonia nitrogen and organic matter from waste reeds in the early phases and achieved low production rate of nitrite nitrogen (0.0004-0.0048 kg/(m3 & sdot;d)) and sulfate (0.09-0.27 kg/(m3 & sdot;d)). For 3 and 5 antecedent dry days, the real-time nitrate nitrogen removal kept at 99.66 +/- 0.25% and 91.26 +/- 7.91%. Microbial communities and functional genes revealed the C, N, S transformation driven by the system improved the responds to dry-wet alterations. The synergy of heterotrophic layer (heterotrophic denitrification and sulfidedriven autotrophic denitrification) and autotrophic layer (sulfur-driven autotrophic denitrification, dissimilatory nitrate reduction to ammonia, and sulfate reduction) promoted the complete denitrification and reduced byproducts formation.
