Unlocking low NO emissions from nitrate-laden wastewater in constructed wetlands: critical role of pyrrhotite substrate layer in mediating nitrate-dependent sulfide oxidation.

Constructed wetlands (CWs) treating nitrate-rich wastewater often face incomplete denitrification and elevated NO emissions due to insufficient electron donors. Pyrrhotite as a CW substrate demonstrated potential for enhancing autotrophic denitrification through coupled sulfur and iron biological oxidation. However, the impact of pyrrhotite layer positioning on regulating NO emissions and underlying mechanisms remains unclear. This study evaluated the effect of pyrrhotite layer placement (top, middle and bottom) on S/Fe-coupled denitrification and NO release under organic carbon-free with varying nitrogen loads. Results showed that the bottom layer achieved 32.36-65.86 % complete denitrification (2.37-5.68 times higher than middle/top layers), while B-CW limited NO emission to only 0.36 % of converted nitrate (39.60-53.60 % lower than M-CW/T-CW). Enhanced performance in B-CW correlated with higher oxidation amounts of reduced sulfur (50.51 vs. 25.27-28.97 mg/L) and ferrous iron (36.83 vs. 18.43-21.12 mg/L), with efficient utilization. Network analysis revealed increased modularity and functional clustering in the bottom layer, with Ralstonia co-occurring with key sulfur/iron-cycling bacteria (Thiobacillus, Undibacterium) to form stable denitrifying consortia. Microbial analysis revealed enrichment of nitrate-reducing bacteria, primarily Ralstonia (14.69 %) in the bottom layer, driving 66.58 % inorganic electron utilization via sulfur oxidation-coupled complete denitrification. Electron and nitrogen mass balances revealed that 81.84 % of reduced nitrate was converted to N Additionally, synergistic interactions among nitrate-reducing bacteria (24.19 %), sulfur-/iron-oxidizing bacteria (16.29 %/4.46 %), organic matter-degrading bacteria (23.23 %), and electroactive bacteria (8.12 %) supported the process. These findings highlight pyrrhotite layer depth as a critical regulator of NO mitigation in CWs, providing a sustainable inorganic strategy for low-carbon and sustainable nitrogen removal.