Performance of sulfur-iron based autotrophic denitrification biofilters for the removal of nitrate and phosphate and mechanisms of electron competition at different S/N

The performance of nitrate and phosphate removal and the mechanism of electron competition in sulfur based (VR-BF) and sulfur-iron based autotrophic denitrification biofilters with sponge iron (SI) as iron substrate (SIVR-BF) were analyzed at different S/N. As S/N from 0.85 to 0.70, SIVR-BF showed higher total nitrogen removal efficiency (90.6 %) and no nitrite accumulation compared to VR-BF (82.2 %), highlighting its stability under fluctuating conditions. At lower S/N, even weakened sulfur autotrophic denitrification (SAD) reduced H⁺ production and limited Fe²⁺ release from SI, SIVR-BF maintained >92 % phosphate removal efficiency. The contribution rate of the SAD pathway to nitrate removal decreased from 96.1 % to 55.1 % in SIVR-BF, while the other pathways increased from 3.8 % to 36 %, which revealed the role of SI as a synergistic electron donor (supplying Fe²⁺ and H₂) in the dynamic regulation of the denitrification pathway. Moreover, the higher extracellular polymeric substances and lower dissolved oxygen in SIVR-BF were beneficial for improving electron transport capacity and maintaining stability. Batch tests confirmed nitrate as the optimal electron acceptor in S₂O₃^2- driven autotrophic denitrification. Coupled SI could alleviate strong nitrite inhibition and electron donor competition under electron donor limited and mixed electron acceptor conditions, improving denitrification rates. Microbial community analysis revealed that the abundance of bacteria related to sulfur-metabolizing genera (Thiobacillus and Sulfurimonas) decreased with reduced S/N in SIVR-BF, and iron/hydrogen-metabolizing genera (Ferritrophicum and norank-f-Hydrogenophilaceae) were enriched. This study elucidates the multi-electron donor synergy in sulfur-iron coupled systems, providing theoretical support for developing efficient and stable low-carbon wastewater treatment technologies.