Revealing Feammox in sulfur/pyrite-based autotrophic denitrification coupled Anammox system: performance and mechanistic insights

Although autotrophic nitrogen removal systems are promising low-carbon technologies for treating low-strength nitrogenous wastewater, their efficiency remains limited and the synergistic roles of different nitrogen transformation pathways in coupled systems are unclear. This study investigated the performance and mechanistic enhancement of nitrogen removal in a sulfur-based autotrophic denitrification (SADn) system integrated with anaerobic ammonium oxidation (Anammox). The coupled system demonstrated high average ammonia removal efficiency (>95 %) under varying hydraulic retention times (HRTs). Isotopic tracer tests (¹⁵N labeling) confirmed synergistic contributions from Anammox (71.63–76.52 %), SADn (14.66–20.24 %), and Feammox (4.16–13.71 %) pathways, with Feammox becoming dominant when the influent NH₄⁺-N concentration increased from 30 to 50 mg/L. Bioelectrochemical and Raman analyses confirmed the enhanced extracellular electron transfer capacity in the coupled system. Thiobacillus was responsible for denitrification and sulfur oxidation, while Candidatus_Brocadia not only had anammox function but also might be related to Feammox. Metagenomic analysis revealed upregulation of genes related to Fe, S, and N metabolism, confirming microbial synergy in N-S-Fe cycling. These findings provide insights into optimizing S/Fe-mediated N removal for low nitrogen-containing wastewater treatment, emphasizing role of Feammox in low-carbon scenarios.