Yifan Zhang, Matthew J Rogers, Chuansheng Wang, Guofang Xu, Kun-Lin Yang, Jianzhong He
Fixed nitrogen and sulfide frequently coexist in anaerobic digestion liquors and other organic-deficient wastewaters. Coupling anaerobic ammonium oxidation (anammox) with sulfide-driven denitrification offers a potential strategy for energy-efficient nitrogen removal, yet rapid integration and stable operation of the coupled process remain challenging. Here a mixotrophic sulfide-driven denitrification-anammox process was established by bioaugmenting established anammox sludge with a mixotrophic sulfide-driven denitrifying culture. The reactor achieved stable total nitrogen (TN) removal efficiencies of 92-97% over 82 days of operation while maintaining complete sulfide removal under increasing sulfide loading. Stoichiometric analysis and batch tests indicated that anammox dominated nitrogen conversion, with an estimated contribution of 88-95% to TN removal, whereas sulfide-driven denitrification primarily contributed to anammox-derived nitrate removal, although some competition for nitrite may occur under specific conditions. This hierarchical partition contrasts with conventional partial denitrification-anammox systems, where excessive denitrification during start-up may compete with anammox for nitrite, thereby delaying the establishment of anammox-dominated nitrogen conversion. The system exhibited low nitrous oxide (N2O) emissions (0.1% of influent TN), which were substantially lower than the default emission factor reported by the Intergovernmental Panel on Climate Change. Functional gene abundance, transcriptional responses, and microbial community analyses supported the persistence of key functional guilds involved in nitrogen transformation. This study highlights that bioaugmentation with mixotrophic sulfide-driven denitrifiers provides a strategy for nitrate recycling in established anammox systems, enabling stable sulfur-nitrogen coupling under organic-deficient and sulfide-rich conditions.