Unveiling the mechanism underlying in-situ enhancement on anammox system by sulfide: Integration of biological and isotope analysis.

The in-situ utilization of sulfide to remove the nitrate produced during the anaerobic ammonium oxidation (anammox) process can avoid prolonged sludge acclimatization, facilitating the rapid initiation of coupled nitrogen removal processes. However, the understanding of in-situ enhancement on anammox system by sulfide remains unclear. Herein, sulfide (NaS) was introduced as an additional electron donor to remove the nitrate derived from the anammox under varying sulfide/nitrogen (S/N, S-S/NO-N, molar ratio) ratios (0.004-4.375). The underlying mechanisms were elucidated by molecular biology techniques including flow cytometry, quantitative polymerase chain reaction, and 16S rRNA amplicon sequencing, alongside isotope tracer analysis. Results revealed that anammox reactors, when operated with in-situ sulfide addition, exhibited a significant enhancement in total nitrogen removal efficiency (NRE) ranging from 11.5 %-41.7 % (achieved 96 %), with the optimal S/N ratios of 0.01-0.8. Isotope tracer analysis indicated the successful coupling of the anammox, sulfur autotrophic denitrification (SADN), and dissimilatory nitrate reduction to ammonium (DNRA) processes within the system, with their contributions to nitrogen removal being 46 %-50 %, 24 %-30 %, and 20 %-22 %, respectively. Moreover, a notable increase in the abundance of sulfur-oxidizing bacteria (SOB) (20 %-40 % increase) and DNRA bacteria (10 %-20 % increase) were observed. Effective collaboration was further supported by the sustained viability of microbial communities. It is speculated that the heightened presence of SOB and DNRA bacteria created a low toxicity environment by converting sulfide to biogenic sulfur, thereby promoting the well-being of anammox bacteria. However, the excessive dosage of sulfide (S/N = 1.8) intensified the DNRA process (contribution>35 %) and weakened the anammox process, leading to an increase in effluent NH-N concentration and a decline in NRE. This study confirms that the in-situ adding an appropriate amount of sulfide favors achieving complete nitrogen removal in anammox system, which provides a novel avenue to resolve the issue of the residual nitrate in anammox process.