Managing microbial sulfur disproportionation for optimal sulfur autotrophic denitrification in a pilot-scale elemental sulfur packed-bed bioreactor.

Elemental sulfur packed-bed (SPB) bioreactors for autotrophic denitrification have gained more attention in wastewater treatment due to their organic carbon-free operation, low operating cost, and minimal carbon emissions. However, the rapid development of microbial S-disproportionation (MSD) in SPB reactor during deep denitrification poses a significant drawback to this new technology. MSD, the process in which sulfur is used as both an electron donor and acceptor by bacteria, plays a crucial role in the microbial-driven sulfur cycle but remains poorly understood in wastewater treatment setups. In this study, we induced MSD in a pilot-scale SPB reactor capable of denitrifying over 1000 m/d nitrate-containing wastewater. Initially, the SPB reactor stably removed 6.6 mg-NO-N/L nitrate at an empty bed contact time (EBCT) of 20 mins, which was designated the S-denitrification stage. To induce MSD, we reduced the influent nitrate concentrations to allow deep nitrate removal, resulted in the production of large quantities of sulfate and sulfide (SO:S 3.2 w/w). Meanwhile, other sulfur-heterologous electron acceptors (SHEAs), e.g., nitrite and DO, were also kept at trace levels. The negative correlations between the SHEAs concentrations and the sulfide productions indicated that the absence of SHEAs was a primary inducing factor to MSD. The microbial community drastically diverged in response to the depletion of SHEAs during the switch from S-denitrification to S-disproportionation. An evident enrichment of sulfur-disproportionating bacteria (SDBs) was found at the S-disproportionation stage, accompanied by the decline of sulfur-oxidizing bacteria (SOBs). In the end, we discovered that shortening the EBCT and increasing the reflux ratio could inhibit sulfide production by reducing it from 43.9 mg/L to 3.2 mg/L or 25.5 mg/L. In conclusion, our study highlights the importance of considering MSD when designing and optimizing SPB reactors for sustainable autotrophic sulfur denitrification in real-life applications.