Managing microbial sulfur disproportionation for optimal sulfur autotrophic denitrification in a pilot-scale...

Elemental sulfur packed-bed (S⁰PB) 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 S⁰-disproportionation (MS⁰D) in S⁰PB reactor during deep denitrification poses a significant drawback to this new technology. MS⁰D, 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 MS⁰D in a pilot-scale S⁰PB reactor capable of denitrifying over 1000 m³/d nitrate-containing wastewater. Initially, the S⁰PB reactor stably removed 6.6 mg-NO₃^--N/L nitrate at an empty bed contact time (EBCT) of 20 mins, which was designated the S⁰-denitrification stage. To induce MS⁰D, we reduced the influent nitrate concentrations to allow deep nitrate removal, resulted in the production of large quantities of sulfate and sulfide (SO₄^2-:S^2- 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 MS⁰D. The microbial community drastically diverged in response to the depletion of SHEAs during the switch from S⁰-denitrification to S⁰-disproportionation. An evident enrichment of sulfur-disproportionating bacteria (SDBs) was found at the S⁰-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 MS⁰D when designing and optimizing S⁰PB reactors for sustainable autotrophic sulfur denitrification in real-life applications.