Overlooked pathways of denitrification in a sulfur-based denitrification system with organic supplementation

Elemental sulfur-driven autotrophic denitrification (SADN) is a cost-effective approach for treating secondary effluent from wastewater treatment plants (WWTPs). Additional organics are generally supplemented to promote total nitrogen (TN) removal, reduce nitrite accumulation and sulfate production, and balance the pH decrease induced by SADN. However, understanding of the impacts of organic supplementation on microbial communities, nitrogen metabolism, denitrifier activity, and SADN rates in sulfur-based denitrification reactors is still limited. Here, a sulfur-based denitrification reactor was continuously operated for 272 days during which six different C/N ratios were tested successively (2.7, 1.5, 0.7, 0.5, 0.25, and 0). Organic supplementation improved TN removal and decreased NO₂^- accumulation, but reduced the relative abundance of denitrifiers and the contribution of autotrophic nitrate-reducing bacteria (aNRB) to TN removal during the long-term operation of reactor. Predictive functional profiling showed that nitrogen metabolism potential increased with decreasing C/N ratios. SADN was the predominant removal process when the C/N ratio was ≤0.7 (achieving 60% contribution when C/N = 0.7). Although organic supplementation weakened the dominant role of aNRB in denitrification, batch tests for the first time demonstrated that it could accelerate the SADN rate, attributed to the improvement of sulfur bioavailability, likely via the formation of polysulfide. A possible nitrogen removal pathway with multiple electron donors (i.e., sulfur, organics, sulfide, and polysulfide) in a sulfur-based denitrification reactor with organic supplementation was therefore proposed. However, supplementation with a high level of organics could increase the operational cost and effluent concentrations of sulfide and organics as well as enrich heterotrophic denitrifiers. Moreover, microbial community had substantial changes at C/N ratios of >0.5. Accordingly, an optimal C/N ratio of 0.25-0.5 was suggested, which could simultaneously minimize the additional operating cost associated with organic supplementation and maximize TN removal and SADN rates.