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Open-water unit process wetlands host a benthic diatomaceous and bacterial assemblage capable of nitrate removal from treated municipal wastewater with unexpected contributions from anammox processes. In exploring mechanistic drivers of anammox, 16S rRNA gene sequencing profiles of the biomat revealed significant microbial community shifts along the flow path and with depth. Notably, there was an increasing abundance of sulfate reducers ( Desulfococcus and other Deltaproteobacteria ) and anammox microorganisms ( Brocadiaceae ) with depth. Pore water profiles demonstrated that nitrate and sulfate concentrations exhibited a commensurate decrease with biomat depth accompanied by the accumulation of ammonium. Quantitative PCR targeting the anammox hydrazine synthase gene, hzsA , revealed a 3-fold increase in abundance with biomat depth as well as a 2-fold increase in the sulfate reductase gene, dsrA These microbial and geochemical trends were most pronounced in proximity to the influent region of the wetland where the biomat was thickest and influent nitrate concentrations were highest. While direct genetic queries for dissimilatory nitrate reduction to ammonium (DNRA) microorganisms proved unsuccessful, an increasing depth-dependent dominance of Gammaproteobacteria and diatoms that have previously been functionally linked to DNRA was observed. To further explore this potential, a series of microcosms containing field-derived biomat material confirmed the ability of the community to produce sulfide and reduce nitrate; however, significant ammonium production was observed only in the presence of hydrogen sulfide. Collectively, these results suggest that biogenic sulfide induces DNRA, which in turn can explain the requisite coproduction of ammonium and nitrite from nitrified effluent necessary to sustain the anammox community. IMPORTANCE This study aims to increase understanding of why and how anammox is occurring in an engineered wetland with limited exogenous contributions of ammonium and nitrite. In doing so, the study has implications for how geochemical parameters could potentially be leveraged to impact nutrient cycling and attenuation during the operation of treatment wetlands. The work also contributes to ongoing discussions about biogeochemical signatures surrounding anammox processes and enhances our understanding of the contributions of anammox processes in freshwater environments.

Sulfide-Induced Dissimilatory Nitrate Reduction to Ammonium Supports Anaerobic Ammonium Oxidation (Anammox) in an Open-Water Unit Process Wetland