Revitalizing simultaneous nitrification, endogenous denitrification and phosphorus removal under carbon starvation:...

The simultaneous nitrification, endogenous denitrification and phosphorus removal (SNEDPR) process provides a resource-efficient paradigm for synergistic nutrient removal. However, its efficacy can be severely constrained by limited electron-donor availability and inefficient intracellular electron transfer under low carbon-to-nitrogen (C/N) ratios. Herein, we demonstrate that anthraquinone-2-sulfonate (AQS) serves as an effective redox mediator to alleviate these electron-transfer limitations. Under severe carbon-limited conditions, with an influent C/N ratio of 3.24±0.11, supplementation with 0.3 mmol/L AQS successfully restored the deteriorated system, recovering total inorganic nitrogen (TIN) removal efficiency to 96.57±0.87% while sustaining robust phosphorus removal (96.47±2.68%). Batch kinetic assays showed that AQS increased electron transfer system activity (ETSA) by over 100%, accompanied by enhanced nitrification-related activity, endogenous denitrification and phosphorus uptake. Integrated metagenomic and metatranscriptomic analyses further revealed transcriptional responses consistent with an electron-shuttling role of AQS, including enhanced ETC-associated electron-transfer potential and increased transcription of downstream denitrification enzymes. ATP synthase-related transcription was also enhanced, indicating an increased potential for PMF-coupled cellular energy conservation. Consequently, increased transcription was observed for key functional genes involved in nitrification (amoABC, nxrAB), denitrification (narGHI, napAB, nirKS, nosZ), and polyphosphate synthesis (ppk). Furthermore, AQS-amended operation was associated with microbial community succession toward a more specialized consortium, in which comammox, Thauera and Candidatus Accumulibacter clade ⅡC were associated with nitrification, endogenous denitrification, and denitrifying phosphorus removal, respectively. Although practical translation will require effective retention or recovery of soluble AQS, these findings demonstrate the potential of AQS-enhanced SNEDPR under carbon-limited conditions and provide multi-omics evidence linking improved nutrient removal with increased electron-transfer activity and coordinated microbial metabolic responses.