Carbon setpoint regulation enables stable endogenous denitrification in an adaptive activated sludge system under low C/N and low-temperature conditions.

Achieving stable and energy-efficient nitrogen removal from low carbon-to-nitrogen (C/N) rural sewage under low-temperature conditions remains a major challenge for decentralized wastewater treatment. In this study, an adaptive activated sludge (AAS) system incorporating a dynamic regulation zone was developed to enhance endogenous carbon management under simultaneous carbon limitation and cold stress. The AAS system maintained efficient nitrogen removal at 10 °C, achieving an average effluent total inorganic nitrogen (TIN) concentration of 12.42 ± 0.59 mg/L at an influent C/N ratio of 3. The dynamic regulation zone buffered hydraulic fluctuations while facilitating intracellular carbon storage and enrichment of endogenous heterotrophs. Despite severe carbon limitation, the combined relative abundance of denitrifying glycogen-accumulating organisms (DGAOs) and denitrifying phosphorus-accumulating organisms (DPAOs) remained as high as 25.73%, supporting sustained endogenous denitrification. Metagenomic analysis revealed adaptive metabolic rerouting under carbon stress, whereby microorganisms appeared to redirect acetyl-CoA-associated metabolic potential away from the tricarboxylic acid (TCA) cycle toward polyhydroxyalkanoate (PHA) synthesis under carbon limitation, suggesting adaptive carbon allocation toward intracellular storage. Based on these findings, a carbon setpoint framework was proposed as a mechanistic generalization describing the adaptive redistribution of carbon flux between energy production and intracellular storage under environmental stress. Overall, the AAS system provides an effective endogenous carbon management strategy for C/N rural sewage treatment while advancing the mechanistic understanding of microbial metabolic adaptation under combined carbon-limited and low-temperature conditions.