Sludge retention time governs microbial selection and metabolic traits in glycerol-driven denitratation under fixed carbon limitation

Stable glycerol-driven denitratation requires a robust feast–famine selection pressure, which is difficult to maintain in low-strength mainstream wastewater. This study investigated sludge retention time (SRT) as an operational lever for reinforcing this selection pressure and elucidated the associated ecological and metabolic mechanisms. Shortening SRT from 20 to 5 d markedly enhanced denitratation performance, achieving a stable nitrate removal efficiency of 92.7% and a nitrite yield of 0.153 g-N g-COD −1 . This optimization originated from a community-level redirection of carbon flux and electron reallocation. The short SRT stimulated a metabolic phenotype favoring intracellular carbon storage, increasing the storage coefficient to 0.498 g-COD g-COD −1 . Meanwhile, the electron supply to downstream denitrification pathways was restricted, manifesting as sequential denitrification mediated by the enzymatic activity mismatch between nitrate and nitrite reductases. In terms of community composition, the deterministic selection pressure imposed by the short SRT simplified the microbial co-occurrence network, directionally enriching a functional consortium centered on Thauera aminoaromatica . This community exhibited a surge in polysaccharide-rich extracellular polymeric substances, which effectively ensured biomass retention under washout pressure. Ultimately, partial least squares path modeling revealed that the regulation of SRT was primarily mediated by the food-to-microorganism ratio, and was transmitted via functional gene potentials and enzyme activities to the final metabolic phenotype, eliciting a positive feedback on the extracellular matrix. These findings identify SRT control as an effective strategy to engineer microbial metabolic traits and provide a cost-effective route for stable nitrite supply for mainstream nitrogen removal.