Adaptive Responses of Salinity-Acclimated Heterotrophic Bacterial Communities to Stepwise Cooling: Implications for Ammonium Assimilation Potential

The adaptive responses of salinity-acclimated heterotrophic bacterial communities with ammonium assimilation potential to prolonged cooling remain poorly understood. In this study, three sequencing batch reactors (SBRs) with salinities of 25 (R1), 50 (R2), and 100 g/L (R3) NaCl were operated continuously for 283 days as the temperature was successively reduced from 25 °C to 8 °C. At 8 °C, the apparent aqueous phase total inorganic nitrogen (TIN) removal efficiencies were 79.99 ± 1.32%, 93.20 ± 8.29%, and 95.66 ± 4.34% in R1, R2, and R3, respectively, with limited net accumulation of NO2−-N and NO3−-N. Cooling was accompanied by distinct changes in sludge properties and microbial communities across the reactors. R1 experienced marked decreases in MLSS and MLVSS and lower community evenness, together with the predominance of Nitrincola (89.42%) and reduced apparent aqueous phase nitrogen removal. In contrast, R2 and R3 retained higher volatile solids and multiple halotolerant heterotrophic genera, while Halomonas remained dominant in R3. Protein-rich EPS increased during cooling, and intracellular ectoine reached 133.03, 215.50, and 443.49 mg/g VSS in R1, R2, and R3 at 8 °C. The continued detection of glnA in metagenomes indicated retained genetic potential for ammonium assimilation. Collectively, these descriptive observations characterize different community responses to cooling and support ammonium assimilation as a plausible contributor to nitrogen transformation.