Combined Effects of Carbon-to-Nitrogen (C/N) Ratio and Nitrate (NO3−-N) Concentration on Partial Denitrification (PD) Performance at Low Temperature: Substrate Variation, Nitrite Accumulation, and Microbial Transformation

In this study, the combined effects of influent carbon-to-nitrogen ratio (C/N = 0.8, 1.5, 2.5, 3.5, 4.5) and nitrate (NO3−-N) concentration (40 and 80 mg/L, labeled as R40 and R80) on the partial denitrification (PD) performance were investigated using an intermittent sequencing batch reactor (SBR) process. With sodium acetate as an additional carbon source, the substrate variation, microbial diversity, and functional bacteria evolution were also explored to reveal the nitrite (NO2−-N) accumulation mechanism at low temperatures (3–12 °C). The results showed that the 3.5-R40 and 2.5-R80 systems both presented the optimal NO2−-N accumulation at a temperature of 10 °C, with the NO2−-N transformation rate (NTR) of 66.89% and 76.79%, respectively. In addition, as the temperature reduced from 10 °C to 5 °C, the NO2−-N accumulation performance was significantly suppressed, where the average effluent NO2−-N of 3.5-R40 (20.00 → 11.00 mg/L) and 2.5-R80 (43.00 → 18.90 mg/L) systems reduced by nearly half. It is worth noting that there was almost no NO2−-N accumulation at a C/N ratio of 0.8, although higher NO3−-N concentration promoted NTR under the same C/N ratio. The high-throughput sequencing showed that the minimum Shannon value of 3.81 and the maximum Simpson value of 0.095 both occurred at a C/N ratio of 2.5, suggesting the downshifted microbial richness. Proteobacteria and Bacteroides increased significantly from 35.31% and 18.34% to 51.69–60.35% and 18.08–35.21%, as compared with the seeding sludge. Thauera and Flavobacterium as the main contributors to NO2−-N accumulation accounted for 31.83% and 20.30% at the C/N ratio of 2.5 under a low temperature of 5 °C. The above discussion suggested that higher temperature (10 °C), lower C/N ratio (2.5–3.5), and higher NO3−-N concentration (80 mg/L) were more favorable for the stable PD formation.