Development of a psychrotolerant composite microbial agent for nitrogen removal and its nitrogen metabolism pathways

Low temperature can suppress biological nitrogen removal (NR) efficiency. Although NR characteristics of single psychrotolerant bacteria have been extensively studied, synergistic interactions between functionally distinct psychrotolerant nitrogen-removing consortia remain unexplored. In this study, a composite microbial agent, designated NDC-6, was generated by coculturing a psychrotolerant nitrifying consortium NC1 (Pseudomonas veronii HN1, P. poae HN2, and P. peli HN3) and an aerobic denitrifying consortium DC1 (Aeromonas sp. AD1, P. extremaustralis AD2, and Serratia liquefaciens AD3) at a 1:1 inoculation ratio, and its NR performance was systematically evaluated. After 3 days of incubation at 10°C, NDC-6 achieved removal efficiencies of 89.3%, 88.1%, 85.5%, and 95.3% for NH₄⁺-N, NO₃⁻-N, total nitrogen (TN), and chemical oxygen demand (COD), which were significantly higher than those of individual strains or single-function consortia. Sodium succinate was identified as the optimal carbon source, which simultaneously improved biomass growth and NR efficacy of NDC-6. Optimal culture conditions determined using response surface methodology were as follows: C/N ratio, 6; temperature, 10.2°C; pH, 7.2; and shaking speed, 156 rpm. Under these conditions, the maximum TN removal efficiency reached 89.8%. Nitrogen balance and functional gene expression (hao, napA, nirS, nirK, cnorB, and nosZ) analyses revealed that NDC-6 achieved complete NR through both assimilatory and dissimilatory pathways. The dissimilatory mechanism relied on synergistic metabolism and functional complementation between NC1 and DC1, mediated by their respective functional genes. This study provides mechanistic insights into the biological treatment of nitrogen-containing wastewater, particularly under low-temperature conditions, and offers a novel strategy for such treatment.