Rational design of a cold-adapted denitrifying consortium reveals synergistic mechanisms for enhanced low-temperature...

Low-temperature conditions severely constrain biological nitrogen removal from nitrogen-containing wastewater, mainly by inhibiting microbial metabolic activity and functional stability. Constructing an efficient and stable denitrifying composite microbial consortium has therefore been proposed as an effective strategy to enhance nitrogen removal. In this study, a bottom-up strategy was employed to construct a low-temperature denitrifying bacterial consortium through statistical screening and partial factorial experiments, resulting in a optimized consortium consisting of strains LTN-2, LTN-3, and LTN-4. At 15 °C and 200  mg L^-1 NO₃^--N, the consortium removed 95.75% nitrate in 48  h, showing markedly enhanced denitrification over individual strains. Genomic analysis combined with qRT-PCR validated a clear synergistic division of labor, where LTN-2 and LTN-4 mainly reduced NO₃^--N to NO₂^--N, while LTN-3 dominated the conversion of NO₂^--N to N₂. Furthermore, metatranscriptomics elucidated five active cold-adaptation pathways, confirming the molecular basis for the consortium's efficient denitrification at low temperatures. Sequencing batch reactor (SBR) experiments confirmed that the experimental group exhibited 28.95%, 52.25%, and 28.57% higher removal efficiencies of NO₃^--N, total nitrogen (TN), and chemical oxygen demand (COD), respectively, compared with the control group. Microbial community analysis further indicated that the constructed consortium enhanced community complexity and cooperative interactions, thereby promoting nitrogen removal under low-temperature conditions. This study provides a scientific basis for the assembly and potential future application of denitrifying microbial consortium in low-temperature wastewater treatment systems.