Quorum sensing (QS) plays a critical role in the enhancement of biofilm performance at low temperatures. In this study, a uni-QS (Sphingomonas rubra) and three bisynergistic-QS [composed of S. rubra and one of the isolated QS aerobic denitrifying bacteria (QS-ADB) Pseudomonas mendocina QQQC, P. mendocina ABDF, and Enterobacter aerogenes] were added to biofilm reactors in the start-up phase at 8 °C. Results indicate that compared to the control reactor, bisynergistic-QS exhibited maximum TN removal efficiency (93.50 ± 5.44, 93.97 ± 3.93, and 92.35 ± 5.04%), largest functional genes amoA, narG, and norB abundance (15-fold, 15-fold, and 75-fold), and uprising C10-HSL and C14-HSL concentrations (22.32-to 39.28-fold and 3.87-to 8.66-fold). Network analysis revealed that the biofilm thickness which possessed strong relationships with its dominant bacteria might contribute to great performance in bisynergistic-QS reactors under low temperature and increased loading rate. Proteobacteria play an important role in the AHL-based QS system. The abundance of keystone taxa Raoultella, Micropruina, and Zoogloea deserved more attention utilizing exogenous aerobic denitrifier addition. This study provided a helpful guideline for the design of exogenously enhancing biofilm denitrification systems at low temperatures.

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