C4-HSL drives rapid biofilm formation in low-temperature aquaculture effluent: strengthening structural stability of biofilm and improving nitrogen removal efficiency

Abstract

During the centralized discharge of winter aquaculture wastewater, low temperature (≤15 °C) and low C/N ratio (≤5) result in delayed initiation of the biofilm process and decreased nitrogen removal efficiency.The quorum sensing (QS) communication system serves as the core mechanism regulating biofilm formation. This study examined how adding N-butyyl-L-homo-serine lactone (C4-HSL) affects the start-up and nitrogen removal in sequential batch biofilm reactors (SBBR) operated at 14 °C with a C/N ≤ 5. Biofilm adhesion tests showed that C4-HSL levels between 400 and 1200 ng/L notably boosted initial adhesion, peaking at 700 ng/L with a 57.5 % increase over the control. The SBBR results indicated that a high concentration of C4-HSL (700–1000 ng/L) effectively removed TN and COD, achieving a TN removal rate of 91.62 %, which was three times that of the control. C4-HSL enhances low-temperature biofilm nitrogen removal by remodeling the structure of functional microbial communities and enriching bacteria along with functional genes involved in biofilm formation and nitrification-denitrification. Additionally, C4-HSL specifically upregulated the abundance of flagella assembly and biofilm matrix synthesis genes (such as virD4, yegE) and quorum sensing-related genes (such as rpfB, bapA), promoting the secretion of extracellular polymeric substances. This drove the transition of biofilm structure from a loose state to a highly organized one, accelerating the maturation of biofilm adhesion and prolonging the stabilization period of the biofilm by 15–20 days. This study revealed the influence of C4-HSL on the initiation mechanism of low-temperature biofilm process from the perspective of quorum sensing regulation, providing a theoretical basis and technical pathway for developing aquaculture effluent treatment technologies based on quorum sensing regulation.