N-acyl-homoserine-lactones as a critical factor for biofilm formation during the initial adhesion stage in drinking water distribution systems

The N-acyl-homoserine-lactone (AHLs)-mediated quorum sensing (QS) system is crucial for the coordination of microbial behaviors within communities. However, the levels of AHLs in biofilms in drinking water distribution systems (DWDSs) and their impact on biofilm formation remain poorly understood. Herein, we simulated DWDSs via biofilm reactors to explore the presence and influence of AHLs during the initial stages of biofilm formation on pipe walls. Glass, polypropylene random copolymer (PP-R) and stainless steel (SS) were used as the coupon materials and the three parallel experimental groups were set up and named accordingly. The glass material is considered to form biofilms only minimally and is therefore used as a negative control. By day 30, the concentration of AHLs in biofilm phase in both PP-R group and SS group reached 1200-1800 ng/L. The predominant AHLs were C6-HSL, C8-HSL, and C10-HSL, with a significant positive correlation between AHLs and biofilm biomass. Metagenomic analysis revealed that microbes exhibiting significant differences among the three groups all demonstrated notable correlations with AHLs. Subsequent analysis of QS genes revealed that the genes associated with AHLs biosynthesis and QS receptors were more abundant in the PP-R and SS groups with biofilm formation. Additionally, we analyzed the abundance of genes related to cell motility, transmembrane transport, tricarboxylic acid cycle, and genetic information synthesis. The co-occurrence network indicates that these processes exhibit a strong correlation with QS genes. This study demonstrates the pivotal role of AHLs in microbial communication during the initial stages of biofilm formation in DWDSs and indicates that the regulatory pathways and mechanisms of AHLs may vary under different environmental conditions.