Abstract

Endotoxins derived from Gram-negative bacteria are increasingly recognized as indicators of microbial lysis in drinking water systems. While many studies have examined endotoxin release from suspended bacteria, its implications for biofilm-associated processes in drinking water distribution systems remain insufficiently understood. In this study, planktonic E. coli suspensions were used to characterize disinfection induced endotoxin accumulation, and a rotating disk reactor biofilm system was employed to evaluate biofilm responses to external endotoxin exposure. Direct RDR biofilm disinfection and concentration gradient exposure were further conducted to connect disinfection-derived endotoxin release with biofilm-associated responses. Chloramine and ultraviolet (UV) disinfection were compared under controlled conditions. Measurable endotoxin accumulation in the liquid phase increased with initial bacterial concentration. When the initial concentration reached 1.2 × 10 5 CFU/mL, chloramine at 5 mg/L increased endotoxin to 3.42 EU/mL after 300 mins. Ultraviolet treatment at 360 mJ/cm produced lower detectable endotoxin levels of 1.63 EU/mL. Chloramine caused oxidative membrane damage with rapid potassium leakage, whereas UV caused DNA damage, rapid loss of culturability, and delayed membrane disruption. The concentration gradient exposure experiment showed that low EU/mL endotoxin caused limited short-term HPC changes, whereas clearer responses occurred under medium and high load exposure. Under the 500 EU/mL high load challenge, biofilm alpha diversity decreased by more than 35 %. Community composition also shifted, with decreased Bacillus and enrichment of Gram-negative genera including Acidovorax and Caulobacter . Overall, chloramine caused continuous endotoxin release, while UV showed lower detectable endotoxin levels but did not eliminate endotoxin activity.