Resuscitation, regrowth, and release of bacteria in biofilm in a river receiving chlorinated reclaimed water

• Culturable E. coli concentration increased in chlorinated reclaimed water. • Riverbed biofilms were the predominant source of E. coli in river water. • Biofilm supported resuscitation and regrowth of E. coli. • RNA concentration was a reliable parameter for viability of E. coli. Reclaimed water provides alternative and sustainable water sources. However, the persistence of bacteria is a significant concern in the use of it. We analyzed the abundance, culturability, viability, regrowth potential, and community composition of bacteria, especially E. coli , in a river receiving chlorinated reclaimed water to reveal the mechanism underlying the increase in bacterial abundance in river water. The culturable (C-) E. coli concentration increased from 55 CFU/L to 2300 CFU/L 850 m downstream, despite the presence of chlorine. The increase rate (Rs) of E. coli in river water was 5.4 h -1 while the Rs of E. coli in biofilm was < 10 6 h -1 , indicating that the growth of C- E. coli and resuscitation of injured E. coli in biofilms were the main causes of the increase in C- E. coli in river water. Beta diversity analysis of bacteria revealed that the bacterial community in the biofilm was more diverse than that in river water. The reduction of total chlorine concentration from 0.87 mg-Cl 2 /L to 0.11 mg-Cl 2 /L led to an increase in orders Enterobacterales and Clostridiales downstream. A study on the effect of chlorine on pure cultures of E. coli showed that RNA analysis might be a better assay for bacterial viability than PCR with propidium monoazide treatment. The results obtained in this study are beneficial to water management authorities in relation to reclaimed water use for preventing increase in pathogenic bacteria downstream.