Study of chlorine disinfection on the formation and resuscitation of Viable but nonculturable (VBNC) bacteria in drinking water distribution systems

Abstract

The presence of Viable but nonculturable (VBNC) bacteria in drinking water distribution systems(DWDS)which can recover and regain pathogenicity under certain conditions, leads to a serious underestimation of the number of microorganisms in the water and consequently microbiological risks. This study investigated E. coli's biofilm integrity and metabolic activity post‑chlorine disinfection and during resuscitation in a Circulating-flow Dynamic biofilm Contactor (CDC) reactor. Chlorine rapidly induced the VBNC state, with concentrations ≥0.3 mg/L causing all respiratory-active bacteria to transition. Higher chlorine levels damaged cell biofilms, reducing intact cell counts by 95 % and depleting ATP. VBNC bacteria in biofilms quickly regained culturability, utilizing available nutrients and ATP. Within 1–4 days, culturable bacteria in the biofilm increased from 2.29 to 4.95 log CFU/mL, significantly faster than in water. This growth corresponded with rising ATP levels, though slower than in aqueous environments. Signaling molecules (C6, C8, C12, C14) regulated gene expression during resuscitation, aiding bacterial recovery and proliferation. Chlorine disinfection disrupted intercellular communication, altering VBNC bacteria's physiological and metabolic characteristics. These findings highlight the complex dynamics of VBNC bacteria in water systems, their resuscitation potential, and the importance of biofilms in bacterial recovery, emphasizing the need for comprehensive water treatment strategies.