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A viable but non-culturable (VBNC) state of bacteria induced by disinfection in water treatment poses serious health risks because of possible resuscitation of VBNC cells during transportation. In this study, a setup using continuous-flow ultraviolet (UVC) irradiation ranging from 0 to 172.2 mJ cm -2  was designed to simulate real-world disinfection in both drinking water (SDW) and reclaimed water (SRW) treatment plants. A systematic investigation of UVC-induced VBNC bacteria, including occurrence, resuscitation, and time-dependent recovery of metabolic activity during post-incubation, was conducted. Different techniques including two new ones of “single cell culture” and D 2 O-labeled single-cell Raman spectroscopy were employed to gain comprehensive insights into VBNC cells. Heterotrophic plate counts (HPC) and 5-cyano-2,3-ditoyl tetrazolium chloride flow cytometry (CTC-FCM) assay demonstrated that exposure to continuous-flow UVC can induce  E. coli  into a VBNC state. Membranes integrity and 16S rRNA transcription level of VBNC bacteria were demonstrated to be unaffected by UVC exposure even at a high dose of 172.2 mJ cm -2 . Resuscitation of VBNC bacteria was identified in a more accurate way based on “single cell culture.” Finally, time-dependent evolution of metabolic activity of UVC-treated cells during post-incubation was examined by D 2 O-labeled Raman spectroscopy at a high-resolution of single-cell level. C-D Raman bands resulting from incorporation of D 2 O-derived D into bacterial biomass were used as a sensitive and quantitative indicator of bacterial metabolic activity. A lower UVC dose, longer post-incubation time, and higher initial number of bacteria were demonstrated to result in a faster recovery of metabolic activity. Heterogeneous metabolic activity and subpopulation with higher metabolic activity were also revealed by single-cell Raman, even for UVC-treated cells losing cultivability. The comprehensive assessment of VBNC bacteria in UVC-disinfected drinking and reclaimed water points out treatment deficiencies of UVC and the necessity to develop more effective strategies to eliminate VBNC cells.

Induction of Escherichia coli Into a VBNC State by Continuous-Flow UVC and Subsequent Changes in Metabolic Activity at the Single-Cell Level