Abstract
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 D2O-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 D2O-labeled Raman spectroscopy at a high-resolution of single-cell level. C-D Raman bands resulting from incorporation of D2O-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.
Highlights
Viable but non-culturable (VBNC) state are bacteria that are living but unable to form colonies when cultured in bacteriological media, but capable of resuscitation under precise stimuli such as nutrients and temperature (Oliver, 2000; Moreno et al, 2007)
Presence of VBNC bacteria in public health-related fields poses a great risk to human health, because their occurrence underestimates the number of total viable bacteria and associated health risk, in addition, their ability to resuscitate endangers human life
UVC disinfection is widely used for water treatment because it is more effective against cysts of Cryptosporidium and Giardia than chlorination and ozonation without producing disinfection byproducts (Belosevic et al, 2001)
Summary
Viable but non-culturable (VBNC) state are bacteria that are living but unable to form colonies when cultured in bacteriological media, but capable of resuscitation under precise stimuli such as nutrients and temperature (Oliver, 2000; Moreno et al, 2007). These advantages indicate great potential of single-cell D2Olabeled Raman in studying heterogeneous metabolic activity of VBNC bacteria during post-incubation. Physiology and metabolism of UVCinduced VBNC state of bacteria were characterized by different methods including HPC (culturability), CTC-FCM (respiratory activity), PMA-qPCR (membrane integrity), and RT-qPCR (gene expression).
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