Journal
FRONTIERS IN MICROBIOLOGY
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2018.02243
Keywords
continuous-flow UVC; VBNC; induction; resuscitation; metabolic activity
Categories
Funding
- Natural Science Foundation of China [51678552, 21777154]
- Science and Technology Project of Fujian Province [2016Y0082]
- Science and Technology Project of Xiamen [3502Z20162003]
- NERC [NE/M002934/1] Funding Source: UKRI
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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 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.
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