The occurrence of viable but non-culturable (VBNC) bacteria will result in significant underestimation of viable bacterial counts in drinking water. Whereas, much is unknown in characterizing their viability. In this study, two environmental isolates (Aeromonas sp. and Pseudomonas sp.) and two model strains (E. coli and S. aureus) were induced into VBNC state by UV irradiation. Then, their metabolic activity was determined by 5-cyano-2,3-ditolyl tetrazolium chloride combination flow cytometry (CTC-FCM) and D2O-labeled Raman spectroscopy, respectively, at both population and single cell levels. The results showed that almost all strains could enter VBNC state irradiated by ≥ 5 mJ/cm2 UV. When determined by CTC-FCM, the population metabolic activity for each strain did not vary significantly (p > 0.05) unless the UV dose reached 200 mJ/cm2. Their single cell activity spectrum narrowed slightly, as indicated by changes in the standard deviation of the logarithmic normal distribution (σ) of 0.015–0.033. This minute difference suggested the CTC-FCM method was suitable for assessing the essential viability of VBNC bacteria. With respect to Raman method, an obvious dose-response effect was recorded. With the UV dosages increased from 10 to 200 mJ/cm2, the CD/(CD + CH) for the four strains were reduced to between 95.7% and 47.9% of unirradiated controls, depending on strain and UV dose. Meanwhile, the single cellular Raman spectrum showed much more heterogeneously metabolic activity distribution, with some cells even entering metabolic “silence”. Considering the ubiquitous participation of water in biochemical processes, the Raman method was more appropriate in assessing the overall metabolic activity. The above findings can not only be a reference for VBNC mechanism studies, but also have the potential in optimizing disinfection and other bacterial removal processes. Keywords: Viable but non-culturable, UV disinfection, CTC-FCM, D2O-labeled Raman, Respiration activity, Metabolic activity
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