Induction of bacteria in biofilm into a VBNC state by chlorine and monitoring of biofilm structure changes by means of OCT

The occurrence of viable but non-culturable (VBNC) bacteria in drinking water may result in significant underestimation of viable cell counts detected by culture-based method, thus raising microbiological safety concern. Chlorine disinfection has been widely used in drinking water treatment to ensure microbiological safety. However, the effect of residual chlorine on inducing bacteria in biofilms into a VBNC state remains unclear. We determined cell numbers of Pseudomonas fluorescence in different physiological states (culturable, viable, dead) by heterotrophic plate count method and flow cytometer in a flow cell system under 0, 0.1, 0.5, 1.0 mg/L chlorine treatment. Numbers of culturable cells were 4.66 ± 0.47 Log10, 2.82 ± 0.76 Log10, 2.30 ± 1.23 Log10 (CFU/112.5 mm3) in each chlorine treatment group. However, viable cell numbers remained at 6.32 ± 0.05 Log10, 6.11 ± 0.24 Log10, 5.08 ± 0.81 Log10 (cells/112.5 mm3). Significant difference between numbers of viable and culturable cells demonstrated chlorine could induce bacteria in biofilms into a VBNC state. In this study, flow cells combination with Optical Coherence Tomography (OCT) were applied to construct an Automated experimental Platform for replicate Biofilm cultivation and structural Monitoring (APBM) system. The OCT imaging results demonstrated that changes of biofilm structure under chlorine treatment were closely related to their inherent characteristics. Biofilms with low thickness and high roughness coefficient or porosity were easier to be removed from the substratum. Biofilm with high rigid properties were more resistant to chlorine treatment. Even though >95 % bacteria in biofilms entered a VBNC state, the biofilm physical structure was still remained. This study revealed the possibility of bacteria to enter a VBNC state in drinking water biofilms and changes of biofilm structure with different characteristics under chlorine treatment, which provide reference for biofilms control in drinking water distribution systems.