From Contamination to Restoration: Microbial Dynamics After Rainwater Backflow into a Pilot-Scale Drinking Water Distribution System.

Abstract

Rainwater backflow caused by system cross-connection into drinking water distribution systems (DWDS), resulting from pipe breaks, leaks, manual installation errors, or misconnections associated with third-pipe systems for rainwater and greywater reuse, can disrupt biostability of the DWDS by introducing microorganisms and nutrients. Using a pilot-scale DWDS with a mature biofilm, this study simulated a conservative worst-case backflow scenario by supplying a 1:1 mixture of rainwater and tap water for two consecutive weeks in recirculation mode, followed by recovery through replacement with uncontaminated tap water without chemical disinfection or hydraulic flushing. This experiment was conducted twice under identical conditions. Microbial abundance, activity, and community composition in bulk water and biofilm were assessed using culture-based methods, total organic carbon (TOC), ATP, online flow cytometry, 16S rRNA gene amplicon sequencing, and continuous by Orbs in-line probe that measures fluorescence-based bio-load. Rainwater backflow caused immediate impact with a one log increase in total cell concentrations, a two-fold increase in TOC values, peaks in ATP concentrations of 75.71 ng/L compared to 3.50 ng/L (experiment I) and 0.913 ng/L (experiment II) for regular tap water, alongside pronounced shifts in bulk water community composition, demonstrating the duration and scale of the backflow event. Rainwater-associated taxa were temporarily detected in the bulk and biofilm, even after 100% tap water was supplied, but remained at low relative abundances. The culture-based methods (CCA, PCN, ADA) demonstrated that regulatory limits regarding were exceeded during and after the backflow, reflecting the delay in compliance after a backflow event. These results highlight the resilience of biostable DWDS to short-term rainwater backflow and demonstrate the value of high-frequency and high-resolution microbial monitoring for rapid detection and management of contamination event.