From suppression to selection: Chlorination drives biofilm adaptation and resistance in raw water distribution...

Pre-chlorination is widely applied to mitigate microbial risks in raw water distribution systems (RWDS). However, the impacts of chlorination on biofilm development and associated microbial risks under long-term and low-dose chlorine exposure remain poorly understood. In this study, a biofilm annular reactor with polyvinyl chloride (PVC) and stainless steel (SS) coupons was used to simulate RWDS, and to evaluate biofilm behavior and microbial risks in response to chlorination. We found that long-term chlorination increased extracellular polymeric substances production and led to a three-phase biofilm development. This included an early buffering phase mainly caused by cell inactivation, a middle reaction phase marked by EPS increase, and a late shedding phase linked to biofilm instability and release. Chlorination also changed the biofilm microbial community, and dominant genera shifted from common freshwater-associated taxa like Sphingomonas to more chlorine-tolerant taxa like Pseudomonas on both materials. 71 and 64 antibiotic resistance genes were detected on PVC and SS coupons, respectively. Most of them were increased on both materials after chlorination, but higher potential resistance shown on SS. In addition, 18 ARGs showed significant positive correlations with one another, and 5 pathogens showed significant associations with specific ARGs. This suggests chlorination can favor the persistence of certain ARGs, which may pose potential risks by contributing to the persistence of resistant microorganisms and genes in downstream systems. These results demonstrate that pre-chlorination can increase microbial and genetic risks in RWDS, underscoring the need to evaluate chlorination strategies and pipe materials for securing drinking water safety.