ProphageActivation as an Overlooked Mechanism Underlyingthe Biocidal Effect of Free Nitrous Acid in Sewers

Abstract Biogenic hydrogen sulfide produced in sewer systems causes odor nuisance and concrete corrosion, necessitating effective biocidal control. Free nitrous acid (FNA) has emerged as a promising biocide, but its unclear mechanisms complicate dosage optimization and risk assessment. Here, using Desulfovibrio vulgaris as a model lysogenic bacterium, we demonstrate that low-dose FNA (0.2–4.0 mg N/L) induces bacterial inactivation via prophage activation-associated lysis in addition to chemical oxidation. Reactive nitrogen species (RNS) scavenging tests revealed that RNS-mediated oxidative stress was closely associated with prophage activation. Activated phages further infected new hosts, reducing the viability of freshly cultured D. vulgaris cells by 25.7% and increasing total phage production 15.2-fold. The phenomenon was further validated in real sewage biofilms, where increased phage production and decreased bacterial viability were observed at a low FNA dose of 0.2 mg N/L, below the dose required for cell destruction by chemical oxidation. Furthermore, metagenomic analysis of 896 sewage samples worldwide revealed that 71.9% of recovered sewage-derived microbial genomes harbor prophages, indicating a widespread genomic basis for prophage activation-mediated bacterial inactivation. Overall, this study expands current understanding of the biocidal mechanisms of FNA and contributes to the development of environmentally sustainable biocidal strategies.