Quantitative Polymerase Chain Reaction-Based UV Biodosimetry Using Indigenous 16S rRNA Genes Enables Dose-Verified Validation of Wastewater Disinfection Reactors

Reliable validation of ultraviolet (UV) reactor performance is essential for effective wastewater disinfection. Because DNA is the primary UV target and ubiquitous in wastewater, intracellular 16S rRNA genes represent promising molecular surrogates for biodosimetry. Here, we developed and field-validated a quantitative polymerase chain reaction (qPCR)-based framework to quantify reduction equivalent dose (RED) in full-scale wastewater UV reactors without spiking external challenge organisms. Across five municipal wastewater treatment plants, a 1.5 kbp 16S rRNA gene amplicon exhibited consistent first-order degradation kinetics at UV fluences of 0–50 mJ/cm2 (R2 > 0.90), with a reproducible fluence-based rate constant (kUV,Amp) of 0.10 ± 0.01 cm2/mJ and <10% variability across sites, seasons, and biomass levels. Applying this kinetic parameter, REDs of 3–57 mJ/cm2 were quantified for full-scale reactors, revealing substantial temporal and regional variability, including periods of insufficient UV dose undetected by routine coliform monitoring. The RED variability correlated strongly with turbidity and UV transmittance, highlighting optical shielding effects. The framework also accurately predicted UV attenuation of representative intracellular antibiotic resistance genes (i-ARGs). Overall, qPCR-based monitoring of intracellular 16S rRNA degradation enables rapid (within hours) and broadly applicable dose-verified UV performance assessment for municipal wastewater.