Degradation of DNA in photolysis and photo-sensitized processes: kinetics, pathways, and implications for water disinfection

Abstract

Waterborne pathogens, antibiotic resistance, and the persistence of viruses in water environments pose significant public health challenges. This dissertation aims to understand the photodegradation kinetics and mechanisms of these contaminants in engineered (UV disinfection systems) and natural (sunlit surface waters) settings, contributing to safer and more effective water disinfection strategies. The first part of this study investigates the UV photolysis kinetics of the intracellular 16S rRNA gene of indigenous bacteria in full-scale municipal wastewater treatment plants (WWTPs). The intracellular 16S rRNA gene exhibited consistent first-order degradation kinetics under UV fluences up to 50 mJ/cm², with an average rate constant of 0.08- 0.11 cm²/mJ, across all samples. In addition, even when the initial bacterial cell concentration was set 20 times higher, no change was observed in the degradation rate constant. This consistency in reactivity supports the application of intracellular 16S rRNA gene photodegradation as a practical biodosimeter for estimating UV dose delivery in WWTP reactors. Measured reduction equivalent doses (REDs) across five WWTPs ranged from 10 to 60 mJ/cm², and showed notable seasonal variability, which shows the necessity of regular monitoring of REDs. Overall, the UV dose estimation approach newly proposed in this study offers a promising on-site biodosimetry tool for WWTPs. In the second part of this study, the reactivity of extracellular antibiotic resistance genes (e-ARGs) with singlet oxygen (1O2)—a key photochemically produced reactive intermediate (PPRI) in sunlit surface waters—was quantitatively evaluated using Rose Bengal as a photosensitizer. The degradation kinetics of the ampicillin resistance gene (ampR) encoded in plasmid pUC19 exhibited a strong dependence on guanine content, indicating that guanine oxidation is the dominant degradation pathway. The second-order rate constants of pUC19 with 1O2 were determined to be 8.7 × 106 M⁻¹s⁻¹ at pH 7.0 and 1.2 × 107 M⁻¹s⁻¹ at pH 8.5, suggesting that deprotonated guanine is approximately six times more reactive with 1O2 than its protonated form. Although 1O2-induced degradation of e-ARG was confirmed, transformation assays indicated partial repair of damaged e-ARG, thereby limiting overall deactivation efficiency. Simulations using a sunlight photolysis model revealed that under natural sunlit surface water conditions, 1O2 plays a relatively minor role in e-ARG degradation. However, in natural systems where 1O2 concentrations exhibit micro-heterogeneity, its contribution may be locally enhanced, when e-ARG is close to the source of 1O2. The third part of this study explored the reactivity of triplet state dissolved organic matter (3DOM*) with e-ARGs, elucidated the underlying degradation mechanism, and assessed the contribution of 3DOM* to e-ARG sunlight photodegradation. Using both DOM proxy compounds, second-order rate constants between 3DOM* and e-ARGs were determined to range from (0.3–8.5) × 10⁹ M⁻¹s⁻¹, with values increasing proportionally with the reduction potential of the proxy, implying that electron transfer is likely the predominant degradation pathway. Notably, the reactivity of 3DOM* was found to be wavelength-dependent, leading to the derivation of a wavelength-dependent quantum yield coefficient of 3DOM*, f(λ). Incorporation of this coefficient into a sunlight photodegradation model under natural sunlit surface water conditions (pH 7) demonstrated that 3DOM* can substantially contribute to e- ARG degradation—accounting for over 90% of the total degradation at a water depth of 10 cm, and showing comparable contribution to hydroxyl radicals even at 100 cm depth. In the final part, the synergistic disinfection effects of direct and indirect inactivation pathways on human adenovirus (HAdV) were investigated. Although HAdV is highly resistant to UV due to efficient host-cell DNA repair, pre-exposure to 1O₂ sensitized the virus to subsequent UV damage, resulting in reduced repair efficiency. Experiments using cell lines with different repair capacities confirmed that combined exposure to UV and PPRIs compromised viral DNA repair. Notably, we newly identified that one of the major inactivation mechanisms of ¹O₂-treated HAdV is the reduction in DNA replication efficiency. These findings highlight a previously unrecognized mechanism by which indirect oxidative damage impairs genome repair processes, enhancing overall virus inactivation under sunlight. Overall, this dissertation provides new insights into the role of UV and PPRIs in degrading/inactivating various types of DNA and pathogenic microorganisms based on several key implications regarding UV DNA photolysis. The findings support the development of improved disinfection validation tools and predictive models for both engineered and natural water treatment systems, ultimately contributing to enhanced protection of public health. Keywords: UV disinfection, sunlight disinfection, photoproduced reactive intermediates, sunlight photodegradation/disinfection model, pathogenic bacterial DNA, extracellular antibiotic resistance genes, dsDNA virus