Employing bacterial mutations for the elucidation of photo-Fenton disinfection: Focus on the intracellular and extracellular inactivation mechanisms induced by UVA and H₂O₂

The bacterial inactivation mechanisms by solar light and the photo-Fenton process is still a matter of debate. In this study, we bring evidence towards the elucidation of the mechanisms that govern photo-Fenton disinfection at near-neutral pH. With the use of porin-deficient and catalase over-producing E. coli strains, in conjunction with measurements of cell wall oxidation and permeability, we are able to i) highlight the role of the aforementioned components in bacterial inactivation and ii) localize the damages in the intracellular domain, despite the addition of the Fenton reagents in the bulk. We report that H₂O₂ oxidizes cell walls but under light the process is of low significance; UVA initiated an intracellular oxidation process based on excess accumulated H₂O₂, while the UVA+H₂O₂ and UVA+H₂O₂+Fe²⁺ processes have the same effect with light, albeit enhanced, as shown by malondialdehyde (MDA) production and ONPG hydrolysis rates. Finally, compared to the UVA-assisted photo-Fenton process, its solar counterpart is enhanced by the direct UVB effects on bacterial DNA. In conclusion, we have sufficient evidence to postulate that the photo-Fenton process is intracellular and propose the pathways that form the integrated bacterial inactivation mechanism by photo-Fenton.