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Identifying the mediators of intracellular E. coli inactivation under UVA light: The (photo) Fenton process and singlet oxygen

Stefanos Giannakis, César Pulgarin

Solar disinfection (SODIS) was probed for its underlying mechanism. When Escherichia coli was exposed to UVA irradiation, the dominant solar fraction acting in SODIS process, cells exhibited a shoulder before death ensued. This profile resembles cell killing by hydrogen peroxide (H2O2). Indeed, the use of specialized strains revealed that UVA exposure triggers intracellular H2O2 formation. The resultant H2O2 stress was especially impactful because UVA also inactivated the processes that degrade H2O2 & mdash;peroxidases through the suppression of metabolism, and catalases through direct enzyme damage. Cell killing was enhanced when water was replaced with D2O, suggesting that singlet oxygen plays a role, possibly as a precursor to H2O2 and/or as the mediator of catalase damage. UVA was especially toxic to mutants lacking miniferritin (dps) or recombinational DNA repair (recA) enzymes, indicating that reactions between ferrous iron and UVA-generated H2O2 lead to lethal DNA damage. Importantly, experiments showed that the intracellular accumulation of H2O2 alone is insufficient to kill cells; therefore, UVA must do something more to enable death. A possibility is that UVA stimulates the reduction of intracellular ferric iron to its ferrous form, either by stimulating O-2(center dot-) formation or by generating photoexcited electron donors. These observations and methods open the door to follow-up experiments that can probe the mechanisms of H2O2 formation, catalase inactivation, and iron reduction. Of immediate utility, the data highlight the intracellular pathways formed under UVA light during SODIS, and that the presence of micromolar iron accelerates the rate at which radiation disinfects water.

PERGAMON-ELSEVIER SCIENCE LTD2022

Mechanistic modelling of solar disinfection (SODIS) kinetics of Escherichia coli, enhanced with H2O2 - part 1: The dark side of peroxide

Ling Feng, Stefanos Giannakis, César Pulgarin

The present bi-partite work describes the development and validation of a mechanistic kinetic model of SODIS E. coli inactivation, enhanced with H2O2. In this first part, the mechanism of the baseline dark phenomena is modelled. A mechanistic model involving E. coli cellular respiration, inactivation due to HO center dot and O-2(center dot-) radicals, and bacterial thermal inactivation, was developed using a series-event model based on the accumulation of damage and cell recovery corrected with the Arrhenius equation for inclusion of the thermal events. The contribution of external H(2)O(2)was included in the internal H2O2 balance, while the balance of extracellular H2O2 considered the consumption caused by its self-decomposition, interactions with cells' membrane, and organic matter from dead cells. Specifically, the kinetic parameters of the external H2O2 sinks, the oxidation reaction of intracellular Fenton, and bacterial thermal inactivation were independently estimated by model regression from experimental data of E. coli inactivation and H2O2 consumption at different controlled conditions of temperature and initial H(2)O(2 )concentration. We complemented the values of the kinetic constants available in the literature with the unknown kinetic parameters estimated from experiemetnal and literature data. The missing kinetic parameters were successfully validated (bacteria error = 4.5%, H2O2 error = 12.9%). This kinetic model helps to understand the intracellular mechanisms and the contributions of each source of inactivation, with the role of radicals' damage being most important at temperatures below 40 degrees C, and the thermal inactivation for temperatures above this value.

ELSEVIER SCIENCE SA2022

Mechanistic modelling of solar disinfection (SODIS) kinetics of Escherichia coli, enhanced with H2O2 - Part 2: Shine on you, crazy peroxide

Ling Feng, Stefanos Giannakis, César Pulgarin

In this second part of the development of a mechanistic kinetic model of the solar inactivation of E. coli enhanced with hydrogen peroxide, we evaluate the mechanisms based on photonic inactivation and integrate them into the kinetic model of the dark process developed in Part 1. The direct photonic inactivation was modelled using a series-event model based on the accumulation of damage by photons and it was coupled with the model used in Part 1 for modelling the damage caused by radicals using a multiple target- multiple hit model, including recovery constant to define the ability of cells to face the specific photonic damage. Catalase and superoxide dismutase inactivation, the intracellular photo-Fenton reaction, and the overproduction of O-2(center dot-) in the NADH/NAD(+) cycle under solar light were included in the model. Finally, the synergistic effect of the photonic damage with thermal inactivation was included in the kinetic constant of the series-event expression in terms of an Arrhenius equation. The kinetic parameters were obtained by model regression using experimental data at different temperatures, solar radiation, as well as initial cellular and H2O2 concentrations. Our model predictions can accurately describe the experimental data of the SODIS process enhanced with H2O2, thus being very useful to estimate disinfection profiles and inactivation routes at different irradiance conditions, water temperature and H2O2 concentration. Finally, an integrated mechanism of E. roll inactivation under the SODIS/H2O2 process is provided.

ELSEVIER SCIENCE SA2022