222 nm causes greater protein damage and repair inhibition of E. coli than 254 nm for water disinfection

David McDonald; Daniel Ma; Natalie M. Hull

doi:10.1371/journal.pwat.0000238

Abstract

Germicidal ultraviolet (UV) light has been widely used to inactivate pathogens in water. Emerging alternatives to conventional low pressure (LP) mercury lamps emitting at 254 nm, such as krypton chloride (KrCl) excimer lamps emitting at 222 nm, are gaining acceptance and popularity due to advantages in human safety and disinfection mechanisms. Cyclobutane pyrimidine dimer (CPD) formation kinetics and photolyase damage kinetics were quantified in E. coli for 222 nm and 254 nm UV. Molecular damage and cell regrowth were also quantified after UV irradiation under photorepair and dark repair incubation conditions using a standardized photorepair fluence response protocol. CPDs and photolyase were measured using enzyme linked immunosorbent assays (ELISA). A novel ELISA for photolyase was developed for this study. Culture-based log inactivation UV fluence responses were similar for 254 nm and 222 nm, with Geeraerd model estimates for rate constants of 1.18±0.09 and 1.24±0.08 cm² mJ⁻¹ for LP and KrCl lamps, respectively. Molecular UV fluence kinetics showed that the rate of CPD formation was greater by LP, but the rate of photolyase damage was greater by KrCl, as supported by the intercepts of repair kinetics. Compared to LP irradiated samples, KrCl irradiated samples exhibited less repair overall. For a given lamp, similar repair was observed between light and dark repair incubations. Percent reactivation rates with respect to photorepair fluence were (3.7±1.4)×10⁻⁵ and (–1.3±2.5)×10⁻⁵ cm² mJ^-1 for LP and KrCl lamps, respectively. CPDs decreased at a higher rate during repair incubations in LP samples than KrCl samples, and photolyase concentration increased in LP samples but decreased in KrCl samples. The results quantify contributions of photolyase protein damage to disinfection and repair prevention mechanism of KrCl lamps. This study mechanistically demonstrates why KrCl lamps can be applied for UV water disinfection to limit photorepair after treatment.

Synopsis: This study used a novel photolyase assay to demonstrate photolyase damage inflicted by krypton chloride excimer lamps contributes to disinfection of bacteria to prevent bacterial photorepair of damaged DNA and regrowth in drinking water treatment.

Citation: McDonald D, Ma D, Hull NM (2024) 222 nm causes greater protein damage and repair inhibition of E. coli than 254 nm for water disinfection. PLOS Water 3(7): e0000238. https://doi.org/10.1371/journal.pwat.0000238

Editor: Ilunga Kamika, University of South Africa, SOUTH AFRICA

Received: March 12, 2024; Accepted: June 18, 2024; Published: July 24, 2024

Copyright: © 2024 McDonald et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Relevant data is available on figshare via the following DOI: 10.6084/m9.figshare.25376107.

Funding: DM was funded by The Ohio State University College of Engineering Undergraduate Research Scholarship and by the Department of Civil, Environmental, and Geodetic Engineering Summer Undergraduate Research Fellowship. DM was supported by a University Fellowship from The Ohio State University and Dr. Natalie Hull’s lab start up fund from The Ohio State University Department of Civil, Environmental and Geodetic Engineering and The Sustainability Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

UV light inactivates many kinds of microorganisms and is often used in drinking water treatment to target pathogens that are resistant to chemical disinfection [1]. Unlike chemical disinfectants, UV does not typically create harmful disinfection byproducts or select for chemical-resistant bacteria [2]. Combining disinfectants in multi-barrier solutions adds redundancy and multi-mechanism disinfection in treatment trains. Monochromatic low pressure (LP) and polychromatic medium pressure (MP) mercury lamps are used in UV treatment systems [3]. Krypton-chloride (KrCl) excimer lamps are emerging, mercury-free UV sources with a peak emission of 222 nm [4–6].

Previous studies have shown that different UV sources produce wavelength-dependent disinfection kinetics for various biological targets [7, 8]. LP lamps emitting 254 nm primarily induce DNA damage by forming cyclobutane pyrimidine dimers (CPDs) [9, 10]. LP lamps form more CPDs in bacterial DNA than far UVC light [11]. Far-UVC light sources, such as KrCl lamps with peak wavelength of 222 nm, can damage proteins [12] at wavelengths <240 nm due to peptide bond absorbance [6, 13, 14]. UV absorption of nucleic acids is comparable for 222 nm and 254 nm, but that of proteins at 222 nm is ~20 fold greater [15–17], suggesting potentially higher inactivation efficiency of far-UVC compared with 254 nm. Compared to 254 nm radiation, 222 nm radiation damages enzymes in vitro more effectively than 254-nm light and inflicts greater damage to proteins in viruses and microorganisms [17, 18]. In addition to protein damage, 222 nm may also result in synergistic disinfection mechanisms and decreased photorepair caused by lipid peroxidation [19]. Some studies have shown that KrCl lamps achieve similar or higher rate of damage of DNA than that achieved by LP lamps [5, 19–21]. However, other studies have indicated that LP lamps require lower fluences (6.9 mJ/cm²) to achieve 4-log inactivation of E. coli ATCC 11229 than for KrCl lamps (10.6 mJ/cm²) [22]. Moreover, LP lamps also greater disinfection at low fluences and can cause more DNA mutations than KrCl [23, 24]. Additionally, 222 nm disinfection efficiency of several bacteria was slightly lower than by 254 nm, respectively [4]. Another study demonstrated that enterohaemorrhagic E. coli was inactivated by at least 4-log by 6 mJ/cm² of 222 nm and 254 nm, but required a UV dose of 24 mJ/cm² by 222 nm to undetectable levels [25]. 222 nm disinfection of several viruses, which are typically more resistant to UV than bacteria, was higher by 222 nm than by 254 nm [4]. Disinfection of 222 nm disinfection was shown to have superior reduction of E. coli DH5α than 254 nm when applied in-situ via electrodes for real-water disinfection [5]. Due to the differences in reports about the effectiveness of 222 nm and 254 nm, more investigation is needed on mechanisms and potential benefits of 222 nm for designing water disinfection systems.

A well-known limitation of UV disinfection is the lack of residual required for maintaining microbial water quality post-treatment. Two potential risks after UV disinfection include the regrowth of bacteria from survivor and resistant populations, and DNA repair followed by reactivation of microorganisms [26–32]. Many microorganisms are capable of light-independent DNA repair through nucleotide excision repair, requiring multiple enzymatic reactions [8, 33]. Some bacteria possess the photolyase enzyme which catalyzes the reaction to cleave CPDs in a process called photorepair, or light repair [34]. The photolysis reaction is activated by UV and visible light with wavelengths in the range of 300–500 nm [35].

Photorepair and reactivation in bacteria after UV treatment by various UV sources, including LP lamps, MP lamps, UV light emitting diodes (LEDs), and excimer lamps is a well-documented phenomenon in the literature [8, 22, 29, 36–38]. E. coli reactivates less after exposure to a MP lamp than a LP lamp, likely due to exposure to polychromatic UV and damage to repair proteins [32, 39–43]. After 4-log inactivation, 24 h photorepair concentrations of E. coli ATCC 11229 calculated using endpoint kinetics were lower when bacteria were inactivated by KrCl disinfection than for LP disinfection [22]. Photorepair in bacteria may peak in less than 30 minutes in bench-scale experiments [38, 44], so endpoint measurements (e.g., 24 h) may not provide sufficient information about repair immediately following UV treatment. E. coli K12 was selected for this experiment because it has previously been studied for its kinetics during LP and UV-LED disinfection [4, 44–46], but not yet for KrCl disinfection and photoreactivation kinetics.

The objective of this research was to compare inactivation kinetics of LP and KrCl lamps and evaluate the wavelength-dependence of molecular mechanisms in photorepair and dark repair kinetics of E. coli K-12. To enhance translatability and reproducibility of results, UV fluence and time-based and fluence-based photorepair kinetics were calculated using standardized protocols [38, 47]. To the authors’ best knowledge, time-based photorepair kinetics for E. coli K-12 have been determined [22, 32, 44], but photorepair fluence kinetics have not yet been determined following KrCl disinfection. It is hypothesized that differences in inactivation mechanisms between 222 nm and 254 nm will account for different photorepair kinetics, specifically that greater photolyase protein damage inflicted by 222 nm will prevent CPD repair. In addition to using standard culture-based enumerations to quantify inactivation and repair kinetics, this study also used molecular methods, including a novel photolyase ELISA developed here, to determine if the hypothesized increase in photolyase damage by KrCl versus LP lamps can inhibit repair of CPDs and suppress reactivation.

2. Methods

2.1 Bacteria preparation and enumeration

Exponential phase E. coli K12 (ATCC #29425) were cultured in Nutrient Broth (Difco BD) for 4 h (180 rpm, 37°C). Cultures were grown from cryo-stocks stored with 25% (w/v) glycerol (Fisher Chemical, G33) and Bacto tryptic soy broth (BD, 211825) at −80°C. Cells were pelleted by centrifugation (10 min, 2,823 × g rcf) and the supernatant discarded. Cells were washed with sterile phosphate buffered saline (PBS), pelleted again, and resuspended in sterile PBS to prepare working stock solutions with target initial concentration of ~10⁷ CFU/mL. The working stock solution was sonicated for 30 s (Branson 2510 Sonicator) before UV exposures [48]. Samples from before and after UV and photorepair exposures were diluted in PBS and spot plated on nutrient broth agar plates made with 8 g/L Difco nutrient broth and 15 g/L Bacto agar (BD PN 214010). After incubation for 12 to 16 hours at 37°C, colony forming units were counted for each dilution.

2.2 UV disinfection experiments

UV exposures were performed with a LP mercury lamp (COOSPIDER, 6W) and a KrCl excimer lamp (USHIO) with peak wavelengths of 254 nm and 222 nm, respectively. The output spectra of the lamps were measured with an HDX Spectroradiometer (Ocean Insight, FL) and are shown in Fig 1A. For E. coli exposures, the lamps were housed in boxes with collimating apertures and a shutter above the sample. The fluence rates emitted from the UV lamps and Petri factor (PF), divergence factor (DF), water factor (WF), and reflection factor (RF) were determined as previously described [49, 50]. An ILT 5000 radiometer with SED 240W detector (International Light Technologies, Inc., MA) was used measure the incident irradiance at the center of sample surface and at 0.5-cm intervals across the length and width of the petri dish. Absorbance scans from 200 to 400 nm were obtained with a 1-cm quartz cuvette (Vernier) with an Agilent Cary 4000 UV-VIS spectrophotometer. The incident fluence rates for all exposures with the LP lamp were within 0.179±0.040 mW/cm² and for the KrCl were within 0.122±0.066 mW/cm². For culture-based disinfection kinetics, three biological replicates were performed. For molecular damage kinetics, two biological replicates were performed.

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Fig 1.

Relative lamp emission of (a) LP and KrCl UV lamps and (b) the fluorescent lamp used for photorepair light exposures.

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UV exposures were performed on 30 to 40 mL of E. coli suspensions in 100-mm polystyrene petri dishes. The larger volume was used for UV exposures performed for repair experiments. Samples were irradiated for calculated exposure times for pre-determined UV fluences. To achieve similar incident irradiances between UV lamps, samples were placed at 40 cm and 22 to 25 cm away from the LP and KrCl lamps, respectively. Samples were continuously stirred by a miniature stir bar (SP Bel-Art, F37121-0006) during exposure. After exposure, 0.11 to 1.1 mL of samples were taken for enumeration by plating and 1.5 mL was stored at −80°C for molecular analyses. All UV and repair experiments were performed in a closed, dark room with minimal red-light lamps for visibility. After UV irradiations, exposure to any unintentional photorepair light was prevented until incubation or cryo-storage.

2.3. Photorepair and dark repair experiments

For photorepair experiments, UV fluences of 12 mJ/cm² were targeted for initial disinfection. A UV fluence of 12 mJ/cm² was chosen to achieve high levels of inactivation without completely inactivating all microorganisms. The actual delivered UV fluences were 13.0±0.8 and 12.7±0.2 mJ/cm² for 254 nm and 222 nm, respectively. The minimal differences in UV fluences (<1 mJ/cm²) contributed to some biological variations in culture-based data.

After UV disinfection, the samples were divided into two 60-mm polystyrene petri dishes (VWR, 25384–092), with 20 mL for the photorepair incubation. One was placed in a box that was completely shielded from light to account for light-independent repair processes (“dark repair”) and the other in a box under a fluorescent lamp to measure photorepair processes (“light repair”). Light repair exposures were performed using a quasi-collimated beam set up with an 18-in., 15 W, daylight fluorescent lamp (Eiko F15T8/D) suspended over a stir plate and Petri dish. The lamp had multiple peaks in the photorepair range (300 to 500 nm), with the largest peak at 436 nm (Fig 1B). The sample surface was placed 12 to 13 cm below the lamp with continuous stirring. 0.2 mL-samples were taken periodically for plating enumeration to determine cell concentration and 1.5-mL samples were stored at −80°C for later DNA and protein analysis. Light and dark experiments were all completed with at least 3 biological replicates.

The photorepair fluence rate was calculated similarly to UV fluence rate and as previously described [38]. Disinfection and photorepair exposures were performed as previously described [51, 52]. The incident irradiance of the lamp was measured from 320 to 475 nm. The absorbance of the sample between 300 and 500 nm was used to determine the water factor. The RF was 0.9786 for these exposures, corresponding to the 436-nm peak of the fluorescent lamp output. The WF and DF were adjusted as the solution depth decreased throughout the exposure due to sampling. The output of the lamp was weighted by the relative absorbance spectrum of E. coli DNA photolyase from 300 to 500 nm [35]. Photorepair fluence rates were within 0.0323±0.003 mW/cm² for all exposures.

2.4 Culture-based inactivation and photoreactivation

Log inactivation was calculated based on the concentrations before (N₀) and after (N) UV exposure (Eq 1). Log repair and percent reactivation [53] were calculated with Eqs (2) and (3), respectively, where N_t was the concentration at time t during repair incubations: Eq (1) Eq (2) Eq (3)

The Geeraerd shoulder + tail model was fitted to the log inactivation data and accounts for a shoulder at low UV fluences and a tail at high UV fluences [54]. The model, originally designed to model bacteria concentration against UV fluence, was adapted to model log inactivation results (normalized to initial concentration, N₀) against UV fluence, as shown in Eq 4. The model has four parameters: inactivation rate (k_max), normalized resistant population concentration (log₁₀[N_res/N₀]), an initial concentration parameter (N_i), and shoulder length (Sl). The Geerard shoulder + tail model initial concentration parameter, N₀, becomes log₁₀[N₀/N₀] (after a transformation necessary for modeling purposes), representing the logarithmic ratio of actual initial concentration for a given experiment to the modeled initial concentration across experiments. The parameter is nearly zero and has been renamed to N_i for clarity. The equation below shows how normalized concentration after a UV exposure (log₁₀[N_i]) is modeled as a function of time (t) when fluence rate is constant, where N_res is the resistant population, N₀ is the initial population, and N is the population of the sample after UV irradiation. The GinaFiT software was used in Microsoft Excel to calculate the inactivation model parameters for each lamp [55].

Eq (4)

2.5. DNA extraction and analyses

DNA was extracted from 500 μL of E. coli samples using the phenol–chloroform method, as previously described [56]. Additional details of DNA extraction are provided in S1 Text. DNA concentrations were measured using a Qubit 4 Fluorometer (Invitrogen) and 1X dsDNA High Sensitivity (HS) assay kit (Invitrogen, Q33230). 20 μL of DNA extract was added to 180 μL of 1X dsDNA HS working solution. CPD-DNA was quantified with an ELISA kit and a 6-point standard curve (11) prepared according to manufacturer instructions (Cell Biolabs, STA-322). Isolated DNA samples were denatured into single-stranded DNA (heat block for 10 min at 95°C followed immediately by 10 min on ice) and then added undiluted to the wells of the microplate with DNA binding solution. After an overnight incubation at room temperature, blocking solution was added. Primary antibody (anti-CPD) was diluted 1:1000 before addition. A secondary antibody (HRP conjugated) was also diluted 1:1000 and added to the wells. Finally, substrate solution was added, followed by stop solution 2 to 30 minutes after the substrate. A BioTek Synergy HTX microplate reader measured the absorbance of the standards and samples at 450 nm. DNA concentrations of repair samples are reported in S2 Fig.

2.6. Protein extraction and analyses

An enzymatic lysis method was used to extract protein from cells. 1 mL of frozen E. coli samples was thawed and pelleted in a Sorvall Legend Micro 21 centrifuge (10 min at 10,000 × g rcf) and the supernatant was discarded. The cells were resuspended in 300 μL of a solution previously described [57], which contained 50 mM Tris-HCl (pH 7) (Fisher Bioreagents, BP152; Fisher Chemical, SA48), 100 mM NaCl, 25 mM MgCl₂ (Amresco, J364), and 1 mM phenylmethylsulfonyl fluoride (PMSF) (RPI, P20270). 0.1 mg/mL lysozyme solution was prepared from lysozyme powder (Sigma, L6876). 1.6 μL of the lysozyme solution was added to the tubes, which were then incubated at 4°C for 4 hours. The tubes were spun in the microcentrifuge to remove debris (30 min at 16,000 × g rcf, 4°C) and the supernatant was transferred to new tubes and stored at −80°.

A Quick Start Bradford Protein Assay Kit (Bio-Rad, 5000201) was used to calculate protein concentration in protein extracts. A micro-assay was performed on 96-well Greiner CELLSTAR microplates (PN 655184). Standards were prepared with 2 mg/mL BSA standards diluted in the solution of Tris-HCl, NaCl, MgCl₂, and PMSF described above. 150 μL of 1X Bradford dye reagent and 150 μL of protein samples or standards were added to wells. The absorbance of the wells at 595 nm were measured by a Biotek Gen5 microplate reader. The absorbances of samples were compared to those of standards to calculate protein concentration. Concentrations of repair samples are summarized in S2 Fig.

2.7. Photolyase enzyme quantification

Photolyase was quantified with a new ELISA targeting the E. coli photolyase enzyme, which was developed for this study using commercially available antibodies and consumables. Immulon 4 HBX microliter plates (Thermo Scientific, 3855) were prepared with 50 μL of 1X ELISA Coating Buffer (BioLegend, 655184) and soaked for 15 minutes. Photolyase standard curves (S1 Fig) were quantified from solutions prepared from stock 0.4 mg/mL E. coli DNA photolyase (BioAcademia, Inc., 01–013) and diluted with the solution of Tris-HCl, NaCl, MgCl₂, and PMSF described above. 50 μL of standards or isolated protein solutions was added with the coating buffer and mixed by pipette. The plates were incubated for 10 to 16 hours at 4°C on an orbital shaker at 110 rpm. After the overnight incubation, the plates were drained and washed twice with 200 μL 1X PBS. 200 μL of assay diluent (Cell Biolabs, 310804) was added to block the wells and the plate was incubated for 1 hour at 25°C. The plate was then drained and 100 μL of primary antibody (polyclonal rabbit anti-E. coli DNA photolyase, BioAcademia, Inc., 61–013) diluted 1:300 in assay diluent to a concentration of 6.7 μg/mL was added to each well. The plate was incubated for 1 h (25°C, 110 rpm), drained, and wells were washed five times with 250 μL of ELISA Wash Buffer (Cell Biolabs, 310806). 100 μL of secondary antibody (HRP conjuated goat anti-rabbit antibody, Millipore, 12–348) diluted 1:1000 in assay diluent to a concentration of 1 μg/mL was added to the wells and the plate was incubated (1 hour, 25°C, 110 rpm). The contents were drained, and the wells were again washed five times with wash buffer. 100 μL of substrate solution (Cell Biolabs, 310807) was added to the wells. The plate was incubated at 25°C, shaking at 110 rpm for 2 to 30 minutes until color appeared in the wells. Then 100 μL of ELISA stop solution (Invitrogen, SS03) was added to the wells. A BioTek Synergy HTX microplate reader measured the absorbance of the standards and samples at 450 nm.

2.8. Analyses of molecular assay results

CPD-DNA and photolyase concentrations from ELISA assays were calculated from standard curves (S1 Fig). ELISA results were normalized by dividing the measured target (CPD-DNA or photolyase; ng/mL) concentrations by initial cell concentration (CFU/mL) of the working stock solution before UV exposure and repair to account for different initial cell concentrations between experiments, resulting in units of ng target per 10⁷ CFU. The log change in concentration for each experiment and biomolecule was also calculated with Eq (5), where C₀ is the initial concentration of the sample before UV irradiation (for UV fluence responses) or before repair (for light and dark repair experiments) and C is the concentration after repair incubations: Eq (5)

2.9. Statistical analyses

Linear regressions were fitted to culture and molecular data. Intercepts were forced through zero for data normalized to initial conditions (e.g., log inactivation, log repair, percent reactivation, and log change) and for fluence based CPD formation because there was no initial CPD DNA, but not for data normalized to total DNA or total protein concentrations or per 10⁷ CFU/mL. Linear regressions for statistical analyses were performed in R Studio (Base R, Version 4.3.1). Throughout, regressions are shown with 95% confidence interval shading and R squared values, and regression parameter estimates are shown with their standard errors and p values.

3. Results and discussion

3.1. Culture-based inactivation kinetics

Culture-based UV fluence responses for E. coli K-12 observed in this study exhibited similar shouldering, log-linear, and tailing regions for both LP and KrCl lamps (Fig 2A), with Geeraerd model parameter estimates summarized in S1 Table. The LP and KrCl lamps both achieved 4-log reduction around 13.6 mJ/cm², as estimated by Geeraerd models. By comparison, 4-log inactivation of ATCC 11229 was achieved at 7 and 11 mJ/cm², respectively, for LP and KrCl [22]. A previous study showed that E. coli ATCC 11229 was more sensitive to LP than KrCl [22], while in this study E. coli K-12 was shown to have similar sensitivities to LP and KrCl. Disinfection kinetics of E. coli K-12 agree with previous results for 222 nm [4] and 254 nm [48]. Previously, 4-log inactivation of E. coli K-12 was achieved with 16–20 mJ/cm² by LP [46, 58], whereas here 4-log inactivation was achieved with 13.6 mJ/cm² in this study. At a higher UV fluence of 40 mJ/cm², 6-log inactivation was observed compared with 5.06 log [59]. Differences in cell preparation, namely multiple washes (i.e., number of centrifuging and pelleting steps) could contribute to cell clumping or aggregation that could lead to decreased UV penetration [60].

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Fig 2.

Culture-based UV fluence–responses for 254 nm LP mercury lamps (blue) and 222 nm KrCl excimer lamps (red) are shown for log inactivation fitted to the Geeraerd model with shouldering and tailing (a). All culture-based UV fluence response data are shown for triplicate experiments, except for fluences of 15 (duplicates) and 16 mJ/cm2 (no replicates) for 222 nm. Slopes for percent reactivation of E. coli as a function of repair time (b) are shown for light and dark repair. Slopes for percent reactivation as a function of photorepair fluence (c) following irradiation by LP and KrCl are shown for light repair.

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Both lamps achieved similar disinfection kinetics, with shouldering around fluences 6–7 mJ/cm² and tailing at fluences >20 mJ/cm² (Fig 2A). The maximum inactivation rates (k_max) for the LP and KrCl lamps were 1.18±0.09 and 1.24±0.08 cm² mJ⁻¹, respectively, and within one standard error of each other. The resistant population log₁₀(N_res/N₀) was slightly smaller (by only 0.36 log₁₀) after LP disinfection than KrCl (Fig 2A and S1 Table). The shoulder lengths (Sl) and the inactivation rates (k_max) of each lamp were estimated to be within a standard error of each other (S1 Table). However, shouldering for E. coli K-12 was not observed in other studies [46, 58]. Another cause of difference may be attributed to initial concentrations in previous studies (~10⁶ CFU/mL) lower than the concentration used in this study (~10⁷ CFU/mL), which can result in slower rate of disinfection [61].

3.2. Culture-based reactivation kinetics

Different light and dark repair kinetics were observed based on UV source. Culture-based repair results as a function of time and photorepair fluence (S3 Fig). Their respective regression rate constants (Fig 2B and 2C) demonstrate that percent reactivation was mostly positive for LP, suggesting reactivation and mostly negative for KrCl suggesting die off. Slopes and their standard error do not overlap between KrCl and LP, indicating statistical significance. Log repair results (S4 and S5 Figs) support these trends. This finding aligns with the hypothesis that less photoreactivation was expected after KrCl disinfection due to damage to repair enzymes [29, 31, 41]. However, log repair results may be more variable than percent reactivation results because the later accounts for initial conditions (i.e., initial concentration before UV exposure), while the former only accounts for conditions immediately following UV exposure.

Time-based kinetic rate constants for percent reactivation (min^-1) are reported as kt. Additionally, rate constants are reported with subscripts l and d signifying light repair and dark repair, respectively, and with wavelengths specifying KrCl (222 nm) or LP (254 nm). Photorepair fluence-based rate constants for percent reactivation (cm²/mJ) are reported as kp. A summary of rate constants is provided in S2 Table.

The order of kt values from highest to lowest was: LP light > LP dark > KrCl dark > KrCl light (Fig 2B and S2 Table). Apparent photoreactivation was greater for LP than for KrCl, as suggested by kt_l,254 > kt_l,222. Slopes were more similar between dark and light repair for LP than KrCl. In our study, 500 mJ/cm² of photorepair fluence for LP resulted in log repair of E. coli K-12 ranging from –0.5 to 0.5 (S5 Fig), which was 0.5 to 1.5 log less than the 1-log repair observed previously at the same photorepair fluence for E. coli ATCC 11229 [38]. One difference between the previous study was the UV fluence applied: Bohrerova and Linden (2007) reported UV fluence of 8 mJ/cm², while this study used 12 mJ/cm². This can be explained by the higher UV dose, where increasing UV dose has been shown to suppress photorepair [30, 62, 63]. This study used an initial concentration above 1×10⁷ CFU/mL to obtain sufficient molecular yields for quantitation (e.g., ELISA), whereas the concentration used in Bohrerova and Linden (2007) was reported 5×10⁶ CFU/mL. Culture media could also affect the kinetics, as Nutrient Broth was used in this study, while Bohrerova and Linden (2007) used Tryptic Soy Agar.

3.3. Detection of UV-induced CPD-DNA damage

For both LP and KrCl lamps, CFU normalized CPD-DNA concentrations increased linearly with UV fluence as shown in S6 Fig and summarized by regression parameters in Fig 3A. The slope ± standard error (ng-cm²/mJ-10⁷CFU) for LP (k_CPD-DNA,254 = 0.086±0.01) was more than double that of KrCl (k_CPD-DNA,222 = 0.038±0.003), confirming the DNA damage mechanism of 254 nm while also demonstrating that DNA damage contributes to the inactivation mechanisms of 222 nm [10, 17, 19]. Together with the culture results from Fig 2, these results indicate that the 222 nm achieves the same level of inactivation as 254 nm with less DNA damage, likely by damaging other molecules. Despite variability between two biological replicates at the highest LP fluence, the slopes and their confidence intervals are far from overlap. These results support previous literature regarding CPD-DNA formation by different UV wavelengths [5, 17].

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Fig 3. Summary of linear regression estimates for molecular UV fluence response kinetics for KrCl (red) and LP (blue) lamps.

Slope estimates (a) for cell-normalized CPD-DNA damage as a function of UV fluence show that CPD-DNA formation rate was higher for LP. Slope estimates (b) for cell-normalized photolyase damage as a function of UV fluence show that less photolyase was detected (i.e., negative slope indicates greater photolyase damage) for KrCl. Orange squares in (b) show slope estimates when the intercepts were forced through mean initial concentrations.

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3.4. Detection of UV-induced photolyase damage

The developed ELISA for photolyase enzyme in E. coli protein extracts from samples demonstrated ability to detect photolyase damage by KrCl disinfection as indicated by decreasing concentrations (S6 Fig) and negative regression slope (k_{Photolyase,222} = –0.0049±0.003, Fig 3B) as a function of UV fluence. Photolyase had no statistically significant trend with increasing LP UV fluence as the slope did not differ from zero (k_{Photolyase,254} = 0.0012±0.006). Initial sample (UV Fluence = 0 mJ/cm²) calculated mean ± standard deviation photolyase concentrations were overlapping (0.58±0.06 and 0.50±0.18 ng per 10⁷ CFU) but modeled intercepts indicating initial concentrations were slightly non-overlapping (0.462±0.055 and 0.662±0.113 ng per 10⁷ CFU) between 222 nm and 254 nm, respectively. When intercepts were forced through mean initial concentrations, differences between modeled slopes between wavelengths were even more apparent.

The results presented here support previous studies with E. coli which demonstrated similar disinfection kinetics between KrCl and LP lamps [19] but lower DNA damage [23] and greater protein damage of KrCl lamps [64]. The combination of the inactivation, DNA, and photolyase results for the same samples in the present study using a novel ELISA corroborate previous results that the mechanism of disinfection for the KrCl lamp relies on protein damage as well as DNA damage. The standardization of fluence-based photorepair kinetics enables calculation of photorepair in real world scenarios, such as in decentralized water systems [47]. In general, these results demonstrate the lower overall photolyase concentration after KrCl UV disinfection. Our results indicate that the photolyase damage may be part of the 222 nm UV disinfection mechanism and could influence regrowth and DNA repair kinetics, which will be discussed in the following section.

3.5. CPD-DNA and photolyase molecular repair kinetics

Molecular analyses performed on repair samples enabled elucidation of UV damage and repair mechanisms by measuring CPD-DNA and photolyase repair enzyme concentrations. CPD-DNA and photolyase concentrations were normalized by cell concentration (ng/10⁷ CFU) and plotted as a function of repair time (S7 Fig) and photorepair fluence (S8 Fig). A summary of time-based regression parameters is shown in S9 Fig while photorepair fluence-based regression parameters are summarized in Fig 4. Despite variability in culture and molecular data in repair experiments, significant differences were still observed. CPD-DNA decreased more quickly, with the negative slope indicating faster repair of DNA damage, in samples irradiated by LP compared than by KrCl, both as a function of photorepair fluence (Fig 4A) and repair time (S9 Fig). The difference in CPD repair was even more apparent when analyzing log change (log₁₀ C/C₀) per min of CPD-DNA concentrations without CFU normalization (Fig 4C and S10 and S11 Figs). Photolyase concentration did not change as a function of repair time or photorepair fluence after exposure to either wavelength as indicated by slopes not different than zero (Fig 4C and S9 Fig), except when normalizing by total protein concentrations which demonstrated a decrease for 254 and an increase for 222 nm (S12 Fig).

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Fig 4. Summary of linear regression parameters for CPD-DNA and photolyase concentrations calculated based on photorepair fluence.

CFU-normalized regression data are summarized as slope (a) and intercept (b) estimates. Log change calculations (c) show that CPD repair is greater for LP light repair than for KrCl light repair.

https://doi.org/10.1371/journal.pwat.0000238.g004

Photolyase mean concentration at repair time = 0 min (i.e., photorepair fluence = 0 mJ/cm²) was lower after KrCl exposure (0.187±0.24 ng/10⁷ CFU) than LP exposure (0.243±0.167 ng/10⁷ CFU) to a target UV fluence of 12 mJ/cm². This is supported by the significant UV dose-dependent decrease in photolyase in KrCl irradiated samples (Fig 3B), and by the significantly lower repair study photolyase intercepts (Fig 4B, see similar in S9 Fig). The lower detection of photolyase, indicating enzyme damage, in KrCl samples compared to LP samples (Fig 3B), despite similar cellular log reduction (Fig 2A) and therefore similar numbers of cells, was expected due to the absorbance of low wavelengths around 222 nm by proteins contributing to their damage [12, 17].

Because less photolyase was available in KrCl samples after UV exposure and there was less CPD to repair, it was expected that the CPD repair in KrCl samples would be less than in LP samples in repair exposures. Additionally, greater intercepts of both photolyase and CPD after LP exposure in photorepair kinetics (Fig 4B and S9 Fig) and greater positive slopes of CPD in UV fluence kinetics (Fig 3) indicated there would be more CPD to repair and more photolyase available for repair in LP samples. For cell-normalized data (S7 and S8 Figs), CPD-DNA repair was greater in LP samples than KrCl samples, especially in samples with light exposure. Light repair had larger decrease in log₁₀ C/C₀ of CPD-DNA for LP compared with KrCl both over time (e.g., at the end of 6 h) (S10 Fig) and as a function of photorepair fluence (S11 Fig). Additionally, the larger decrease of log₁₀ C/C₀ CPD-DNA for LP light than for LP dark (S10 Fig) supports that light repair due to photolyase was at least partially responsible for reversing CPD-DNA damage [26]. Differences in intercepts that were more pronounced for both photolyase and CPD, along with time- and photorepair fluence- based variations in data used for normalizations, may have confounded ability to observe strong differences, such that the known phenomenon of increased photolyase gene expression per cell after LP UV exposure [65] could not be differentiated from after KrCl exposure.

4. Wavelength selection implications for water disinfection

Our results provide evidence that using different UV wavelengths for water treatment can be advantageous due to different disinfection mechanisms. Previous studies show that disinfection by combinations of wavelengths is more likely to have disinfection synergy when the wavelengths have different inactivation mechanisms [10, 66]. Sequential exposure by LP and KrCl light was shown to inactivate MS2 with electrical energy efficiency similar to that of a medium pressure (MP) lamp, with polychromatic emission [67]. Furthermore, it is well established that MP UV damages proteins and inhibits photorepair of bacteria [31, 32, 39, 40, 42, 68]. Therefore, further research should investigate the comparison of KrCl and MP lamps for disinfection and inhibition of photorepair. The present study showed that LP lamps can form more CPDs in DNA with lower doses, while the KrCl lamp damaged the photolyase enzyme, which can prevent risk of repair following UV disinfection. Combining LP and KrCl may result in potential synergy by using both DNA-damaging wavelengths (e.g. 254 nm) and protein-damaging wavelengths (e.g. 222 nm) to efficiently inactivate microorganisms and prevent photorepair and deserves future study. Improving our understanding of suppressing DNA repair processes will make UV treatment more applicable for water systems. Specifically, future research should investigate water matrix effects (e.g., tap water, natural water) on disinfection and repair, as previously studied [69–72]. The use of UV disinfection in drinking water treatment has potential to reduce public exposure to chlorine-resistant pathogens in drinking water [2]. By preventing repair and inducing die-off, water treatment systems using UV disinfection can add less residual chlorine to the treated water while maintaining residual disinfection, which could result in formation of fewer toxic disinfection byproducts from chemical disinfection [73, 74]. Future research could also examine the synergistic effects of the KrCl and LP lamps being used together to inactivate bacteria through DNA damage and inhibit repair through protein damage. Further optimization of UV inactivation by tailoring wavelength combinations for disinfection will improve protection of public health. More detailed studies with water containing organic matter, natural water samples, and other pathogens will be necessary to elucidate effectiveness of KrCl disinfection for water and wastewater.

5. Conclusions

This research compared the UV inactivation and damage mechanisms of E. coli K-12 by LP and KrCl lamps. We observed that the lamps had similar culture-based disinfection kinetics. A Geeraerd shoulder + tail model estimated that the inactivation rate constants of E. coli in the log-linear region of the dose response curves were 1.18±0.09 and 1.24±0.08 cm² mJ⁻¹ following shoulder lengths of 6.0±0.46 and 6.82±0.51 mJ/cm² for LP and KrCl, respectively. Molecular assays revealed DNA-damage and protein-damage were the primary mechanisms of inactivation for LP and KrCl, respectively, while DNA-damage may contribute to the inactivation mechanism of KrCl. Molecular UV fluence–responses showed that CPD-DNA concentrations increased more over time, for the same UV fluence, in samples exposed to LP than KrCl. CPD-DNA repair kinetics indicated that photorepair fluence-based light repair rate was greater for samples treated by LP compared to KrCl. In this study, we also developed a new ELISA to detect photolyase enzyme in E. coli. Using this ELISA, less photolyase was detected in samples exposed to KrCl than unexposed E. coli and LP samples. Thus, damage to the photolyase enzyme responsible for DNA repair was quantified. Culture-based results showed that photorepair after LP exposure was greater than after KrCl exposure, with cells dying after KrCl exposure. These results were expected because photolyase requires light to repair CPDs, but the KrCl lamp damaged photolyase during UV exposure. The ELISA developed in this study was used to demonstrate KrCl lamps inflict greater damage to photolyase in bacteria than LP lamps. Due to the suppression of DNA repair and therefore cell regrowth through protein damage, KrCl lamps are a promising treatment alternative for water disinfection to prevent microbial regrowth and DNA repair.

Supporting information

S1 Text. DNA extraction details.

https://doi.org/10.1371/journal.pwat.0000238.s001

(DOC)

S1 Table. Summary of Geeraerd model parameters.

A Geeraerd shoulder + tail model was applied to log inactivation data from fluence–responses with the LP and KrCl lamps. The four parameters in this model describe the inactivation kinetics of E. coli with both lamps.

https://doi.org/10.1371/journal.pwat.0000238.s002

(XLSX)

S2 Table. Observed rate constants for time-based and photorepair fluence based repair kinetics.

https://doi.org/10.1371/journal.pwat.0000238.s003

(XLSX)

S1 Fig. Standard curves for CPD-DNA and photolyase.

Data points shown are means and standard deviations of technical triplicate absorbance measurements for each ELISA plate. Linear regressions are shown for each plate.

https://doi.org/10.1371/journal.pwat.0000238.s004

(TIFF)

S2 Fig. Total DNA and protein linear regression parameters calculated based on repair time.

Total DNA concentrations are measured by Qubit (1x dsDNA High Sensitivity Kit, Invitrogen) and total protein concentrations were measured by Bradford Assay (Bio-Rad). Closed squares indicate samples in which DNA or protein were detected by the respective assays. Open squares indicate samples in which DNA or protein was not detected.

https://doi.org/10.1371/journal.pwat.0000238.s005

(TIFF)

S3 Fig.

Percent reactivation as a function of repair time (a) and photorepair fluence (b). Panel (a) shows culture-based percent reactivation values over time for E. coli during photorepair (closed symbols, solid lines) and dark repair (open symbols, dashed lines) incubations following irradiation by LP UV 254 nm (LP, blue) and KrCl 222 nm (KrCl, red). All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1). Panel (b) shows culture-based percent reactivation of E. coli as a function of photorepair fluence.

https://doi.org/10.1371/journal.pwat.0000238.s006

(TIFF)

S4 Fig. Log repair as a function of repair time.

Culture-based log repair values over time for E. coli during photorepair (closed symbols, solid lines) and dark repair (open symbols, dashed lines) incubations following irradiation by LP UV 254 nm and KrCl 222 nm. All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1).

https://doi.org/10.1371/journal.pwat.0000238.s007

(TIFF)

S5 Fig. Log repair as a function of photorepair fluence.

Culture-based photorepair fluence response of E. coli log repair following irradiation by LP UV 254 nm and KrCl 222 nm. Mean values bars are shown for at least experimental duplicate or triplicate, except for data at 5 and 340 min (n = 1). All experimental data are shown for LP and KrCl.

https://doi.org/10.1371/journal.pwat.0000238.s008

(TIFF)

S6 Fig.

Molecular-based UV fluence-responses are shown for (a) CFU normalized CPD-DNA concentration and (b) CFU normalized photolyase concentration. Points represent means of experimental duplicates for CPD and photolyase.

https://doi.org/10.1371/journal.pwat.0000238.s009

(TIFF)

S7 Fig. Cell-normalized concentrations of CPD-DNA and photolyase as a function of repair time.

Cell-normalized concentrations of CPD-DNA and photolyase protein during photorepair (closed symbols, solid lines) and dark repair (open symbols, dashed lines) incubations following irradiation by LP UV 254 nm (blue) and KrCl 222 nm (red). All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1).

https://doi.org/10.1371/journal.pwat.0000238.s010

(TIFF)

S8 Fig. Cell-normalized concentrations of CPD-DNA and photolyase as a function of photorepair fluence.

Quantities of CPD-DNA (closed squares, solid lines) and photolyase protein (closed triangles, dashed lines) during photorepair incubations following irradiation by LP UV 254 nm (blue) and KrCl 222 nm (red). Quantities of molecular targets were normalized to the initial cell concentration for each experimental replicate (photorepair fluence = 0 mJ/cm2). All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1).

https://doi.org/10.1371/journal.pwat.0000238.s011

(TIFF)

S9 Fig. Summary of CPD-DNA and photolyase linear regression parameters calculated based on repair time.

Slope (a) and intercept (b) estimates, respectively, and standard errors for CFU-normalized data for time-based linear regressions. Slope (c) estimates and standard errors for log-change normalized data for time-based linear regressions.

https://doi.org/10.1371/journal.pwat.0000238.s012

(TIFF)

S10 Fig. Log10 C/C0 of CPD-DNA and photolyase as a function of repair time.

The log10 C/C0 values for CPD-DNA and photolyase during photorepair (closed symbols, solid lines) and dark repair (open symbols, dashed lines) incubations following irradiation by LP UV 254 nm (blue) and KrCl 222 nm (red). Kinetics were calculated relative to the DNA and protein concentrations measured immediately after UV exposure (repair time, t = 0 min). All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1).

https://doi.org/10.1371/journal.pwat.0000238.s013

(TIFF)

S11 Fig. Log10 C/C0 of CPD-DNA and photolyase as a function of photorepair fluence.

The log10 C/C0 values for CPD-DNA (closed squares, solid lines) and photolyase protein (closed triangles, dashed lines) during photorepair incubations following irradiation by LP UV 254 nm (blue) and KrCl 222 nm (red). All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1).

https://doi.org/10.1371/journal.pwat.0000238.s014

(TIFF)

S12 Fig. Molecular-normalized quantities of CPD-DNA and photolyase as a function of photorepair fluence.

Quantities of CPD-DNA (closed squares, solid lines) and photolyase protein (closed triangles, dashed lines) during photorepair incubations following irradiation by LP UV 254 nm (blue) and KrCl 222 nm (red). CPD-DNA and photolyase quantities were normalized to DNA and protein concentrations, respectively, for each sample. All data are either duplicate or triplicate, except for data at 5 and 340 min (n = 1). Linear regressions shown are forced through zero with 95% confidence interval shading.

https://doi.org/10.1371/journal.pwat.0000238.s015

(TIFF)

S1 Graphical abstract.

https://doi.org/10.1371/journal.pwat.0000238.s016

(TIFF)

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Figures

Abstract

1. Introduction

2. Methods

2.1 Bacteria preparation and enumeration

2.2 UV disinfection experiments

2.3. Photorepair and dark repair experiments

2.4 Culture-based inactivation and photoreactivation

2.5. DNA extraction and analyses

2.6. Protein extraction and analyses

2.7. Photolyase enzyme quantification

2.8. Analyses of molecular assay results

2.9. Statistical analyses

3. Results and discussion

3.1. Culture-based inactivation kinetics

3.2. Culture-based reactivation kinetics

3.3. Detection of UV-induced CPD-DNA damage

3.4. Detection of UV-induced photolyase damage

3.5. CPD-DNA and photolyase molecular repair kinetics

4. Wavelength selection implications for water disinfection

5. Conclusions

Supporting information

S1 Text. DNA extraction details.

S1 Table. Summary of Geeraerd model parameters.

S2 Table. Observed rate constants for time-based and photorepair fluence based repair kinetics.

S1 Fig. Standard curves for CPD-DNA and photolyase.

S2 Fig. Total DNA and protein linear regression parameters calculated based on repair time.

S3 Fig.

S4 Fig. Log repair as a function of repair time.

S5 Fig. Log repair as a function of photorepair fluence.

S6 Fig.

S7 Fig. Cell-normalized concentrations of CPD-DNA and photolyase as a function of repair time.

S8 Fig. Cell-normalized concentrations of CPD-DNA and photolyase as a function of photorepair fluence.

S9 Fig. Summary of CPD-DNA and photolyase linear regression parameters calculated based on repair time.

S10 Fig. Log10 C/C0 of CPD-DNA and photolyase as a function of repair time.

S11 Fig. Log10 C/C0 of CPD-DNA and photolyase as a function of photorepair fluence.

S12 Fig. Molecular-normalized quantities of CPD-DNA and photolyase as a function of photorepair fluence.

S1 Graphical abstract.

References