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Inactivation of E. coli, Bacteriophage MS2 and Bacillus Spores under UV/H2O2 and UV/Peroxydisulfate Advanced Disinfection Conditions Peizhe Sun, Corey Tyree, and Ching-Hua Huang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06097 • Publication Date (Web): 25 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016
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Inactivation of E. coli, Bacteriophage MS2 and Bacillus Spores under
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UV/H2O2 and UV/Peroxydisulfate Advanced Disinfection Conditions
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Peizhe Sun, *,a,b Corey Tyree,b Ching-Hua Huang*,a
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a
School of Civil and Environmental Engineering, Georgia Institute of Technology,
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Atlanta, Georgia 30332, United States b
Division of Energy and Environment, Southern Research Institute, Birmingham, Alabama 35205, United States
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*Corresponding Authors.
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Phone: 404-894-7694. E-mail:
[email protected] 12
Phone:404-358-4858. E-mail:
[email protected] 13 14 15 16 17
Revised manuscript submitted to
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Environmental Science & Technology
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Abstract: Ultraviolet light (UV) combined with peroxy chemicals, such as H2O2 and
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peroxydisulfate (PDS), have been considered potentially highly effective disinfection
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processes. This study investigated the inactivation of E. coli, bacteriophage MS2 and
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Bacillus subtilis spores as surrogates for pathogens under UV/H2O2 and UV/PDS
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conditions, with the aim to provide further understanding of UV-based advanced
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disinfection processes (ADPs). Results showed that one additional log of inactivation of
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E. coli was achieved with 0.3 mM H2O2 or PDS at 5.2×10-5 Einstein·L-1 photo fluence (at
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254 nm) compared with UV irradiation alone. Addition of H2O2 and PDS greatly
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enhanced the inactivation rate of MS2 by around 15 folds and 3 folds, respectively,
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whereas the inactivation of B. subtilis spores was slightly enhanced. Reactive species
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responsible for the inactivation were identified to be ·OH, SO4·- and CO3·- based on
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manipulation of solution conditions. The CT value of each reactive species was calculated
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with respect to each microbial surrogate, which showed that the disinfection efficacy
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ranked as ·OH > SO4·- > CO3·- >> O2·-/HO2·. A comprehensive dynamic model was
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developed and successfully predicted the inactivation of the microbial surrogates in
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surface water and wastewater matrices. The concepts of UV-efficiency and EE/O were
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employed to provide a cost-effective evaluation for UV-based ADPs. Overall, the present
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study suggests that it will be beneficial to upgrade UV disinfection to UV/H2O2 ADP for
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the inactivation of viral pathogens.
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INTRODUCTION
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Increasing water pollution by infectious biological contaminants is a serious health
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risk around the globe. Recent monitoring studies have detected pathogens in urban
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wastewater influents, including but not limited to adenovirus, enterovirus, and norovirus.1
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Several outbreaks of biological contaminants have also been documented,2-4 among
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which, Escherichia coli (E. coli), Listeria, Salmonella, Heptatis A and Cyclospora were
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the most reported. The U.S. Environmental Protection Agency (USEPA) has listed twelve
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microbial contaminants, including both bacterial and viral pathogens, on the 3rd version
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of the Drinking Water Contaminant Candidate List (CCL), a list of unregulated chemical
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and biological contaminants known or anticipated to occur in public water systems.5
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Some of the microbial contaminants can survive conventional water treatment processes
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(e.g., coagulation/flocculation, activated sludge, and filtration processes) and are likely to
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persist in freshwater with the potential for transmission by a waterborne route.6-8
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Ultraviolet (UV)-based disinfection techniques are being increasingly applied in
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water and wastewater treatment facilities worldwide due to their lower tendency to form
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harmful disinfection byproducts (DBPs) compared to chemical disinfection processes,9-15
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and the ability of UV irradiation to chemically modify the DNA or RNA of
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microorganisms and thus inactivate them.9,16 However, studies have shown that a number
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of microbial contaminants and their surrogates are resistant to UV disinfection including
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adenovirus, bacteriophage MS2, Bacillus subtilis spores, and some antibiotic-resistant
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bacteria and genes.17-21 Therefore, it will be advantageous to develop means to enhance
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the efficacy of UV-based processes for pathogen elimination.
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UV-based
advanced
oxidation
processes
(AOPs),
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such
as
UV/H2O2,
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UV/peroxydisulfate (UV/PDS), and UV/TiO2, have been shown to be highly effective in
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degrading organic micropollutants (e.g., pesticides, pharmaceuticals, and chlorinated
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solvents) in drinking water and wastewater.22-29 Organic contaminants are degraded much
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more effectively and extensively by AOPs than UV alone because highly reactive radical
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and oxidizing species are generated under these designed conditions. In analogy, AOPs
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may hold promise to be more effective than conventional water disinfection processes in
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inactivating pathogens due to these more powerful oxidation capabilities.19,20,30-33
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Especially, radical species, such as hydroxyl radical and carbonate radical, generated in
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AOP or natural conditions, are able to inactive E. coli and MS2.19,30,31,34 Several studies
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have evaluated UV/H2O2 for disinfection purposes. For example, Mamane et al.19
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investigated the inactivation of E. coli, B. subtilis spores, and bacteriophages MS2, T4,
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and T7 using UV (>295 nm) compared to UV/H2O2 AOP. The authors found that
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UV/H2O2 AOP led to an additional one log of inactivation for T7 and additional 2.5 logs
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of inactivation for MS2 phage compared to UV alone. In contrast, inactivation efficiency
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of the other microorganisms was not significantly affected by AOP. Bounty et al.35
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examined the inactivation of adenovirus, one of the most UV-resistant pathogens, using
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UV/H2O2 AOP, and reported that UV inactivation of adenovirus can be significantly
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enhanced by addition of H2O2 due to formation of hydroxyl radicals. The above varying
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outcomes of different microorganisms were likely the results of specific disinfection
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conditions and mechanisms between particular microorganisms and radicals, implying
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that process optimization based on each pathogen of interest would be necessary. Indeed,
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previous studies suggest that viruses are more susceptible to radical attack than bacteria
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and spores.19,34 Whether it is beneficial to upgrade UV disinfection process to UV-based
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advanced disinfection process (ADP) is still not clear based on previous research. In
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addition, there has been little research on applying PDS with UV, another powerful AOP
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option, for disinfection purposes.
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To assess the disinfection potential of UV-based ADP, this study employed three
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microbial surrogates, including E. coli, bacteriophage MS2 and Bacillus subtilis spores as
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representatives for pathogenic bacteria, viruses and protozoa, respectively, to investigate
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the disinfection potency of different radical species generated by UV/H2O2 and UV/PDS
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processes. Dynamic kinetic models were developed to quantitatively evaluate the
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inactivation kinetics of microbial surrogates by the UV-based ADPs to gain mechanistic
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insight. An energy-cost assessment was also conducted to optimize the disinfection
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efficiency and assess the overall performance of the UV-based ADPs.
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MATERIAL AND METHODS Chemicals. Sources of chemical and reagents are provided in the Supporting Information (SI) Text S1.
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Microbial surrogates. E. coli (ATCC 15597), bacteriophage MS2 (ATCC 15597-B1)
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and Bacillus subtilis spores (ATCC 6633) were selected as surrogates of pathogenic
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bacteria, viruses and protozoa. Dry powder form of each microbial surrogate was
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purchased from ATCC and revived accordingly. Culture preparation followed the
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methods by Cho et al.32 Details of stock preparation are described in SI Text S2.
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Experimental setup. The UV-based ADP experiments were conducted with a bench-
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scale collimated-beam UV apparatus (SI Figure S1) equipped with a 4W low-pressure
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UV lamp emitting light predominantly at 254 nm (Philips Co., Netherlands). The
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spectrum emission of the lamp was characterized by a spectroradiometer (Spectral
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Evolution, SR-1100) (shown in SI Figure S2). The reaction solution (10 mL) was put into
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a glass petri dish (inner diameter = 5.4 cm) which was placed on a stir plate,
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perpendicular to the incident light. Therefore, the optical path length was 0.44 cm. The
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UV fluence rate received in the reaction solution was measured to be 2.2×10-7 Einstein·L-
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1
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1.0 mJ·cm-2 was equivalent to 5.5×10-6 Einstein·L-1 (SI Text S3).
·s-1 using potassium ferrioxalate as chemical actinometer. For this experimental setup,
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Most of the experiments were conducted in phosphate buffer solution (3.0 mM PBS
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at pH 7.0) containing 0.3 mM H2O2 or PDS and microbial surrogates, except where stated
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otherwise. The initial densities of E. coli and B. subtilis spores for each disinfection
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experiment were around 4×106 CFU/mL and for MS2 were around 3×106 PFU/mL.
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Preliminary tests using dynamic light scattering (Zetasizer Nano ZS instrument, Malvern
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Instruments) showed that the microbial surrogates (up to ~108 CFU/mL or PFU/mL) were
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at dispersed state not forming clumps.
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In the experiments to test the inactivation of microbial surrogates in real water
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matrices, samples of surface water (SW) from a river source and wastewater (WW) from
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secondary effluent were collected locally at a drinking water treatment plant and a
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municipal wastewater treatment plant, respectively. The water samples were filtered by
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glass fiber filters prior to use. The characteristics of the water samples are summarized in
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SI Table S1, in which inorganic ions were measured by a Dionex DX-100 ion
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chromatography instrument with conductivity detection and dissolved organic carbon
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(DOC) was measured by a Shimadzu TOC analyzer. The colony-forming-unit (CFU) and
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plague-forming-unit (PFU) of SW and WW were determined, which showed no
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interference with the counting of the microbial surrogates.
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Modeling. Kinetic modeling of radical species was simulated using Simbiology
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application in Matlab 2014b. Over one hundred elementary reactions (SI Table S2) were
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considered with rate constants obtained from literature.29,36,37 This model takes into
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account the effects on ADP from most of inorganic ions, including chloride, sulfate,
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nitrogen species, carbonate species, and from DOC present in the water matrices. The
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scavenging effects of pathogens on the radicals were considered much lower than those
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by the water matrix components and thus neglected in the simulations. Major radical
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concentrations predicted by this model (under UV/H2O2, UV/PDS and UV/H2O2/NaCO3
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conditions) were validated using radical probes, such as p-nitroaniline, anisole and
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nitrobenzene (details shown in SI Text S4, Table S3). The concentrations of radical
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species presented in this study were all obtained by model simulation at the end of 2 min
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reaction time. Preliminary test runs showed that the concentrations of major radicals
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reached pseudo-steady-state within 2 min reaction time.
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RESULTS AND DISCUSSION
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Inactivation of microbial surrogates by UV, UV/H2O2 and UV/PDS. The E. coli,
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bacteriophage MS2 and B. subtilis spores were treated under UV, UV/H2O2 and UV/PDS
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conditions (Figure 1). Control experiments were conducted under the same conditions
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without UV irradiation, which showed that the microbial surrogates were not inactivated
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by H2O2 or PDS within 30 min (data not shown). Experiments showed that 4-log
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inactivation of E. coli was achieved by UV exposure at 10.6 mJ·cm-2 (i.e., 5.2×10-5
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Einstein·L-1) (Figure 1A). The overall inactivation of E. coli by UV alone presented a lag
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phase for around 4 mJ·cm-2 followed by a linear loss of bacteria viability over exposure
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time, which was consistent with literature.9,38 The application of 0.3 mM H2O2 or PDS
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statistically significantly enhanced the inactivation of E. coli (P < 0.005 by paired t-test).
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Additional 1-log reduction was achieved at 10 mJ·cm-2, whereas the lag phases were only
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slightly shortened. The UV dose required to achieve 4-log inactivation were 8.6 and 8.8
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mJ·cm-2 for UV/H2O2 and UV/PDS, respectively.
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As for bacteriophage MS2, 4-log inactivation was not achieved till at 85 mJ·cm-2 (i.e.,
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4.1×10-4 Einstein·L-1) by UV irradiation alone (Figure 1B). The loss of MS2 viability
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exhibited pseudo-first-order kinetics over UV exposure time. The addition of 0.3 mM
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H2O2 greatly enhanced the inactivation rate by almost 15 folds, reducing the UV intensity
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to achieve 4-log inactivation to 6 mJ·cm-2. The inactivation was also enhanced under
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UV/PDS condition, but less effective than UV/H2O2 condition.
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B. subtilis spores were inactivated by UV alone, with 4-log inactivation at around 30
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mJ·cm-2 (i.e., 1.5×10-4 Einstein·L-1) (Figure 1C). The overall inactivation kinetics
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presented a lag phase followed by a linear relation between –log(N/N0) and UV exposure
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time. The inactivation of spores was enhanced under UV/H2O2 (P < 0.005), whereas the
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addition of PDS did not achieve observable difference from UV alone.
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The inactivation of microbial surrogates by UV irradiation was extensively
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investigated in prior studies and nicely summarized in a literature review.38 A range of
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inactivation rates (i.e., log of inactivation per mJ·cm-2 obtained from linear range data)
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were reported and summarized (E. coli 0.506, MS2 0.055, B. subtilis spores 0.059),38
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which are close to the inactivation rates measured in this study (E. coli 0.550, MS2 0.047,
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B. subtilis spores 0.128). UV irradiation is effective to inactivate bacteria, primarily due
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to dimerization of adjacent thymine molecules in their DNA.39 On the other hand,
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viruses, such as bacteriophage MS2, are known to much less susceptible to UV radiation
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than bacteria.38 The application of the same amount of H2O2 led to different enhancement
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of the inactivation for E. coli, MS2 and B. subtilis spores, which suggested they have
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different sensitivity toward reactive species produced under UV/H2O2 condition.
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Similarly, E. coli and MS2 were subject to inactivation by the radicals produced under
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UV/PDS condition, whereas the inactivation of B. subtilis spores was not enhanced.
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Therefore, further studies were conducted in the aim of identifying what reactive species
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was/were responsible for the inactivation of the microbial surrogates.
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Contribution of reactive species. In UV/H2O2 and UV/PDS systems, multiple
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reactive species are generated. The photolysis of H2O2 and PDS produces primary
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radicals, i.e., hydroxyl radical and sulfate radical. These primary radicals react with water
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components yielding secondary radicals, such as carbonate radical and superoxide
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radical. Based on the simulation results (Table 1), hydroxyl radical, sulfate radical,
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carbonate radical and superoxide radical are present at significantly high concentrations
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(> 10-13 M) under UV/H2O2 and UV/PDS conditions, which make them potentially
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important to inactivate microbial pathogens. To elucidate the role of each radical,
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different combinations of oxidants (i.e., H2O2 and PDS) and radical scavengers (i.e., t-
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butal alcohol (TBA) and NaHCO3) were employed to create conditions where only one
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radical species was dominantly higher in concentration (Figure 2). Such conditions were
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also validated using the kinetic model to calculate the predicted pseudo-steady-state
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concentrations of radicals (Table 1). Statistical analysis using t-test was employed to
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assess the experimental results between the radical-dominant conditions and control
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groups for any significant differences.
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Hydroxyl radical. It is commonly considered that the dominant radical species
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generated by UV/H2O2 in a clean water system, such as PBS, is hydroxyl radical due to
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the direct photolysis of H2O2 (eqn. 1).
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−
hυ H 2O2 /HO2 → 2 • OH
(1)
•
OH + HCO3- /CO32− → CO3•− + H2O
(2)
•
OH + H2O2 → HO•2 /O•2− + H2O
(3)
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However, with the presence of (bi)carbonate from dissolved CO2, carbonate radical
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will be produced from the reaction between hydroxyl radical with (bi)carbonate (eqn. 2).
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Additionally, H2O2 can react with hydroxyl radical yielding superoxide radical (eqn. 3).
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Although carbonate radical and superoxide radical are known not very reactive towards
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common organic compounds,37 it has been suggested that carbonate radical can inactivate
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MS234 and superoxide radical contributed to the photoinactivation of E. coli.40 Therefore,
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the enhancement of the inactivation of microbial surrogates under UV/H2O2 (Figure 1)
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may not be (only) attributed to hydroxyl radicals. The application of TBA as the hydroxyl
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radical scavenger is a common approach to study the contribution of hydroxyl radical,
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whereas TBA also prevents the formation of carbonate radical and superoxide radical
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(reaction system 3 in Table 1). On the other hand, it is very difficult to create conditions
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where only hydroxyl radical dominates because: (1) the elimination of (bi)carbonate is
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difficult to achieve in an open reactor; (2) superoxide radical scavengers, such as 4-
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hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl
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hydroxyl radical as well. Therefore, in order to elucidate the contribution of hydroxyl
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radical, the role of carbonate radical and superoxide radical should be determined first.
(TEMPOL),
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potentially
react
with
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Superoxide radical. Applying eqn. 4-6, a system (UV/10×H2O2/PDS/TBA) where
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superoxide radical was the major reactive species was created (reaction system 7 in Table
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1). Essentially, the photolysis of PDS produced sulfate radical (eqn. 4), which reacted
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with H2O2 yielding superoxide radical (eqn. 5). TBA was used to quench the hydroxyl
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radical generated from the photolysis of H2O2 (eqn. 6). The application of H2O2 was to
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produce superoxide radical and to suppress sulfate radical concentration as well (eqn. 5).
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Concentrations of H2O2 (3 mM) and PDS (0.3 mM) were selected in order to produce a
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significantly high amount of superoxide radical with minimal hydroxyl and sulfate
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radical production. •−
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hν S2O8 → 2 ⋅ SO4
(4)
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SO•4− + H2O2 → HO•2 /O•2− + SO24−
(5)
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TBA+ • OH → product
(6)
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As Table 1 shows, in the superoxide radical dominated condition, the predicted
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superoxide radical concentration was close to that in the UV/H2O2 system (i.e., ~2×10-8
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M), while the concentrations of hydroxyl radical and sulfate radical were 2-3 orders of
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magnitude lower than those in the UV/H2O2 and UV/PDS systems, respectively (reaction
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system 7 versus 1 and 2). Therefore, negligible inactivation was expected from hydroxyl
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radical and sulfate radical. For comparison, control experiments (i.e., UV irradiation
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only) were conducted in PBS with addition of TBA. Preliminary experiments confirmed
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that TBA (0.1 M) did not have detectable impact on the microbial surrogates (data not
246
shown). Results showed that the superoxide radical dominated condition did not have
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better inactivation efficiency than the control groups for all microbial surrogates (Figure
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2A), indicating that superoxide radical has little disinfection potency.
2-
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Carbonate radical. A carbonate radical dominated system was created by adding 0.1
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M NaHCO3 into the UV/H2O2 system. The solution pH was maintained at 8.5. The
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amount of (bi)carbonate ions were sufficient to suppress hydroxyl radical concentration
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to around 10-15 M (reaction system 6 in Table 1), more than two orders of magnitude
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lower than that in the UV/H2O2 system. At such concentration, hydroxyl radical was
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expected to have negligible effects on the inactivation of microbial surrogates. In other
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words, the contribution from hydroxyl radical was less than 1% of that in the UV/H2O2
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system. Therefore, carbonate radical was the only important reactive species in the
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UV/H2O2/NaHCO3 system. To elucidate the contribution of carbonate radical, control
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groups (i.e., UV irradiation) were conducted in PBS with the addition of NaHCO3.
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Preliminary experiments confirmed that NaHCO3 (0.1 M) had little impact on the
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microbial surrogates (data not shown). As shown in Figure 2B, carbonate radical
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enhanced the inactivation of E. coli and MS2 (P < 0.005), whereas B. subtilis spores were
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resistant to carbonate radical. The disinfection role of carbonate radical was further
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confirmed by adding 0.01 mM p-nitroaniline, a carbonate radical scavenger, in the
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UV/H2O2/NaHCO3 system, in which the inactivation of MS2 was greatly inhibited (SI
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Figure S3). The carbonate radical concentration in the UV/H2O2/NaHCO3 system was
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8.64×10-12 M, which was around one order of magnitude higher than that in the UV/H2O2
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system. Therefore, the inactivation by carbonate radical should be less significant in a
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low-carbonate UV/H2O2 system. Nevertheless, this result indicates that the inactivation of
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E. coli and MS2 under UV/H2O2 conditions was partly attributed to carbonate radical.
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Quantitative discussion is performed in a later section after obtaining CT values.
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Sulfate radical. Under UV/PDS conditions, both sulfate radical and hydroxyl radical
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were produced due to trace amounts of chloride in PBS (eqn. 7 and 8, Table 1). The
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concentrations of chlorine-containing radicals (i.e., Cl· and Cl2·-), also simulated by the
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model, were 1-2 orders of magnitude lower than those of sulfate radical and hydroxyl
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radical and thus could be neglected (data not shown). The reaction between sulfate
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radical and H2O/OH- can also generate hydroxyl radical (eqn. 9).
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SO•4− + Cl− → Cl• + SO24−
(7)
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Cl• + H 2 O/OH− → ClOH•− →• OH + Cl−
(8)
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SO•4− + H2 O/OH− →• OH + SO24−
(9)
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To create a condition where sulfate radical is the only important reactive species, 0.1
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M TBA was added to quench hydroxyl radical formation because TBA reacts with
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hydroxyl radical ( k = 7.6×108 M-1·s-1) much faster than with sulfate radical ( k = 9.1×105
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M-1·s-1).37 A higher initial PDS concentration (i.e., 3 mM) was applied to compensate the
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sulfate radical consumed by TBA (see reaction system 5 versus 4 in Table 1). Control
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groups (i.e., UV irradiation) were conducted in PBS with TBA but without PDS. As
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Figure 2C shows, sulfate radical only enhanced the inactivation of MS2 (P < 0.005)
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whereas no observable contribution was achieved for the inactivation of E. coli and B.
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subtilis spores. This result suggests that the enhancement of the inactivation of E. coli by
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UV/PDS in PBS was attributed to the hydroxyl radical produced by the reactions in eqn.
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7-9.
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Overall, the results showed that the major reactive species for the inactivation of
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microbial surrogates are hydroxyl radical, sulfate radical and carbonate radical. Indeed,
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the second-order rate constants between radicals and biomolecules, including
294
saccharides, amino acids and lipids, suggest that hydroxyl radical and sulfate radical are
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the most reactive species (k = ~107–1010 M-1·s-1) whereas carbonate radical can react with
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biomolecules at the rate 104–108 M-1·s-1.37 Superoxide radical, on the other hand, only
297
reacts with amino acids, and the rates are lower than 10 M-1·s-1.37
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CT values. The observed loss of viability (-log(N/N0)obs) of pathogens can be
299
expressed as the sum of inactivation contributed by UV irradiation and radical attacks, as
300
shown in eqn. 10, in which, the inactivation by radical attacks was related to the radical
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concentration multiplied by exposure time (CT, M·min).
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N − log N0
N = − log N obs 0
N − log UVC N0 •
= f1 ( I , t ) + f 2 ([ OH ], t ) +
N • − log N OH 0
N • − − log N SO4 0
f 3 ([ SO4•− ], t ) +
f 4 ([CO3•− ], t )
•− CO3
(10)
303
In order to quantitatively express the contribution of each reactive species to the
304
inactivation of microbial surrogates, CT profile for each reactive species was obtained by
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subtracting the inactivation by UV alone from the overall inactivation. Radical
306
concentrations were calculated based on the kinetic modeling results (Table 1). As
307
discussed above, it was difficult to create a system only dominated by hydroxyl radical
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whereas it was possible for sulfate radical and carbonate radical. Therefore, CT profiles
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of sulfate radical and carbonate radical were first obtained. The CT profile of hydroxyl
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radical was then obtained based on the results subtracted of carbonate radical
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contribution. It is recognized that eqn. 10 is a simplified assumption that does not
312
consider potentially interactive impacts of disinfectant species on the pathogens.
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However, due to the limited knowledge on the disinfection effects of radical species, eqn.
314
10 served as an initial attempt to quantify the contributions from different radical species.
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The CT values from eqn. 10 obtained in PBS matrix were then tested in real water
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matrices to assess the robustness of this simplified model (next section).
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The CT profile of carbonate radical was obtained from the inactivation results in
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carbonate radical dominated system (UV/H2O2/NaHCO3), in which carbonate radical
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concentration was 8.64×10-12 M. Carbonate radical inactivated E. coli and MS2. For E.
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coli, the CT profile has a slope of 4.35×1010 M-1·min-1 with a lag phase of 5.32×10-12
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M·min. For MS2, the CT profile has a slope of 2.62×1010 M-1·min-1 (SI Figure S4C).
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After obtaining the CT profile of carbonate radical, the CT profile of hydroxyl radical
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can be calculated by subtracting the contribution from carbonate radical from the
324
observed inactivation of microbial surrogates. The slope and lag phase of CT profiles of
325
hydroxyl radical was calculated and shown in Table 2.
326
The CT profile of sulfate radical was obtained from the inactivation results in sulfate
327
radical dominated system (UV/10×PDS/TBA), in which sulfate radical concentration was
328
2.13×10-13 M. As discussed above, sulfate radical only inactivate bacteriophage MS2
329
within the disinfection time. A linear relation was obtained between the loss of MS2
330
viability and CT of sulfate radical (SI Figure S4B). The slope of the CT profile was
331
1.61×1012 M-1·min-1 (Table 2), meaning that exposure of MS2 at (1.61×1012)-1 M sulfate
332
radical for one minute could achieve one log of inactivation.
333
The slope values derived from literature19,30,34 are also included in Table 2 for
334
comparison. Significant differences exist among the studies, possibly due to the
335
employment of different microbial strains or measurement approaches for radical
336
concentrations. It is also worthwhile to note that some of the slope values are higher than
337
the commonly referred diffusion-controlled limits (i.e., 109–1010 M-1·s-1). However, for a
338
reaction between two reactants which are significantly different in size, such as
339
microorganism and radical, the rate limits should be higher than 1010 M-1·s-1. For
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example, the diffusion-controlled limits for MS2 and hydroxyl radical was estimated
341
above 6.6×1011 M-1·s-1 (i.e., ~4×1013 M-1·min-1). Detailed calculations are shown in SI
342
Text S5.
343
The slopes of CT profiles are indicators of disinfection potency of each radical
344
species. E. coli is more efficiently inactivated by hydroxyl radical than carbonate radical.
345
As for MS2, hydroxyl radical is more efficient than sulfate radical followed by carbonate
346
radical. B. subtilis spores can only be inactivated by hydroxyl radical. The different
347
disinfection efficacy of radicals is expected to partly relate to their oxidizing power and
348
radical charge. Indeed, hydroxyl radical and sulfate radical have higher oxidizing power
349
((E° (•OH/H2O) = 1.9−2.7 V; E° (SO4•−/SO42−) = 2.5−3.1 V) than carbonate radical (E°
350
(CO3•−/CO32−) = 1.63 V at pH 8.4),41 indicating that hydroxyl radical and sulfate radical
351
are more reactive towards biomolecules. However, although sulfate radical has higher
352
oxidizing power than hydroxyl radical, it is less efficient to inactivate microbial
353
surrogates. This difference may be due to the electrostatic repulsion between the surface
354
of microbial surrogates and sulfate radical because they are both negatively charged. In
355
contrast, the non-charged hydroxyl radical likely can more easily attack the
356
microorganism surfaces. Besides oxidizing power and radical charge, hydroxyl radical’s
357
low selectivity in reactions with organic molecules may render higher disinfection
358
potency compared to sulfate radical and carbonate radical, which selectively react with
359
electron-rich molecules.
360
Regarding the difference among the microbial surrogates, MS2 is more vulnerable to
361
radical attack followed by E. coli and B. subtilis spores, meanwhile a lag phase in CT
362
profiles is only observed for E. coli and B. subtilis spores. These observations can be
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explained by the differences in their repair mechanisms and outer structures. The capsid
364
of MS2 is primarily consisted of proteins, which react with radical species at higher rates
365
than other biomolecules, such as polysaccharides and lipids,37 which are the main
366
building blocks of the outer membranes of E. coli. As for B. subtilis spores, it was
367
suggested that some enzymes on the outer layer can generate pigments which potentially
368
deactivate reactive oxygen species.42. The overall experimental results suggest that it
369
would be beneficial to apply oxidants to enhance UV disinfection of viruses.
370
Inactivation in real water matrices. The inactivation of microbial surrogates was
371
further examined in real water matrices under the similar UV fluence rate (2.2×10-7
372
Einstein·L-1·s-1) and oxidant dose (0.3 mM). Different water samples, including surface
373
water and wastewater secondary effluent, were tested (SI Table S1). Control experiments
374
counting CFU and PFU of spiked microbial surrogates in the SW and WW samples
375
showed comparable results to those spiked in PBS, indicating that the microbial
376
surrogates were negligibly affected by SW or WW components.
377
Experiments showed that, in surface water matrix, the inactivation profiles of E. coli
378
by UV/H2O2 and UV/PDS processes were almost identical to that by UV only (Figure
379
3A), whereas substantially enhanced inactivation of MS2 was achieved with the addition
380
of H2O2 or PDS to UV (Figure 3B). However, in wastewater matrix, the inactivation of
381
either E. coli or MS2 was at similar rates under UV with or without the addition of
382
oxidants (Figure 3C,D). Dynamic kinetic simulations with the input of the real water
383
component data were conducted to predict the concentrations of radical species (Table 1).
384
Specifically, photo-decomposition of nitrate was considered as a source for hydroxyl
385
radical; DOM was considered as scavengers for hydroxyl radical, sulfate radical and
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386
carbonate radical; radical reactions involving chloride, (bi)carbonate and sulfate were all
387
included in the kinetic simulations (SI Table S2). The calculated radical concentrations
388
were applied to eqn. 10 to obtain the predicted inactivation profiles of microbial
389
surrogates in the real water matrices. As Figure 3 shows, the predictions agreed well with
390
the experimental results. The good agreement between the predicted and experimental
391
inactivation indicates that the eqn. 10 captures the majority of disinfection actions for the
392
microbial surrogates investigated in this study.
393
Optimization of UV-based ADP. Oxidant dose effect on UV efficiency. Experiments
394
with the real water matrices showed that the UV-based ADP only enhanced the
395
inactivation of MS2 significantly in surface water matrix and great scavenging effects
396
occurred in wastewater matrix. The concentration of oxidants (0.3 mM) did not achieve
397
observable enhancement of the inactivation of E. coli and B. subtilis spores (Figure 4).
398
However, one may assume that a higher oxidant dose would overcome the scavenging
399
effects and achieve measurable inactivation enhancement. To test this hypothesis, model
400
simulations were performed by varying the dose of H2O2 or PDS in UV in surface water
401
and wastewater matrices. The term, UV efficiency (in log·(mJ·cm-2)-1), is defined as the
402
log inactivation of microbial surrogates normalized by the UV dose. The prediction of
403
UV efficiency was performed based on the kinetic model for radical concentrations and
404
eqn. 10 for microbial inactivation.
405
As Figure 4 shows, the increase of H2O2 dose or PDS dose has a great effect on the
406
UV efficiency for MS2, but has little effect on E. coli and B. subtilis spores, suggesting
407
the increase of oxidant dose would not be beneficial to enhance the inactivation efficacy
408
for E. coli and B. subtilis spores. The increase of H2O2 dose up to around 0.25 mM and
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409
0.5 mM enables the UV efficiency for MS2 to surpass that for B. subtilis spores in
410
surface water and wastewater, respectively (Figure 4A,C). H2O2 dose up to around 1 mM
411
(for surface water) and 2 mM (for wastewater) renders the UV efficiency for MS2 similar
412
to that for E. coli, which is four times higher than the UV efficiency for MS2 by UV only.
413
Further increase of H2O2 dose yields a tapering increase of UV efficiency for MS2 due to
414
the scavenging effects from H2O2 itself. A similar trend of UV efficiency profile for MS2
415
was also observed at varying PDS dose (Figure 4B,D). However, the effectiveness of
416
PDS is quite different in surface water versus wastewater matrices. Although significant
417
enhancement of UV efficiency for MS2 is obtained in surface water matrix at increasing
418
PDS dose (Figure 4B), the increase of PDS yields limited enhancement in wastewater
419
matrix (Figure 4D). Indeed, the addition of up to 10 mM PDS only triples the UV
420
efficiency for MS2 in wastewater, which is still lower than that for E. coli. Comparing all
421
the simulation results in Figure 4, the investigation suggests that the disinfection efficacy
422
of UV/PDS process is more susceptible to real water matrix effect than the UV/H2O2
423
process.
424
Overall, the change of UV efficiency suggests that it would be beneficial to add a
425
certain amount of H2O2 or PDS to boost the inactivation of MS2 in ADPs. However, the
426
cost of oxidants should also be considered in order to systematically optimize the
427
UV/oxidant ADP.
428
Energy optimization. Based on the discussion above, the application of additional
429
oxidants, especially H2O2, to create ADP condition can achieve substantial enhancement
430
on the inactivation of MS2, but not for E. coli and B. subtilis spores. Therefore, energy
431
optimization was only performed for MS2. In order to optimize ADPs, an economic
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432
analysis using the EE/O concept was performed. EE/O is defined as the electric energy to
433
achieve one order of inactivation, which gives a quantitative cost-effective evaluation of a
434
given condition. The electrical energy of UV lamp and the consumption of oxidants
435
would be considered in EE/O evaluation. The overall EE/O can be expressed using eqn.
436
11. EE/O =
437
(P/V) + α ⋅ [Oxidant] N −log N 0 t
(11)
438
where, P/V is the energy input of UV lamps with the unit of kWh·L-1; [Oxidant] is the
439
applied concentration of H2O2 or PDS (mM); α is the unit convertor to translate oxidant
440
amount to energy unit (i.e., 2.27×10-4 kWh·mmole-1 H2O2; 1.64×10-3 kWh·mmole-1 PDS,
441
the unit conversion is detailed in SI Text S6). The term, -log(N/N0)t is the inactivation of
442
MS2 under conditions corresponding to [Oxidant] and P/V values. Therefore, EE/O is
443
with the unit of kWh·L-1.
444
In order to achieve the most cost-effective condition, two alternative strategies are
445
commonly considered, installation of more UV lamps and addition of more oxidant dose.
446
Therefore, P/V and [Oxidant] were varied to predict the EE/O at each given UV and
447
oxidant doses. Applying the scan function of variables in Simbiology, radical
448
concentrations were predicted at UVC dose ranging from 2.4×10-5 to 4.8×10-4 Einstein·L-
449
1
450
by eqn. 10. A detailed demonstration of the calculation of EE/O is provided in SI Text S7.
451
The log(EE/O) at varying UVC doses and oxidant doses in surface water and
452
wastewater were calculated and shown in Figure 5, where cooler colors represent lower
453
EE/O while hotter colors represent higher EE/O. Without addition of oxidants (i.e., at x-
and oxidant dose from 0 to 1 mM. Then, the overall inactivation of MS2 was calculated
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454
axis = 0), the EE/O of UV alone is around 5.7×10-5 kWh·L-1. With the addition of H2O2
455
(Figure 5A,B), EE/O shifts to lower values in both surface water and wastewater, with the
456
exception that EE/O decreases then increases at the turning point around 0.05 mM H2O2
457
in wastewater below UVC dose of 1.0×10-4 Einstein·L-1. In UV/PDS ADP, the influence
458
of PDS to decrease EE/O starts to reverse at below 5×10-5 Einstein·L-1 of UVC dose in
459
surface water (Figure 5C), whereas EE/O always increases with the increase of PDS dose
460
in wastewater (Figure 5D). These trends have confirmed that it would be beneficial to
461
add a certain amount of oxidants to improve energy efficiency in the inactivation of MS2.
462
The solid line in Figure 5 presents the UV and oxidant dose combination to achieve
463
certain extent of inactivation. To meet a criteria of 4-log inactivation, certain oxidants
464
must be added at the UV irradiation below 4.2×10-4 Einstein·L-1. In the UV/H2O2 ADP,
465
the most energy-efficient condition is at around 5×10-5 Einstein·L-1 UV and 0.1 mM
466
H2O2 with the EE/O of 1.4×10-5 kWh·L-1 in surface water, and around 1.5×10-4
467
Einstein·L-1 UV and 0.08 mM H2O2 with the EE/O of 2.2×10-5 kWh·L-1 in wastewater.
468
As for UV/PDS ADP, the most energy-efficient condition is at around 1.0×10-4
469
Einstein·L-1 UV and 0.05 mM PDS with the EE/O of 3.5×10-5 kWh·L-1 in surface water,
470
whereas there is no optimal conditions within 5×10-4 Einstein·L-1 UV as the increase of
471
PDS always yields higher EE/O in wastewater. Therefore, based on the overall EE/O
472
evaluation, the UV/H2O2 ADP is more favorable than the UV/PDS ADP in both surface
473
water and wastewater matrices. The typical reduced equivalent dose in the UV
474
disinfection process is below 90 mJ/cm2 (i.e., ~5×10-4 Einstein·L-1), which is just
475
sufficient to achieve 4-log inactivation of MS2. However, the EE/O value (5.8×10-5
476
kWh·L-1) is much higher than the optimal points of UV/H2O2 ADP (i.e., 1.4×10-5 kWh·L-
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1
for surface water; 2.2×10-5 kWh·L-1 for wastewater). Therefore, by combining UV
478
irradiation and H2O2, nearly two thirds of the energy consumption would be saved.
479
Environmental Implications. This study has demonstrated the disinfection potency
480
of novel UV-based ADPs, as well as provides new information on the effects of radical
481
species on microorganisms. This study is among the first to quantitatively investigate
482
sulfate radical and carbonate radical as reactive species that can inactivate
483
microorganisms. It was also elucidated that superoxide radical exerts negligible effect to
484
inactivate microorganisms. The new knowledge gained from this study is useful for
485
designing optimized UV-based ADPs for water treatment. Specifically, this study
486
suggests that UV/H2O2 is more cost-effective than UV disinfection for virus removal,
487
whereas it is not beneficial to upgrade UV disinfection to UV-based ADP for the
488
inactivation of bacteria and spores. For water treatment facilities to achieve lower EE/O,
489
one can adjust the number of UV lamps and oxidant doses based on the specific water
490
quality using the kinetic model provided in this study.
491
Results of this study are also relevant for certain natural sunlit water systems, where
492
radical species are generated by sunlight-excited photosensitizers. The difference in the
493
resistance of bacteria, viruses and spores to radical species also suggests that the presence
494
of radicals is likely to impact the microorganism community in water systems. In water
495
systems with a higher radical concentration, the microorganisms which are more resistant
496
to radical attack are likely to accumulate over the ones that are more vulnerable.
545 546
ASSOCIATED CONTENT
547
Supporting Information. Text S1-S7, Tables S1−S3 and Figures S1−S4. This material is
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Environmental Science & Technology
available free of charge via the Internet at http://pubs.acs.org.
549 550
ACKNOWLEDGMENTS
551
This project was support by the Southern Research Institute. The authors would like to
552
thank Dr. Brian Mastin for his efforts on the initiation of this research.
553 554
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Figure 1. Inactivation of (A) E. coli, (B) bacteriophage MS2 and (C) Bacillus subtilis spores under UV, UV/H2O2 and UV/PDS conditions ([H2O2] = 0.3 mM, [PDS] = 0.3 mM, [PBS] = 3 mM at pH 7.0). UV fluence rate at 2.2×10-7 Einstein·L-1·s-1 was employed. Error bars represent one standard deviation of the means (n = 3).
671 672 673 674 675 676 677 678 679 680
Figure 2. Comparison of the inactivation of microbial surrogates under UV irradiation and UV with different dominated reactive species. Numbers on the bars indicate the corresponding conditions shown in Table 2. (A) superoxide radical dominated condition ((7*): 0.1 M TBA in PBS; (7): 3 mM H2O2, 0.3 mM PDS and 0.1 M TBA in PBS); (B) carbonate radical dominated condition ((6*): 0.1 M NaHCO3 in PBS; (6): 0.3 mM H2O2 and 0.1 M NaHCO3 in PBS); and (C) sulfate radical dominated condition ((5*): 0.1 M TBA in PBS; (5): 3 mM PDS and 0.1 M TBA in PBS). Conditions (5-7) were all under UV fluence rate of 2.2×10-7 Einstein·L-1·s-1. UV fluence (8.9×10-7 – 4.4×10-6 Einstein·L1 -1 ·s ) varied slightly for different conditions as detailed in SI Table S4. Error bars represent one standard deviation of the means (n = 3).
681 682 683
Figure 3. Inactivation of E. coli and bacteriophage MS2 in surface water (SW) or wastewater (WW) (UV fluence rate = 2.2×10-7 Einstein·L-1, [H2O2]0 = 0.3 mM, [PDS] = 0.3 mM). Error bars represent one standard deviation of the means (n=3).
684 685
Figure 4. UVC efficiency (log of inactivation normalized by UV dose) in surface water (SW) and wastewater (WW) at various levels of H2O2 or PDS dose.
686 687 688
Figure 5. Log(EE/O) (in kWh·L-1) for the inactivation of MS2 in surface water (SW) and wastewater (WW) with UV/H2O2 ADP or UV/PDS ADP. Solid lines represent log of inactivation.
689 690
Table 1. Simulated Molar Concentrations (in M) of Reactive Species with Various Components in Solutions
691 692
Table 2. The Disinfection CT (Concentration × Exposure Time) Values of UVC Irradiation and Radical Species.
693
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694 1
1
(B) MS2
(A) E. coli
0
0
-log (N/N0)
-log (N/N0)
-1 -2 -3 UV UV/H2O2
-4
UV UV/H2O2
-2
UV/PDS
-3
UV/PDS
-4
-5 -6
-5 0
695
-1
2
4
6
8
10
12
0
20
UV dose (mJ/cm2)
40
60
80
100
UV dose (mJ/cm2)
1
(C) B. subtilis spores
-log (N/N0)
0 -1 -2 -3
UV UV/H2O2
-4
UV/PDS
-5 0
696 697 698 699 700 701
5
10
15
20
25
30
2
UV dose (mJ/cm )
Figure 1. Inactivation of (A) E. coli, (B) bacteriophage MS2 and (C) Bacillus subtilis spores under UV, UV/H2O2 and UV/PDS conditions ([H2O2] = 0.3 mM, [PDS] = 0.3 mM, [PBS] = 3 mM at pH 7.0). UV fluence rate at 2.2×10-7 Einstein·L-1·s-1 was employed. Error bars represent one standard deviation of the means (n = 3).
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(A) UV/TBA(/H2O2/PDS)
-log (N/N0)
6
E. coli
4
B. subtilis spores
MS2
2
7*
7
7*
7
7*
7
0
702 (B) UV/NaHCO3(/H2O2)
-log (N/N0)
6
MS2 E. coli B. subtilis spores
4
2
6*
6
6*
6
6*
6
0
703
-log (N/N0)
6
(C) UV/TBA(/PDS)
E. coli
4
B. subtilis spores
MS2
2
5*
704 705 706 707 708 709 710 711 712 713 714
5
5*
5
5*
5
0
Figure 2. Comparison of the inactivation of microbial surrogates under UV irradiation and UV with different dominated reactive species. Numbers on the bars indicate the corresponding conditions shown in Table 2. (A) superoxide radical dominated condition ((7*): 0.1 M TBA in PBS; (7): 3 mM H2O2, 0.3 mM PDS and 0.1 M TBA in PBS); (B) carbonate radical dominated condition ((6*): 0.1 M NaHCO3 in PBS; (6): 0.3 mM H2O2 and 0.1 M NaHCO3 in PBS); and (C) sulfate radical dominated condition ((5*): 0.1 M TBA in PBS; (5): 3 mM PDS and 0.1 M TBA in PBS). Conditions (5-7) were all under UV fluence rate of 2.2×10-7 Einstein·L-1·s-1. UV fluence (8.9×10-7 – 4.4×10-6 Einstein·L1 -1 ·s ) varied slightly for different conditions as detailed in SI Table S4. Error bars represent one standard deviation of the means (n = 3).
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1 0
(A) E. coli, SW
0
(B) MS2, SW -2
-log (N/N0)
-log (N/N0)
-1 -2 UV UV/H2O2
-3
-4
UV/PDS UV (model, R2 = 0.9699) UV/H2O2 (model, R2 = 0.9325)
-4 -5
UV UV/H2O2
-6
UV/PDS UV (model, R2 = 0.9133) UV/H2O2 (model, R2 = 0.9501)
-8
UV/PDS (model, R2 = 0.9864)
UV/PDS (model, R2 = 0.8741)
-6
-10 0
1
2
3
4
5
6
0
5
10
15
Time (min)
715
20
25
30
35
Time (min)
1
1
(D) MS2, WW
(C) E. coli, WW
0
0
-log (N/N0)
-log (N/N0)
-1 -2 -3
UV UV/H2O2
-4
UV/PDS UV (model, R2 = 0.9913) UV/H2O2 (model, R2 = 0.9464)
-5
-1 -2 UV UV/H2O2
-3
UV/PDS UV (model, R2 = 0.8066) UV/H2O2 (model, R2 = 0.9420)
-4
UV/PDS (model, R2 = 0.8901)
2
UV/PDS (model, R = 0.9325) -6
-5 0
716 717 718 719 720
1
2
3
Time (min)
4
5
6
0
5
10
15
20
25
30
35
Time (min)
Figure 3. Inactivation of E. coli and bacteriophage MS2 in surface water (SW) or wastewater (WW) (UV fluence rate = 2.2×10-7 Einstein·L-1, [H2O2]0 = 0.3 mM, [PDS] = 0.3 mM). Error bars represent one standard deviation of the means (n=3).
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721 1.0
2.0
-log(N/N0) / (I0t) (cm /mJ)
0.8
2
0.6 E. coli MS2 B. subtilis spores
0.4
E. coli MS2 B. subtilis spores
1.8
2
-log(N/N0) / (I0t) (cm /mJ)
(A) SW, UV/H2O2
0.2
1.6
(B) SW, UV/PDS
1.4 1.2 1.0 0.8 0.6 0.4 0.2
0.0 0
2
4
6
8
0.0
10
0
722
4
-log(N/N0) / (I0t) (cm /mJ)
2
2
-log(N/N0) / (I0t) (cm /mJ)
0.5 0.4
(C) WW, UV/H2O2
0.3 0.2 0.1 0.0 0
2
4
6
8
10
0.30
E. coli MS2 B. subtilis spores
0.6
6
PDS (mM)
0.7
723
2
H2O2 (mM)
8
10
0.25 E. coli MS2 B. subtilis spores
0.20 0.15 0.10
(D) WW, UV/PDS
0.05 0.00 0
2
H2O2 (mM)
4
6
8
10
PDS (mM)
724 725 726
Figure 4. UVC efficiency (log of inactivation normalized by UV dose) in surface water (SW) and wastewater (WW) at various levels of H2O2 or PDS dose.
727 728 729 730
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(A) SW, UV/H2O2
731
(B) WW, UV/H2O2
732
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(C) SW, UV/PDS
733
(D) WW, UV/PDS
734 735 736 737
Figure 5. Log(EE/O) (in kWh·L-1) for the inactivation of MS2 in surface water (SW) and wastewater (WW) with UV/H2O2 ADP or UV/PDS ADP. Solid lines represent log of inactivation.
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738 739
Table 1. Simulated Molar Concentrations (in M) of Reactive Species with Various Components in Solutions Reaction System
740 741 742 743
•
OH
(M)
SO •4− (M)
CO •3− (M)
HO •2 /O •2− (M)
(1)
UV/H2O2
1.78×10-13
1.64×10-19
4.28×10-13
2.83×10-8
(2)
UV/PDS
3.47×10-13
2.09×10-13
3.53×10-14
6.84×10-14
(3)
UV/H2O2/TBA
2.09×10-17
3.12×10-24
5.67×10-17
2.17×10-11
(4)
UV/PDS/TBA
2.25×10-18
2.17×10-14
2.26×10-15
3.06×10-24
(5)
UV/10×PDS/TBA
2.21×10-17
2.13×10-13
2.22×10-15
3.20×10-22
(6)
UV/H2O2/NaHCO3
1.19×10-15
1.21×10-25
8.64×10-12
8.95×10-8
(7)
UV/10×H2O2/PDS/TBA
2.09×10-16
1.52×10-14
2.59×10-15
2.05×10-8
(8)
UV/H2O2 in SW
1.36×10-14
9.14×10-23
3.86×10-13
(9)
UV/PDS in SW
7.30×10-15
6.60×10-14
1.30×10-13
(10)
UV/H2O2 in WW
2.31×10-15
3.60×10-23
1.20×10-14
(11)
UV/PDS in WW
1.50×10-16
4.17×10-15
2.15×10-13
Solution medium (1-7) was 3.0 mM phosphate buffer at pH 7.0; oxidant concentration was 0.3 mM in most cases or was 3.0 mM in where 10× was indicated; TBA concentration was 0.1 M; NaHCO3 concentration was 100 mM; background total inorganic carbon = 4.78×10-5 M; UV fluence rate was 2.2×10-7 Einstein·L-1·s-1; total simulation time was 120 s.
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744 745
Table 2. Disinfection CT (Concentration × Exposure Time) Values of UVC Irradiation and Radical Species Determined in This Study. E. coli
UVC (254 nm) Hydroxyl radical Sulfate radical
B. subtilis spores
CTlag
Slope
CTlag
Slope
CTlag
Slope
1.56×10-5
9.20×104
0
9.10×103
2.96×10-5
4.14×104
2.50×1012 7.55×1012 a 4.25×109 b
0
9.30×1012 4.17×1012 a 1.93×1011 c
1.62×10-13
6.23×1011