Comparison of Antibiotic Resistance Removal Efficiencies Using

Article pubs.acs.org/est

Comparison of Antibiotic Resistance Removal Efficiencies Using Ozone Disinfection under Different pH and Suspended Solids and Humic Substance Concentrations Gijung Pak,† Dennis Espineli Salcedo,‡ Hansaem Lee,§ Junsik Oh,‡ Sung Kyu Maeng,∥ Kyung Guen Song,# Seok Won Hong,# Hyun-Chul Kim,⊥ Kartik Chandran,∇ and Sungpyo Kim*,†,‡,¶ †

Department of Environmental Engineering and ‡Program in Environmental Technology and Policy, Korea University, Sejong 30019, Republic of Korea § Water & Environment R&D Team, Research & Development Division, Hyundai Engineering & Construction Co., Ltd., Seoul 110-920, Republic of Korea ∥ Department of Civil and Environmental Engineering and ⊥Water Resources Research Institute, Sejong University, Seoul 05006, Republic of Korea # Center for Water Resource Cycle Research, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea ∇ Department of Earth and Environmental Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, United States ABSTRACT: This study mainly evaluated the effectiveness of ozonation toward the enhancement of the removal efficiencies of antibiotic-resistant bacteria (ARB), pB10 plasmid transfer, and pB10 plasmids under different pH and suspended solids (SS) and humic acid concentrations. First, chlorination was tested as a reference disinfection process. Chlorination at a very high dose concentration of Cl2 (75 mg L−1) and a long contact time (10 min) were required to achieve approximately 90% ARB and pB10 plasmid transfer removal efficiencies. However, even these stringent conditions only resulted in a 78.8% reduction of pB10 plasmid concentrations. In case of ozonation, the estimated CT (concentration × contact time) value (at C0 = 7 mg L−1) for achieving 4-log pB10 plasmid removal efficiency was 127.15 mg·min L−1, which was 1.04- and 1.25-fold higher than those required for ARB (122.73 mg·min L−1) and a model nonantibiotic resistant bacterial strain, E. coli K-12, (101.4 mg·min L−1), respectively. In preventing pB10 plasmid transfer, ozonation achieved better performance under conditions of higher concentrations of humic acid and lower pH. Our study results demonstrated that the applicability of CT concept in practice, conventionally used for disinfection, might not be appropriate for antibiotic resistance control in the wastewater treatment process. Further studies should be conducted in wastewater engineering on how to implement multiple barriers including disinfection to prevent ARB and ARG discharge into the environment.

1. INTRODUCTION

ARG dissemination before they are discharged into the environment from sources such as wastewater treatment plants. Traditionally, disinfection processes such as chlorination have been used in controlling pathogens and indicators of fecal contamination in treated wastewater effluent before discharge to the environment. However, according to recent studies, these disinfection processes are not reliable for removing ARB or ARGs.8,9 In some cases, chlorination can select for ARB in the wastewater microbial community.13 Also, free chlorination could result in the formation of potentially carcinogenic disinfection byproducts. Therefore, more powerful and sophisticated disinfection technologies and strategies are needed in controlling not just conventional indicators of fecal contamination, but also ARB and ARGs in treated wastewater.

Recently, there have been elevated concerns about the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) via environmental sources.1,2 ARB and ARGs are hazards to public health and have been increasingly detected in the environment both in terms of their diversity3,4 as well as their levels because of the continuous transport and/or stimulation of ARGs by various human activities including domestic wastewater discharge.5 Wastewater treatment plants (WWTPs) are the primary point sources of environmental ARB and ARGs.6−10 ARGs have been shown to persist after their introduction into the environment.11 In addition, ARGs are usually contained in plasmids or integrons, transmissible genetic elements that can be transferred to other organisms via horizontal gene transfer (HGT), which is believed to be one of the major drivers of the distribution of ARGs in the environment.1 In addition, even naked DNA encoding ARGs can be transferred to other bacteria via transformation.12 Therefore, it is important to control the pathways of ARB and © XXXX American Chemical Society

Received: March 17, 2016 Revised: June 9, 2016 Accepted: June 23, 2016

A

DOI: 10.1021/acs.est.6b01340 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

2. MATERIALS AND METHODS 2.1. Bacterial Culture Preparation. E. coli DH5α (pB10) was selected for use as an antibiotic-resistant strain and a plasmid donor in this study. On the basis of the comprehensive study, pB10 has several transposons which contain ARGs. Aside from a Tn501-like mercury resistance (mer) transposon, there are other transposons that contain ARGs for streptomycin (Tn5393c streptomycin-resistance transposon), tetracycline (Tn1721-like tetracycline-resistance transposon), and one encoding small protein sul1, a dihydropteroate synthetase conferring sulfonamide resistance. The oxa2 cassette encodes a β-lactamase of the oxacillin-hydrolyzing type, conferring resistance to amoxicillin.29 For the pB10 plasmid transfer study, Pseudomonas aeruginosa PAK exoT, which is resistant to gentamycin,30 was used as a recipient. DH5α (pB10) and P. aeruginosa cultures were grown in lysogeny broth (LB) (Difco Laboratories, Sparks, MD 21152 USA) medium, supplemented with appropriate antibiotics (donor: amoxicillin (50 μg mL−1) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany), streptomycin (50 μg mL−1) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany), sulfamethoxazole (150 μg mL−1) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) and tetracycline (20 μg mL−1) (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany); recipient: gentamycin (50 μg mL−1) (Duchefa Biochemie, Haarlem, The Netherlands)), on a rotary shaker at 150 rpm at 30 °C for 24 h. E. coli K-12 was also selected as a non-antibiotic-resistant strain for comparison with the antibiotic-resistant strains.31,32 Before the experiment, tetracycline sensitivity was tested for the E. coli K-12; we confirmed that this species is not resistant to tetracycline. For disinfection tests, cultures were harvested at an optical density (OD) value of 1.3. 2.2. Disinfection Experiments. Each disinfection experiment setup using chlorination and ozonation was separately designed and conducted in a triplicate manner in calculating the removal efficiencies of antibiotic resistance. 2.2.1. Free Chlorination. Cl2 refers to free chlorine, which is the sum of Cl2, HOCl, and OCl− concentrations. Sodium hypochlorite (NaOCl) (Samchun Pure Chemical Co., Ltd., Korea) stock solution was used to investigate the disinfection efficiency of the chlorination process. An appropriate amount of stock solution was added to the E. coli DH5α cultures in phosphate buffer (0.63 mM) solution to achieve various final concentrations of chlorine (Cl2) (3, 7.5, 20, and 75 mg L−1). Samples were collected at several time intervals (1, 3, 5, and 10 min). In determining the antibiotic doses, our previous study was followed.33 The used disinfectant concentrations ranged from the one used in practice to very high concentrations to observe the significant differences of antibiotic resistance removal.34 Residual chlorine was quenched by adding sodium thiosulfate (Na2S2O3) prior to the evaluation of the efficiency of disinfection.35 2.2.2. Ozonation. Ozone was generated from pure oxygen (99.9%) by using an ozone generator (LAB 2B, Ozonia, Korea). The flow rate of pure oxygen to the ozone generator was maintained at 4−5 L min−1. The ozone−oxygen mixture was introduced at a constant rate to the bottom of the reactor via a porous gas diffuser. The concentration of ozone gas introduced was measured using an ozone analyzer (Orbisphere Model 3600, Switzerland). The initial ozone concentrations used in the experiments were 3, 5, 7, and 10 mg L−1. Samples

Advanced oxidation processes (AOPs) are designed to remove toxic or nonbiodegradable organic compounds through the action of a number of reactive oxygen species (ROS) including ozonide radical (O3−•), hydrogen trioxyl radical (HO3•), hydroperoxyl radical (HO2•), and hydroxyl radical (•OH).14 Recently, several studies have shown that AOP technology shows promise for removing micropollutants such as pharmaceuticals and microbial contents.15−18 Among these AOP options, ozone processing has gained attention for removing micropollutants not only in drinking water treatment processes but also in WWTPs.19,20 McKinney and Pruden, however, showed that UV disinfection has limited potential to damage ARGs in water and wastewater effluents.21 Michael et al. demonstrated the capacity of solar-driven AOP (solar photo Fenton) to not just reduce wastewater toxicity but also completely eliminate ARB.22 These examples of AOPs can produce •OH that enhances the oxidation potential to increase effectivity against micropollutants. Although several studies have recently been conducted on ARB and ARG removal efficiencies during water or wastewater disinfection processes,15,22−24 few comprehensive studies have been conducted using ozonation. Alexander et al. recently conducted an investigation on an ozone treatment system and its ARB and ARG reduction.17 Ferro et al. used UV/H2O2 process in the investigation of antibiotic resistance removal, but neither paper addresses ARG transfer efficiencies.18 Plasmids carry a wide range of genes that are involved in bacterial social behavior.25 As mentioned above, they can carry ARGs that are of particular importance to public health because they have the potential to spread ARGs to pathogens.1 One case study showed the risk of the transmission of an antibioticresistant Escherichia coli through household contact and plasmid transfer.26 Plasmids act as vehicles for the HGT of genes essential for the evolution of bacterial traits, which have an impact to human health.25 However, the efficacy of AOP treatment options on the rates and extents of removal of plasmid, which has ARGs, and plasmid transfer has not been elucidated, especially, under various water quality conditions. For example, it is a wellknown fact that free radical formation is dependent on pH and dissolved organic matter (DOM) such as humic substances, usually contained in wastewater, could also affect oxidation reaction of ozone.27,28 Accordingly, it is expected that antibiotic resistance removal efficiency should be affected by these factors. Furthermore, it has not been clearly studied whether the CT (disinfectant concentration × contact time) values, which are commonly used and accepted in disinfection practice, is still valuable for the proper reduction of antibiotic resistance including plasmid transfer rate and plasmid abundance. Therefore, the main objective of this research was to quantitatively investigate the effects of ozonation as a disinfection process to the removal efficiencies of ARB, pB10 plasmid transfer, and pB10 plasmid concentrations under different pH and suspended solids (SS) and humic acid concentrations. Chlorination was tested as a reference disinfection process because it is widely used for wastewater disinfection.34 To our knowledge, this is the first comprehensive study that has evaluated the ozonation disinfection efficiencies on antibiotic resistance control under varying pH, SS, and humic acid conditions. B


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survive. After overnight incubation, transconjugants were counted, and averages were calculated using triplicate plates. 2.4.3. pB10 Plasmid Measurement. The decrease in pB10 plasmid levels after disinfection was evaluated by quantitative polymerase chain reaction (q-PCR). After disinfection, the culture was centrifuged at 3500 × g for 5 min at room temperature, and the pellet was used for DNA extraction by using a NucleoBond PC100 Kit and AX 100 columns (Macherey-Nagel, Dü r en, Germany), according to the manufacturer protocol. The concentration and purity of the extracted DNA from E. coli was evaluated by ultraviolet absorbance spectrophotometry at 260 nm. The presence of the extracted pB10 plasmid was confirmed by PCR, using a highly specific primer set (F5′-CAATACCGAAGAAAGCATGCG-3′ and R5′-AGATATGGGTATAGAACAGCCGTCC-3′). The qPCR conditions were similar to those employed in a previous study.38 2.5. 3D Fluorescence EEM Spectrophotometry. Humic acid concentration was measured using 3D fluorescence excitation−emission matrix (EEM) spectrophotometric technique (PerkinElmer LS-50B, USA). For the spectroscopic analysis, the initial concentration of humic acid was controlled at 20 mg L−1 to minimize the inner filter effect, which is a problem in fluorescence measurement when the absorbing light at one end of the cuvette is not available on the other end. 3D fluorescence EEM spectrophotometry was used to measure DOM. Fluorescence spectra were collected using a PerkinElmer LS-50B luminescence spectrometer, which uses a 450W xenon lamp source. The acquisition interval and integration time were maintained at 1 nm and 0.1 s, respectively. Right-angle geometry was used for liquid samples in a 10 mm fused-quartz cuvette. 3D spectra were obtained by repeatedly measuring the emission spectra within the range of 200−600 nm, with excitation wavelengths from 200 to 400 nm, spaced at 5 nm intervals in the excitation domain. Spectra were then concatenated into an EEM. 2.6. Data Analysis. The inactivation rate constant (k) for E. coli K-12, ARB, pB10 plasmid transfer, and pB10 plasmid by ozone inactivation was calculated using the first-order Chick− Watson expression ln (N/N0) = −kCT, in which N/N0 is the survival ratio of E. coli K-12, ARB, pB10 plasmid transfer, and pB10 plasmid concentrations after contact with the disinfectant at an average concentration (C) for a particular period (T) and k is the inactivation rate constant.39 CT values were calculated at 2-log, 3-log, and 4-log. During ozone generation, the residual ozone concentration was frequently checked and the value was maintained as initial concentrations (C0) 3, 5, 7, and 10 mg L−1. To get the ARB removal efficiencies, the results of ARB removal percentage (%) of the average CFU mL−1 were plotted against times of disinfection. pB10 plasmid removal efficiencies were obtained by plotting the results of the qPCR against times of disinfection. Lastly, to compute the pB10 plasmid transfer efficiencies, the CFU mL−1 of the transconjugants P. aeruginosa cells that were able to receive the copies of pB10 plasmid were enumerated and plotted as removal percentage (%) against times of disinfection. Error bars correspond to 95% confidence intervals. 2.7. Statistical Analysis. The purpose of this study is to evaluate ozone disinfection efficiencies on antibiotic resistance removal, so statistical analysis was conducted to compare the removal efficiencies among different pH, different SS concentrations, and different humic acid concentrations. Using the t test results, statistical significances were calculated

were collected at several time intervals (1, 3, 5, and 10 min) for evaluation of the efficiency of disinfection. 2.2.3. Effects of pH, SS, and Humic Acids. The pH of the samples was measured using an Orion 3 Star pH meter (Thermo Scientific, Waltham, MA, USA). SS concentrations were measured using Standard Methods 2540D and 2540E.36 SS stock solution (5 g L−1) was prepared from activated sludge collected from an aeration basin in the Cheongwon Wastewater Treatment Plant located at Osong in Chungbuk, Korea. Collected activated sludge was autoclaved at 121 °C, 15 psi for 20 min, and centrifuged at 3500 × g for 5 min at room temp. Centrifuged solids were diluted with 0.63 mM phosphate buffer solution to achieve SS stock solution concentrations (5, 10, 15, and 20 mg L−1). Humic acid stock solutions (1 g L−1) were prepared by suspending 0.1 g of commercially obtained humic acid (Wako Pure Chemical Industrial Co., Osaka, Japan) in 100 mL of distilled water and dissolving it with continuous stirring. The pH was adjusted to 11.0 using 0.5 M NaOH, and the solution was stirred continuously for 24 h. The solution was filtered using a 0.2 μm membrane filter. The pH was then lowered to 7.0 using 0.5 M H3PO4, and the solution was continuously stirred for another 24 h. The solution was filtered again using a 0.2 μm membrane filter. 2.4. Antibiotic Resistance. The removal efficiencies of antibiotic resistance in each disinfection process was evaluated via the removal of ARB, pB10 plasmid transfers, and pB10 plasmid copies. 2.4.1. Measurement of ARB Concentrations. The decrease in the number of viable E. coli colonies following chlorination or ozonation was measured using a culture-based technique. Disinfected (for all the collection times) or nondisinfected samples containing E. coli DH5α (pB10) were serially diluted with phosphate buffer (0.63 mM) to produce between 30 and 300 colonies on each LB agar plate. After 16 h of incubation at 37 °C, when pB10 plasmid transfer efficiencies were maximized, the colonies on the plates were counted (data is not shown here). For each sample, triplicate plates were prepared, and the results were averaged. A total of 0.1 mL of diluted sample was then transferred onto the LB agar plates. 2.4.2. pB10 Plasmid Transfer Efficiency Analysis. The impact on pB10 plasmid conjugation transfer following disinfection was evaluated using a modified plasmid transfer mating technique.37 Disinfected (or nondisinfected) E. coli DH5α (pB10) and P. aeruginosa PAK exoT cultures were harvested, and each culture was centrifuged at 3500 × g for 5 min. After discarding most of the supernatant, each culture (donor and recipient) was resuspended in the remaining liquid. Then, cultures were mixed together, inoculated on LB media plates, and incubated for 16 h at 37 °C. After incubation, the pellets on plates were collected and resuspended in 1 mL of LB broth, transferred to a tube, and vortexed. The mixed cultures were serially diluted and spread on LB media plates containing tetracycline (2 mg L−1) and gentamycin (10 mg L−1). In counting only transconjugants on media and minimizing the usage of combination of antibiotics, preliminary experiments were conducted to confirm which antibiotic combinations and what concentrations should be used.37 On the basis of the experimental results, 2 mg L−1 tetracycline and 10 mg L−1 gentamycin concentrations were determined. This combination kills E. coli (donor) and P. aeruginosa (recipient) while allowing the transconjugants (P. aeruginosa PAK exoT (pB10)) to C


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Environmental Science & Technology based on the p value at 95% confidence level with one-tail, to determine if two sets of data are significantly different from each other.

3. RESULTS AND DISCUSSION 3.1. Efficacy of Disinfection Processes for Controlling ARB and Plasmid. Recently, several studies have evaluated the efficacy of conventional disinfection processes for the removal of ARB and ARGs in water environment.22,40,41 However, few studies have been conducted on tracking in parallel the fate of ARB, plasmid transfer, and plasmid abundance after disinfection. Before testing the ozonation process, chlorination was tested as a reference disinfection process because it is commonly used in practice. The removal efficiencies of ARB, pB10 plasmid transfer, and pB10 plasmid at different concentrations of free Cl2 with varying reaction times (maximum 10 min) are presented in Figure 1. To achieve approximately 90% removal efficiency of ARB and pB10 plasmid transfer, a very high concentration of Cl2 (75 mg L−1) and a long contact time (10 min) were required. However, even these stringent conditions resulted in only a 78.8% removal in ARB and pB10 plasmid concentrations In typical engineering practice, the Cl2 dosage used in treated domestic wastewater is lower than 10 mg L−1.34 In addition, the estimated CR × T (residual disinfectant concentration × contact time) values with Cl2 are approximately 0.1−0.8 mg min L−1 for 90−99% removal of bacteria (1−2 log removal).34 In our study, however, 99% (2-log removal) removal of ARB, pB10 plasmid transfer, and pB10 plasmid concentration was not achieved even at a CT value of 750 mg min L−1. The CT values in this study might be overestimated because the calculated CT values were based on the initial disinfectant concentration (C), not residual disinfectant concentration (CR). It is clear that the required CT value for pB10 plasmid removal is more than that needed for ARB removal. For example, if we assume that the residual chlorination concentration (CR) is following a first-order equation (dCR/dt = −αCR) and decay value (α) as 0.001−0.01 min−1 in our batch reactor, the expected CR concentration ranged from 67.8 to 74.2 mg/L and estimated CRT will be 678−742 mg min L−1.42 Although CRT values are more representative than CT values, there is no attempt to calculate real residual chlorine concentration in this study because the primary purpose of the CT value calculation by chlorination is to compare it with the CT values used in practice. The lack of measured residual concentrations precludes extension of these results compared to other conditions and studies. However, within this study, valid inferences can still be made based on the administered dose relating to reduction of ARB, conjugative plasmid transfer, and pB10 plasmid instead of calculating real CT values. Table 1 summarizes the inactivation rate constant (k), initial chlorine concentration (C0), and estimated contact time (T) at 2-log, 3-log, and 4-log to achieve inactivation of antibiotic resistance by chlorination under our experimental setup. As mentioned above, because we do not attempt to find the exact residual concentration CR in the batch reactor, we just tried to compare the required contact time (T) for each removal using Figure 1 data. As estimated in the previous section, longer chlorination contact time is required for removal efficiencies of ARB, pB10 plasmid transfer, and pB10 plasmid. Previous studies have similarly reported that ARB and/or ARGs are more resistant to conventional disinfection processes, such as chlorination or UV treatment, than are non-ARB. In

Figure 1. Removal efficiency profiles of (a) ARB, (b) pB10 plasmid transfer, and (c) pB10 plasmid using chlorination. Performed in triplicates; error bars correspond to 95% confidence intervals.

some cases, the percentage of antibiotic-resistant phenotypes increased in the total bacterial cells remaining using chlorination or UV disinfection.43,44 In other studies, the abundance of ARGs was not significantly reduced after UV or Cl2 disinfection.6,24 Fiorentino et al. recently showed complete inactivation of multidrug-resistant (MDR) E. coli by chlorination and H2O2/sunlight processes.45 In their paper, MDR E. coli showed much higher resistance to disinfection processes compared to a significant fraction of the total E. coli population. When MDR E. coli population was exposed chlorination and H2O2 sunlight processes, there was a slower rate of inactivation compared to that achieved with the total E. coli population. D


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Environmental Science & Technology Table 1. Inactivation Rate Constants and Contact Time (T) to Achieve 2-log, 3-log, and 4-log Inactivation by Chlorination T = ln(N/N0)/k (min)a

ARB

pB10 plasmid transfer

pB10 plasmids a

C0 (mg L−1)a

k (min−1)

2 log10 (99%)

3 log10 (99.9%)

4 log10 (99.99%)

3 7.5 20 75 3 7.5 20 75 3 7.5 20 75

0.0208 0.0401 0.0591 0.1057 0.0175 0.0384 0.0548 0.0987 0.0192 0.0248 0.0411 0.0741

274.22 165.09 128.61 84.41 325.93 172.40 138.70 90.40 297.07 266.94 184.94 120.41

384.92 222.51 167.57 106.20 457.51 232.36 180.72 113.73 417.00 359.78 240.96 151.49

495.62 279.93 206.53 127.98 589.08 292.32 222.74 137.06 536.92 452.63 296.98 182.56

C0 = initial concentration; T = contact time

Roller et al. observed that the DNA-transforming activity of Haemophilus inf luenzae was resistant up to 6-log inactivation of H. inf luenzae by chlorine dioxide (ClO2).46 In addition, they reported that a very high concentration (20 mg L−1) of ClO2 with a 5 min contact time only slightly inhibited the transformability of partially purified DNA. These studies imply that ARGs can be disseminated into the environment via transformation in which free DNA from one organism is taken up by another organism and as a result develop antibiotic resistance,47 even if the ARB has already been killed or inactivated by the disinfection process.48 Ozone, as a strong oxidant, can effectively inactivate many kinds of microorganisms. It has a stronger oxidizing ability (as compared to that of chlorine), reacting with the organics on the cell wall and causing severe surface damage before penetrating into the cell plasma.49 The removal profiles for ARB, pB10 plasmid transfer, and pB10 plasmids as a function of ozone concentration and contact time are presented in Figure 2. Similarly, for disinfection, when ozone was applied to the reactor, a more effective removal in ARB, pB10 plasmid transfer, and pB10 plasmids was achieved than when chlorination was applied; removals of 105−106 (−log(N/N0) = 5−6) were achieved when high concentrations of ozone (7− 10 mg L−1) were applied for contact times of 10 min. For quantifying the inactivation rate of antibiotic resistance by ozonation under our experimental setup, inactivation rate constant (k) and CT values were calculated and summarized in Table 2. The CT concept is a simple approach that was used to develop disinfection requirements under the Surface Water Treatment Rule (SWTR), which established minimum treatment requirements for public water systems using surface water as supply source.50 If this concept is valid, then the log inactivation is proportional to the product of disinfectant concentration and contact time.51 As shown in Table 2, the inactivation rate constants k and T of ARB, pB10 plasmid transfer, and pB10 plasmids were increasing as C0 increased. Contrary to these results are the corresponding CT values where the lowest values can be obtained at C0 = 7 mg L−1. At C0 = 3 mg L−1, the k value was highest for the control, E. coli K12 (0.305 min−1), which is a nonantibiotic resistance representative. Correspondingly, CT values of ARG, pB10

Figure 2. Removal efficiency profiles of (a) ARB, (b) pB10 plasmid transfer, and (c) pB10 plasmid using ozonation. Performed in triplicates; error bars correspond to 95% confidence intervals.

plasmid transfer, and pB10 plasmids were more than twice of that of the control. The same trend was observed when the required CT values were calculated for each targeting reduction (1-log or 2-log, etc.) of ARB, pB10 plasmid transfer, and pB10 plasmids (Table 2). For example, to achieve 4-log inactivation of plasmid transfer at C0 = 7 mg L−1, the estimated CT value required was 127.15 mg·min L−1, which was 1.04- and 1.25-fold higher than those required for ARB (122.73 mg·min L−1) and E. coli K-12 non-ARB (101.4 mg·min L−1), respectively. Again, the direct CT value comparison between this study and other could misread the results because the required CT value of E. coli K12 in this study seems too high. Taylor et al. reported a 0.02 mg·min L−1 CT value for 2-log inactivation.52 However, it is E


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Environmental Science & Technology Table 2. Inactivation Rate Constants (k) and CT Values of Inactivation by Ozonationa T = ln (N/N0)/k (min)

ARB

pB10 plasmid transfer

pB10 plasmids E. coli K-12b

CRT (mg·min/L)

C0 = CR (mg L−1)

k (min−1)

2 log10 (99%)

3 log10 (99.9%)

4 log10 (99.99%)

3 5 7 10 3 5 7 10 3 5 7 10 3

0.1661 0.373 0.6363 0.7353 0.1507 0.3638 0.6142 0.7529 0.1272 0.3695 0.7 0.8033 0.305

34.34 16.66 10.30 9.39 37.85 17.08 10.67 9.17 44.84 16.82 9.36 8.60 18.70

48.20 22.83 13.91 12.53 53.13 23.41 14.41 12.23 62.94 23.05 12.65 11.47 26.25

62.06 29.01 17.53 15.66 68.41 29.74 18.16 15.29 81.05 29.28 15.94 14.33 33.80

2 log10 (99%) 3 log10 (99.9%) 103.02 83.31 72.07 93.94 113.55 85.41 74.66 91.75 134.52 84.09 65.51 85.99 56.10

144.61 114.17 97.40 125.26 159.38 117.06 100.90 122.33 188.83 115.25 88.54 114.66 78.75

4 log10 (99.99%) 186.19 145.04 122.73 156.57 205.22 148.71 127.15 152.91 243.14 146.41 111.56 143.32 101.40

T = contact time; C0 = initial concentration; CT = disinfectant concentration × contact time bE. coli K-12 was used as a control, representing nonantibiotic-resistant bacteria a

still noteworthy that the required CT values for ARB, pB10 plasmid transfer, and pB10 plasmids are 2 or 3 times higher than those of non-ARB. In addition, k values obtained from the experimental data of inactivation by chlorine were 0.01−0.1 min−1, which is 10−80 times lower compared to inactivation by ozonation, which were 0.1−0.8 min−1. One of the interesting findings from our study is that the decrease of pB10 plasmid transfer or pB10 plasmid itself might not be sufficient in practice even though the target ARB removal can be achieved (e.g., 2-log removal) under a certain ozone disinfection condition (i.e., C0 = 3 or 5 mg L−1 from Table 2) if a certain ARB regulation was set in the future. This observation can be plausible by the culture-based ARB detection method and ARG transformation. During ozone disinfection, a number of ROS including H2O2 can be produced.53 For many years, it has been recognized that the H2O2 molecule could induce “viable but non-culturable” (VBNC) status of bacteria including E. coli.54 This implied that some ARB VBNC survivors after ozonation might not be detected by culture-based methods but would still be involved in transferring the ARGs. Also, there is a possibility that the even though the ARB cell surface was damaged by ozonation the plasmid on ARGs were not inactivated because the mechanism of ozone disinfection was caused by cell wall disruption rather than penetration into the cell.55 If relatively intact ARGs are released from the cells, then these can be transferred to other microorganisms, called transformation.12 The resistance mechanisms of ARB to ozonation are not yet well understood, but ARB might respond better under stress conditions rather than non-ARB because ARB were cultivated under the antibiotic presence in our study. In general, ARB are more responsive to the stress-related conditions such as high concentrations of antibiotics.56 This observation indicates that the ARB strains could adapt to ozone-related oxidative stress conditions. 3.2. Factors Affecting the Efficiency of Removal of ARB, pB10 Plasmid Transfer, and pB10 Plasmids by Ozonation. 3.2.1. pH. The impact of pH on the disinfection capacity of ozone is complex. It is well-known that the ozone decomposition rate increases as the concentration of hydroxide ions increases (eq 1) [O3] + [OH−] → [HO2−] + [O2 ]

Hydroxide ions react with ozone molecules and result in the production of hydroxyl radicals (•OH) (eq 2).57 [O3] + [HO2−] → [•OH] + [O2•−] + [O2 ]

(2)

•

Because hydroxyl radicals ( OH) have a stronger oxidation potential (2.86 V) than ozone itself (2.07 V),58 the effect of pH on microorganism inactivation is a trade-off situation; increased pH reduces the ozone concentration but produces more radical species. Thus, the effect of pH on microbial disinfection has remained poorly understood.27,59 In the present study, it was observed that the lower the pH the higher the antibiotic resistance removal efficiencies in ARB, pB10 plasmid transfer, and pB10 plasmid (Figure 3a) especially at 3 mg O3 L−1. The same pattern was observed for pB10 plasmid transfer and ARGs (Figure 3b,c). The results of antibiotic removal efficiencies at different pH were tested with t test at 95% confidence level whether they are statistically different or not. On the basis of the results, the ARB removal efficiencies, pB10 plasmid transfer, and pB10 plasmid removal efficiencies at pH 6 were higher than those at pH 9, with p values of 0.04, 0.03, and 0.06, respectively. Although every p value is not lower than 0.05, the visual removal efficiencies of antibiotic resistance by disinfection is increased as pH is decreased. Hunt and Marinas reported that ozone molecules are primary players in E. coli inactivation because different pH conditions (pH 6 and 8) cause no kinetic differences, regardless of the presence or absence of the radical scavenger tertbutanol.59 In a recent study, Zuma et al. reported that E. coli inactivation proceeded more rapidly at an acidic pH than at a basic pH. The calculated kinetic value at pH 4.93 was 2.209 (min−1) and only 1.126 (min−1) at pH 9.16.27 The authors explained that maintaining the molecular ozone concentration is more effective for E. coli inactivation than by free radicals formed by ozone decomposition at high pH. They suggested that hydroxyl radical scavengers such as bicarbonate ions present in microorganisms might quench the free radical reaction. Accordingly, reducing the concentration of ozone at high pH decreases the CT value of ozone. In our study, the ozone concentration (7 mg L−1) was monitored as a function of pH variance, and as expected, ozone concentrations were maintained at lower pH but dropped at higher pH. In this way, our results indicate that the higher ARB disinfection capacity

(1) F


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Figure 3. Removal profiles of (a) ARB, (b) pB10 plasmid transfer, and (c) pB10 plasmid, at different pH values using ozonation. Performed in triplicate; error bars correspond to 95% confidence intervals.

Figure 4. Removal profiles of (a) ARB, (b) pB10 plasmid transfer, and (c) pB10 plasmid, with different SS concentrations using ozonation. Performed in triplicate; error bars correspond to 95% confidence intervals.

observed at lower pH was more likely caused by the maintenance of a high concentration of ozone molecules rather than a direct effect of the pH. 3.2.2. Effect of SS and Humic Acids. In this study, SS and humic acid are selected as model solid organic matter and DOM in wastewater, respectively. Humic acid was chosen as model DOM because humic substances represent 50−90% of the DOM pool and they are reactive in ozonation processes that can be a problem in wastewater treatments using ozone disinfection.60,61 Humic acids contain volatile aromatic compounds that can be attacked by ozonation and reduce their concentration.62 Initially, we thought that there will be a negative impact on both the addition of SS and humic acids because they are scavengers to radicals, but the results proved otherwise. As already described on previous studies for wastewater, we also observed a decrease in the ozone concentration as the SS concentration increased (Figure 4a−c).63 This figure also

shows that the nonantibiotic resistant E. coli K-12 has 1-log higher reduction compared to that of ARB after 7 min with 5 mg L−1 SS. In agreement, our results for removal of ARB, pB10 plasmid transfer, and pB10 plasmids showed a decreasing in the ozone disinfection efficiency as the SS concentration increased, suggesting that ozone was consumed by the SS (Figure 5b). According to Kawara, SS are first decomposed into highmolecular-weight compounds and then further into lowmolecular-weight compounds by ozone oxidation.64 As the SS concentration increases, the disinfection efficiency of ozone decreases because the SS consume various types of radicals produced by ozonation. Figure 6 shows the removal efficiencies of ARB, pB10 plasmid transfer, and pB10 plasmids by ozonation with different humic acid concentrations. A greater removal in antibiotic resistance was observed at higher humic acid concentrations. G


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Figure 6. Removal profiles of (a) ARB, (b) pB10 plasmid transfer, and (c) pB10 plasmid, with different humic acid concentrations using ozonation. Performed in triplicate; error bars correspond to 95% confidence intervals. Figure 5. Profiles of ozone decomposition in terms of (a) pH, (b) SS, and (c) humic acid. Performed in triplicate; error bars correspond to 95% confidence intervals.

(7 mg L−1) for 15 min. Humic acid before and after ozonation (humic acid/ARB contact with humic acid after ozonation/ humic acid after ozonation) were then compared using EEM analysis. The fluorescence EEM of humic acid shows a more intense peak associated with a higher level of excitation energy in the wavelength range of 220−240 nm and a peak with lower intensity associated with the lower excitation energy between 260−280 nm (Figure 7). Ozonation for up to 15 min almost completely removed the fluorophores corresponding to humic acid, but a negligible change in dissolved organic carbon (DOC) was found upon ozonation. Figure 7 also shows that the ozonation resulted in a blue shift, i.e., the emission shifts toward a shorter wavelength, indicating a reduction in the π-electron system in humic molecules. This result suggests that ozone molecules and/or • OH preferentially reacted with abundant electron-rich sites

Ozone reacts with humic substances (Figure 5c) to produce oxygenated byproducts of low molecular weight, which are generally more easily biodegradable, polar, and hydrophilic than their precursors.28 In addition, non-antibiotic-resistant E. coli K12 is 3-log more susceptible to ozonation after 7 min, with 5 mg L −1 humic acid, compared to ARB. Although visual observations are clear, the results of antibiotic removal efficiencies at different SS and humic acid concentrations with multiple t test at 95% confidence level did not support our observations. This might be a result of the small sample numbers. A separate experiment was conducted to monitor ARB survival after mixing with humic acid and treatment with ozone H


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Figure 7. Photographs of EEM spectra of (a) humic acid at a concentration of 20 mg L−1, (b) humic acid mixed with ARB after 15 min of ozonation, and (c) humic acid after 15 min of ozonation.

(e.g., aromatic functional groups and double-bonded Ccontaining groups in the organic molecules) that can cause a high level of UV absorbance, which coincided with a dramatic decrease in the specific UV absorbance (SUVA) upon the ozonation. SUVA is calculated from the UV254 absorbance divided by the DOC of the water sample and has been used as an indicator of the humic content in water environmental systems.65,66 Consequently, it can be speculated that ozonation of humic acid results in a decrease in the number of aromatic rings or conjugated bonds in a chain structure and/or the conversion of a linear to a nonlinear ring system. Ozone decomposition can be initiated by substances capable of inducing formation of a superoxide anion (O2−) such as hydroxide ions, peroxides, ferrous iron, or humic substances.53 Humic substances are also known to be promoters, which are molecules capable of regenerating superoxide anions from hydroxyl radicals.67,68 Accordingly, when humic substances are present, both ozone and humic substances can act as initiators of hydroxyl radicals, thereby achieving an improved oxidative effect as the concentration of humic acid increases.69 As shown above, CT values used in real practice might not prevent ARB spread and plasmid which still can transfer to pathogens. However, it is also true that the suggested CT values in this study might not be achievable in practice. Because multidrug resistance issue is getting serious, the potential risk of antibiotic resistance from wastewater treatment plants should be properly handled.1,32,36 More discussions should be addressed in wastewater engineering on how to make multiple barriers including disinfection preventing hazard bacteria discharge into the environment.45,70

Furthermore, more comprehensive economic analysis is needed for not just the costs of advanced treatment processes of these emerging pollutants but also how serious an impairment would occur if we could not prevent the antibiotic resistance spread in the environment. ARGs released into the natural environment can be acquired by both pathogenic and environmental microorganisms that can threaten the future of antibiotic therapy.70 The development and discovery of new pharmaceuticals are very costly and slow, but the increase of antibiotic resistance through HGT has been increasing in the past 10 years. Even though there have been a lot of proposed policies and strategies to address the spread of antibiotic resistance, the response of scientists, engineers, policymakers, and other stakeholders has been very slow because they are hindered by current regulations and policies and the inability to implement proposed regulations regarding antibiotic resistance.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +8210-7181-2469. E-mail: [email protected]. Present Address ¶

S.K.: Korea University, Sejong Campus, Science and Technology Building, Room 509, 2511 Sejong-ro, Sejong City 30019, Republic of Korea. Author Contributions

G.P. and D.E.S. contributed equally to this study. Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS This research was financially supported by the Korea Institute of Science and Technology (KIST) Institutional Program (2E26252) and Supported by a Korea University Grant.

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