The Role of Reactive Oxygen Species in the Electrochemical

The high oxidation potential of ROS makes them more effective disinfectants than chlorine for all kinds of microorganisms (9). For example, the C̄T v...
0 downloads 0 Views 208KB Size
Download PDF
Environ. Sci. Technol. 2006, 40, 6117-6122

The Role of Reactive Oxygen Species in the Electrochemical Inactivation of Microorganisms JOONSEON JEONG, JEE YEON KIM, AND JEYONG YOON* School of Chemical and Biological Engineering, College of Engineering, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742, Korea

Electrochemical disinfection has emerged as one of the most promising alternatives to the conventional disinfection of water in many applications. Although the mechanism of electrochemical disinfection has been largely attributed to the action of electro-generated active chlorine, the role of other oxidants, such as the reactive oxygen species (ROS) •OH, O3, H2O2, and •O2- remains unclear. In this study, we examined the role of ROS in the electrochemical disinfection using a boron-doped diamond (BDD) electrode in a chloride-free phosphate buffer medium, in order to avoid any confusion caused by the generation of chlorine. To determine which species of ROS plays the major role in the inactivation, the effects of several operating factors, such as the presence of •OH scavenger, pH, temperature, and the initial population of microorganisms, were systematically investigated. This study clearly showed that the •OH is the major lethal species responsible for the E. coli inactivation in the chloride-free electrochemical disinfection process, and that the E. coli inactivation was highly promoted at a lower temperature, which was ascribed to the enhanced generation of O3.

Introduction Electrochemical disinfection has been growing in importance for the treatment of not only drinking water and sewage water, but also processed water in industry because it is inexpensive, easy to perform, and is known to inactivate a wide variety of microorganisms (1-2), even though the problems of toxic byproducts generation have to be carefully examined before any real application (3-4). Many studies have been conducted to develop an effective electrochemical disinfection process that can be used as an alternative to conventional treatment. The most common methods of electrochemical disinfection have been based on electro-chlorination, wherein “active chlorine” species (HOCl, OCl-) are generated in-situ by electrolyzing saline water (5). However, the enhanced inactivation of microorganisms observed with electrochemical disinfection cannot be fully explained based only on the action of the electro-generated chlorine. Recently, as another potential disinfecting agents in the electrochemical disinfection, increasing attention has been given to reactive oxygen species (ROS), such as •OH, O3, H2O2, and •O2-, which can be produced along with chlorine during electrolysis (1, 6-8). As evidence for the role of ROS, the morphological change of cells induced by electrochemical * Corresponding author phone: +82-2-880-8927; fax: +82-2-8768911; e-mail: [email protected]. 10.1021/es0604313 CCC: $33.50 Published on Web 08/30/2006

 2006 American Chemical Society

disinfection, observed by scanning electron microscopic analysis, was reported to be similar to that induced by the • OH generated from Fenton reaction (1). The high oxidation potential of ROS makes them more effective disinfectants than chlorine for all kinds of microorganisms (9). For example, the C h T value for •OH is approximately 105 times lower than that for chlorine, thus only a small concentration can inactivate microorganisms to a considerable extent (9-10). Although a number of studies have been conducted to investigate the role of ROS in electrochemical disinfection, most of them focused on the possibility that ROS might enhance the inactivation efficiency in the electrochemical disinfection process by producing chlorine, thus failing to provide experimental results which are relevant to the direct role of ROS. Few studies have been specifically directed at investigating the role played by ROS in the inactivation of microorganisms. In addition, determining the exact mechanism by which ROS enhance the inactivation process is complicated by the presence of the active chlorine species generated from the electrolysis chloride ion present in treated water (1). Furthermore, it was also poorly understood which species of ROS is more significant for inactivating microorganisms when the reaction conditions are varied. In this study, we attempted to investigate the role of ROS as a disinfecting agent in electrochemical disinfection using a chloride-free phosphate buffer solution as an electrolyte, so as to exclude the formation of oxidants other than ROS. A boron-doped diamond (BDD) electrode was used as the anode material, since it is known to achieve high efficiency in producing ROS due to its high O2 overpotential, and Escherichia coli was used as an indicator microorganism. In addition, it was aimed to clarify which ROS species (OH, O3, H2O2, •O2-) was mainly responsible for the inactivation of microorganisms as the various operating factors, such as the presence of the radical scavenger, the initial population of the microorganism, pH, and temperature, were varied.

Materials and Methods Chemicals and Analysis. All chemicals were of reagent grade and used without further purification. Potassium dihydrogen phosphate (KH2PO4), sodium hydroxide (NaOH), tert-butyl alcohol (t-BuOH), methanol (CH3OH), sodium chloride (NaCl), N,N-dimethyl-p-nitrosoaniline (RNO), and sodium thiosulfate (Na2S2O3) were purchased from Aldrich Co. (USA). 0.2 M phosphate buffer prepared with the distilled and deionized water by a Millipore purification system (Barnstead NANO Pure, U.S.) was employed as a supporting electrolyte. The ROS, such as O3, H2O2, •OH, and •O2- were identified by direct or indirect methods. The concentration of O3 was measured using the indigo method with a UV-vis spectrophotometer (Hewlett-Packard 8453, U.S.) and 10 cm cuvettes. This method is based on the quantitative decolorization of indigo trisulfonate as a result of its reaction with O3, which is observed at 600 nm and whose detection limit is about 0.01 mg/L (11). The formation of H2O2 was determined by a colorimetric method using copper(II) ions and 2,9-dimethyl1,10-phenanthroline (DMP) at 454 nm (12). The literature and our preliminary tests confirmed that these analytical methods have no significant interference toward each other under the experimental conditions in this study (11-12). For the analysis, an appropriate amount of sample taken from the electrolytic solution was injected into the analytical reagents as quickly as possible to minimize the decay of oxidants. Two different methods were employed to assess the production of •OH, which is difficult to measure directly VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6117

because of its short lifetime. The first method involved the use of t-BuOH, a well-known •OH scavenger. An excess amount of t-BuOH (0.03 M) was introduced to verify the presence of •OH. It was preliminarily confirmed that the 0.03 M t-BuOH itself had no effect on the inactivation of E. coli under the conditions used in this study. In the second method, N,N-dimethyl-p-nitrosoaniline (RNO) was used as an indicator for •OH production, since it is known to react selectively with •OH to form a more stable radical (13). The advantages of using RNO as a tool for detecting •OH, are its electrochemical inertness, high rate of reaction with •OH (k ) 1.2 × 1010 M-1s-1), and large light absorptivity (440 nm ) 3.44 × 104 M-1cm-1). The yellow tinted RNO was decolorized by its reaction with the •OH formed from the oxidation of water, and the bleaching of the color was measured every 3 min during the 15 min period of electrolysis. To investigate the role of •O2- on the inactivation of E. coli, the excess amount of methanol (0.03 M) was used. No lethal effect of 0.03 M methanol alone was confirmed. Preparation and Analysis of Microorganisms. For all disinfection experiments conducted in this study, E. coli (ATCC 8739) was employed as an indicator bacterium. The stock suspension of E. coli was prepared following the procedures described elsewhere in the literature (9). Most of the initial population of E. coli was adjusted to approximately 105 CFU/mL, except in those experiments where the effect of the initial population was investigated, in which case they varied from approximately 103 to 108 CFU/mL. During the experiments, 1 mL of suspension was withdrawn at each sampling time and was immediately quenched with excess Na2S2O3 (10 mM) to eliminate the residual disinfectants in the sample solution. Then, the sample was diluted accordingly, depending on the initial number of viable cells. Three replicates of the diluted and undiluted 0.1 mL suspensions were used for counting by the spread plate method with nutrient agar grown at 37 °C for 24 h and showed good reproducibility within a standard deviation of 10%. For selected experiments, this procedure was repeated three times to examine the level of reproducibility of experimental data, providing error bars displaying the standard deviation in the figures. Investigation of Morphological Change of E. coli Cells. To study the morphological change of the E. coli cells during the electrolysis, two analytical methods involving transmission electron microscopy (TEM) and atomic force microscopy (AFM) were employed in this study. The TEM specimen was prepared following the procedures described by Feng et al. (14), and observed using a transmission electron microscope (JEM 1010, JEOL, Japan). For the AFM specimen, 40 mL of E. coli suspension was concentrated to 4 mL with a 0.2 µm Teflon syringe filter (Advanced Microdevices, India), followed by mounting 0.3 mL of the concentrated E. coli suspension on a glass slide. After air-drying for 24 h, the samples were observed using an atomic force microscope (Dimension 3100, Digital Instruments, U.S.) operating in tapping mode. Electrochemical Cell and Apparatus. All electrochemical experiments were carried out in an 80 mL open and undivided Pyrex cell. The anode was a boron doped diamond film on a niobium substrate (Nb/BDD) with a surface area of 6 cm2 (CONDIAS GmbH, Germany) and the cathode was a platinum sheet (Princeton Applied Research, U.S.). The two electrodes were installed parallel to each other and kept 20 mm apart by a Teflon cell top. To measure the anodic potential during electrolysis achieved by constant current, the Ag/AgCl electrode (Princeton Applied Research, U.S.) was introduced as the reference electrode only in a separate experiment. All potential values quoted in this study were with reference to the Ag/AgCl electrode. The correction for ohmic drop was carried out with using IR compensation technique (positive feedback or current interruption) following the literature (15). 6118

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 19, 2006

FIGURE 1. Effect of current density on E. coli inactivation ([E. coli]0 ) 105 CFU/mL, [KH2PO4]0 ) 0.2 M, pH ) 7.1, 25 °C). All disinfection experiments were performed without the Ag/ AgCl electrode under galvanostatic conditions to avoid any chloride contamination caused by the Ag/AgCl electrode. A computer controlled potentiostat-galvanostat (PARSTAT 2273A, Princeton Applied Research, U.S.) was used to apply constant current. The temperature of the electrolytic solution was maintained at the desired value using a thermostatic water bath (model RW-2025G, JEIO TECH, Korea), and the variation did not exceed (0.2 °C during electrolysis. To investigate the effect of pH, the stock solutions of phosphate buffers having three different pH values (pH 5.6, 7.1, 8.2) were diluted to 0.2 M and used as the electrolyte. It was confirmed by a separate experiment that no significant change in pH was observed before and after electrolysis, and no pH measurement was done during all disinfection experiments due to the chloride contamination problem. Stirring was carried out using an immersible stirrer unit (ColeParmer Co., U.S.) with a magnetic bar. Experimental Procedures. Prior to the experiments, the electrodes were sonicated for 10 min (60 kHz and 500 W, Powersonic 410, Whasin Tech Co., Korea). Then, in order to condition the electrodes, anodic polarization was performed for 10 min at 120 mA/cm2 in 1 M H2SO4. Next, the electrodes and reactor were washed several times with sterile water and irradiated with a sufficient UV intensity for 5 min (IT ) 225 mJ/cm2, 6 W × 2 low-pressure mercury lamp, Philips Co., Netherlands) to entirely eliminate any residual microbial cells adsorbed on the electrodes and reactor surface (16). After that, the electrodes were immersed in the electrolytic solution, and the resultant electrochemical cell was placed in the thermostatic water bath (model RW-2025G, JEIO TECH, Korea). The appropriate amount of E. coli stock was suspended in the electrolytic solution and stirred for at least 10 min prior to electrolysis. The electrolysis was begun by applying a constant current density ranging from 0 to 100 mA/cm2. Five to seven samples were withdrawn at timed intervals to count the number of viable cells or to analyze the concentration of oxidants.

Results and Discussion Effect of Electrical Current on E. coli Inactivation. Figure 1 shows the electrochemical inactivation of E. coli in the chloride-free phosphate buffer as the current density was varied from 33 to 100 mA/cm2. The preliminary test confirmed that no inactivation of E. coli was achieved without applying a current. As shown in Figure 1, even without adding any chemicals, the successful inactivation of E. coli was observed at all current densities, with the inactivation increasing with increasing current density, although not linearly. The possibility of chlorine production by chloride impurity, if it may exist in the phosphate buffer, was examined by adding a

trace amount of chloride ion (2 µM as NaCl) into the electrolytic solution, which is presumed to be an upper limit of chloride concentration in the reagent grade phosphate buffer medium. No significant variation in the inactivation was observed with adding chloride, indicating that the action of chlorine was not responsible for the inactivation of E. coli in Figure 1. In addition, the inactivation of E. coli had a lag phase at the beginning of the electrolysis, which is similar to the previous observations with other chemical disinfectants (9-10), and the period of lag appeared to shorten with increasing current density. Two possible mechanisms to explain the behavior of E. coli inactivation shown in Figure 1 were taken into account, involving direct oxidation and indirect oxidation mediated by reactive oxidants generated during the electrolysis. A microbial inactivation by a direct electron-transfer reaction between the cells and electrode, which was ascribed to the dimerization of coenzyme A in the cell wall, was reported as the anodic potential, which is low so that no oxidants can be formed, were applied (17). Since the corresponding anodic potentials measured at each current density in Figure 1 lie in the region of water oxidation (3.0∼3.5 V), the direct oxidation of cells cannot be excluded at this moment, although the experimental design of this study differs from that employed in the previous one (17). However, in a subsidiary experiment, no remarkable inactivation of E. coli was achieved at anodic potentials below 1.2 V, for which water oxidation cannot occur, during 15 h of electrolysis (data not shown), indicating that the contribution of direct oxidation is not significant in the inactivation of Figure 1. The other possible mechanism responsible for the inactivation of E. coli is indirect oxidation mediated by several oxidants produced from the oxidation of water. ROS such as •OH, O , H O , and •O - (or HO •) can be considered as 3 2 2 2 2 candidate oxidants, as described by eqs 2-9 (18-19). The most common oxidant is the •OH formed by the one-electron oxidation of water (eq 1). At the same time, O3 can be generated by the reaction of O2 with •O, which is the oneelectron oxidation form of •OH (eqs 3 and 4), and H2O2 is formed by the combination of two •OHs (eq 5). Further reactions of OH with O3 or H2O2 yield •O2- (eqs 6-8).

•

H2O f •OH + H+ + e-

(1)

OH f 1/2O2 + H+ + e-

(2)

•

OH f •O + H+ + e•

(3)

O + O2 f O3

(4)

OH + •OH f H2O2

(5)

OH + O3 f HO2• + O2

(6)

OH + H2O2 f HO2• + H2O

(7)

•

•

•

HO2• S •O2- + H+

(pKa ) 4.8)

(8)

To examine whether the •OH is generated during electrolysis in this study, the formation of H2O2 (eq 5), whose presence is widely accepted as evidence for the production of •OH (18), was measured while varying the current density. The possible formation of H2O2 as the cathodic reaction was ruled out due to the use of platinum cathode where H2O2 formation is known to be unfavorable (20). As shown in Figure 2, the amount of H2O2 produced increased with increasing current density and gradually leveled off as the electrolysis proceeded. Adding excess t-BuOH (0.03 M) as an •OH scavenger markedly

FIGURE 2. Effect of •OH scavenger on H2O2 formation ([KH2PO4]0 ) 0.2 M, pH ) 7.1, 25 °C, Inset: Effect of •OH scavenger on E. coli inactivation, [E. coli]0 ) 105 CFU/mL). inhibited the formation of H2O2, thus confirming the production of •OH during the electrolysis in this study. The leveling off of the H2O2 formation as the electrolysis proceeded can be explained by the further oxidation of H2O2 to O2 (eq 9) (18).

H2O2 f O2 + 2H+ + 2e-

(9)

The role of •OH in the inactivation of E. coli was investigated by adding excess t-BuOH. As shown in the inset of Figure 2, the presence of 0.03 M t-BuOH completely halted the inactivation of E. coli, implying that the electro-generated •OH is a major lethal species responsible for inactivating E. coli. The possible role of phosphate radical generated from the reaction of •OH with phosphate buffer solution could be excluded by the subsidiary experimental finding that no remarkable change resulting from high concentration of phosphate buffer (1 M) was observed in the inactivation of E. coli (data not shown). However, it is still unclear whether the •OH is the sole oxidant responsible for the inactivation of E. coli shown in the inset of Figure 2, because other ROS species such as H2O2, O3, and •O2- can be concurrently generated along with •OH (eqs 4-7). Thus, further investigation is required to elucidate their possible roles. The role of H2O2 was directly examined by adding an appropriate amount of H2O2 to the E. coli suspension without applying any current, viz. 1 mM, which is a much higher concentration than that formed by electrolysis as shown in Figure 2. The presence of 1 mM H2O2 made no significant inactivation of E. coli after a contact time of 10 min (data not shown), indicating that the lethality of H2O2 alone was negligible in the experimental conditions used herein, which is consistent with the results reported in the literature (9). The amount of O3 that may have accumulated in the electrolyzed solution was not measurable. The possibility of O3 consumption by the buffer solution can be excluded because the phosphate buffer used in this study is known not to react with ozone (21). However, even a tiny presence of O3 concentration not measured by reasonably sensitive analytical method such as an indigo method cannot exclude the possibility causing a considerable amount of inactivation, because of its high reactivity toward E. coli (2-log inactivation C h T ) 0.04 mg min/l) (9), the residual disinfecting activity of the electrolyzed water was measured by injecting E. coli into the electrolyzed solution obtained after the electrolysis stopped. This idea is based upon that, unlike •OH with its extremely short lifetime, a trace amount of O3, if generated during electrolysis, is likely to affect the inactivation of E. coli by remaining in the electrolyzed solution even after the VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6119

FIGURE 4. Effect of initial population of E. coli on the inactivation (83 mA/cm2, [KH2PO4]0 ) 0.2 M, pH ) 7.1, 25 °C).

FIGURE 3. Morphological change of E. coli cells resulting from the electrolysis at 100 mA/cm2 for 5 min ([E. coli]0 ) 108 CFU/mL, [KH2PO4]0 ) 0.2 M, pH ) 7.1, 25 °C, (A) before electrolysis (TEM), (B) after electrolysis (TEM), (C) before electrolysis (AFM), (D) after electrolysis (AFM)). electrolysis stopped. No remarkable inactivation of E. coli was observed for up to 1 h in the electrolyzed water which is obtained after the electrolysis stopped (data not shown), indicating that the trace O3, if it exists, does not play any significant role in the inactivation of E. coli under the experimental conditions used herein. To verify the role of •O2- on the inactivation of E. coli, a separate experiment was carried out with using excess methanol (0.03 M) as another •OH scavenger. Unlike to t-BuOH, which scavenges the hydroxyl radical to form a stable RO•, methanol reacts with •OH to produce •O2- in the presence of oxygen (eq 10) (21).

As shown in the inset of Figure 2, no significant inactivation was observed with adding 0.03 M of methanol, indicating that the role of •O2- is negligible in the experimental conditions used herein. Morphological Change of E. coli Cells with Electrolysis. The morphological change of the E. coli cells as a result of the electrochemical disinfection was analyzed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Figures 3A and B show the TEM images of the untreated and treated E. coli cells. In the untreated E. coli cells, the cell walls of both the outer and inner membranes were intact (Figure 3 A). However, after electrolysis for 5 min at 100 mA/cm2 (achieving approximately one log inactivation), the drastic changes in the nature of the contents of cell, as well as in the structure of the cell walls, were observed (Figure 3B). The cell walls appear to be no longer uniform and to have become torn in places, and the cells are mostly empty. This may be one of typical appearances in the cells damaged by strong oxidants such as O3 (22-23). In the AFM images shown in the Figures 3C and D, which show the cells before and after electrolysis, the surface of the untreated cells appears to be smooth and flat, whereas the treated cells have a rough and sunken surface, as if they had shrunk when the inner contents escaped from the cells. These morpho6120

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 19, 2006

logical changes can be interpreted by the attack of ROS involving •OHs disrupting the integrity of the cell membrane, leading to the lysis of the cells. It needs to be noted that no O3 was measurable in the experimental condition used herein. Effect of Initial Population of E. coli. Figure 4 shows the effect of the initial population of E. coli on the inactivation, which was significantly reduced as the initial population increased. This behavior can be explained in two ways. First, as the initial population of microorganism increases, the decay of oxidants is expected because the microorganism itself can act as a consumer for oxidants, causing the reduction of inactivation kinetics. This is supported by the report of the previous study that the significant decay of oxidants such as ozone ([O3]initial ) 0.065 ∼ 0.27 mg/L) is closely related to the population of E. coli ranging around ∼ 106 CFU/mL (22), although no ozone was measured at the condition used in Figure 4 (data not shown). Second explanation for the effect of initial population in Figure 4 can be found from the heterogeneous nature of the electrochemical system under the level of significant difference of inactivation. Since the •OH is very unstable, inactivation reactions involving •OH tend to take place in a region very close to the electrode surface rather than in the bulk solution. The •OH-based electrochemical disinfection is a surface-layer-based process, whose reaction characteristics would quite differ from the electro-chlorination that is a volume-based chemical process. Based on the fact that the diffusion coefficient of bacteria is ∼10-8 cm2/s (24), the diffusion distance of E. coli is approximately ∼2 µm per unit time (second). Considering the thickness of diffusion layer ranging to several tens of micrometers with a vigorous mixing, the diffusion of E. coli cells in the diffusion layer can be regarded as a major ratelimiting step within the time scale of inactivation in this study. Since the electrode has a fixed surface area, increasing the initial population results in greater competition between the individual E. coli cells as they try to approach the electrode surface, leading to less inactivation. The result shown in Figure 4 partly implies that an electrode with a larger surface area is more favorable in the case of electrochemical disinfection. Effect of Temperature and pH. Figure 5 shows the effect of temperature and pH on the E. coli inactivation. First, a larger enhancement in the E. coli inactivation was observed at lower temperature at same pH (pH 7.1). This observation is opposite to the general disinfection behavior, in that the microbial inactivation with chemical disinfectants decreases at a lower temperature (25). To ascertain whether the change in temperature affects the production of •OH, the decolorization of RNO (2 × 10-5 M) as an indicator of •OH production

FIGURE 5. Effect of temperature and pH on E. coli inactivation (68 mA/cm2, [E. coli]0 ) 105 CFU/mL, [KH2PO4]0 ) 0.2 M). was measured at several temperatures, and the results can be expressed as first-order kinetics described by eq 11.

-

d[RNO] ) k[•OH][RNO] ) kobs[RNO] dt

(11)

where k (M-1s-1) is the second-order rate constant for the reaction of RNO with •OH, and kobs (s-1) is the observed pseudo first-order rate constant. A good linear relationship for the logarithm of RNO decolorization versus time was obtained with r2 > 0.99 during the whole period of electrolysis (15 min). As shown in Table S1 in the Supporting Information, the temperature change had little effect on kobs, indicating that the temperature dependence of E. coli inactivation in Figure 5 cannot be ascribed to the enhanced production of •OH. Since temperature is known to affect the electrochemical generation of O3 (15), the role of O3 was examined as another potential cause of the temperature dependence. The concentrations of O3 formed after 10 min under conditions identical to those shown in Figure 5 were measured to be 0.14 and 0.05 mg/L at 4 and 15 °C, respectively (see Table S1 in the Supporting Information). However, no measurable O3 concentration was observed at temperatures higher than 25 °C, indicating that the O3 may be involved in the inactivation of E. coli at temperatures below 15 °C in the results shown in Figure 5. This observation is consistent with previous studies which found that a greater amount of O3 can be formed at lower temperatures in electrochemical systems (15), while increasing the temperature can significantly reduce the solubility of O3 and accelerate its decomposition rate (26). The concurrent formation of O3 in electrochemical disinfection can be beneficial, because the disinfecting action of O3 can occur in the bulk solution as well as in the vicinity of the electrode surface, in contrast with the short-lived •OH, for which the reaction only takes place in the vicinity of the electrode surface. The pH dependence of E. coli inactivation was also observed in Figure 5, which increases with lowering pH. It was preliminarily confirmed that the pHs of 5.6 or 8.2 had no detrimental effect on the E. coli inactivation. The tendency observed in Figure 5 would be in the correct order if chlorine was the major disinfectant, since HOCl (pKa ) 7.4) is a more effective germicide than OCl- (27). However, no chlorine species can be formed in this study, because chloride-free phosphate buffer was used as the electrolyte. As one approach to explain this pH effect, the variation in the amount of •OH produced with pH was also examined by RNO decolorization. As shown in Table S1 in the Supporting Information, lowering the pH resulted in the

production of a larger number of •OH, supporting the hypothesis in part that the inactivation of E. coli was enhanced by decreasing the pH. However, other factors influencing pH effect cannot be excluded, since the difference in the RNO decolorization rate with pH was not large enough to interpret the pH effect observed in Figure 5, requiring further examination. The other possible causes for pH dependences were also examined as follows. First, the concentration of O3 produced was not measurable at all pHs at 25 °C (see Table S1 in the Supporting Information), so that its contribution to pH dependency in Figure 5 was excluded, although the electrochemical generation of O3 is known to depend on the pH of the electrolyte (15). Second, the possible influence caused by changing species of H2O2 and •OH with pH could be also ruled out with considering their extremely high pKa values (11.6 and 11.9 for H2O2 and •OH, respectively). On the other hand, the pKa value for •O2- (4.8) is near pH ranges considered here, but it appear to be negligible in explaining pH dependency because no significant effect of •O2- on the inactivation of E. coli was previously found in the result of Figure 2. Third, since the electrostatic attraction between the negatively charged E. coli cells and the positively charged anode surface is a critical parameter for the inactivation of E. coli, the variation in the surface charge of the E. coli cells with pH was examined. However, the analysis of the zeta potential of the E. coli suspension showed no notable variation in the surface charge of the E. coli cells with pH (data not shown). Overall, a more detailed study is needed to fully explain this pH effect. From an engineering point of view, this study clearly shows that ROS as the additional disinfectants, such as •OH and O3 formed by electrolyzing water, can cause a significant inactivation of microorganism, as much as chlorine in the electrochemical disinfection. Furthermore, the potential role of •OH out of ROS, which has oxidizing potentials higher than that of chlorine, deserves to be underlined in treating the spore forming microorganisms that are difficult to inactivate by only chlorine.

Acknowledgments This research was partially supported by the Brain Korea 21 Program of the Ministry of Education. This support was greatly appreciated.

Supporting Information Available Summary of kobs and O3 formation as a function of pH and temperature. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Diao, H. F.; Li, X. Y.; Gu, J. D.; Shi, H. C.; Xie, Z. M. Electron microscopic investigation of the bactericidal action of electrochemical disinfection in comparison with chlorination, ozonation and Fenton reaction. Process Biochem. 2003, 39 (11), 1421-1426. (2) Drees, K. P.; Abbaszadegan, M.; Maier, R. M. Comparative electrochemical inactivation of bacteria and bacteriophage. Water Res. 2003, 37, 2291-2300. (3) Jackson, A.; Arunagiri, S.; Tock, R.; Anderson, T.; Rainwater, K. Technical note: electrochemical generation of perchlorate in municipal drinking water systems. J. Am. Water Works Assoc. 2004, 96 (7), 103-108. (4) Bergmann, H.; Koparal, S. The formation of chlorine dioxide in the electrochemical treatment of drinking water for disinfection. Electrochim. Acta 2005, 50, 5218-5228. (5) Park, J.; Lee, M. S.; Han, D.; Lee, D. H.; Park, B. J.; Lee, I.; Uzawa, M.; Aihara, M.; Takatori, K. Inactivation of Vibrio parahaemolyticus in effluent seawater by alternating-current treatment. Appl. Environ. Microbiol. 2004, 70 (3), 1833-1835. VOL. 40, NO. 19, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6121

(6) Kerwick, M. I.; Reddy, S. M.; Chamberlain, A. H. L.; Holt, D. M. Electrochemical disinfection, an environmentally acceptable method of drinking water disinfection? Electrochim. Acta 2005, 50, 5270-5277. (7) Liang, W.; Qu, J.; Chen, L.; Liu, H.; Lei, P. Inactivation of Microcystis aeruginosa by continuous electrochemical cycling process in tube using Ti/RuO2 electrodes. Environ. Sci. Technol. 2005, 39 (12), 4633-4639. (8) Son, H.; Cho, M.; Chung, H.; Choi, S.; Yoon, J. Bactericidal activity of mixed oxidants: Comparison with free chlorine. J. Ind. Eng. Chem. 2004, 10 (5), 705-709. (9) Cho, M.; Lee, Y.; Chung, H.; Yoon, J. Inactivation of Escherichia coli by photochemical reaction of ferrioxalate at slightly acidic and near-neutral pHs. Appl. Environ. Microbiol. 2004, 70 (2), 1129-1134. (10) Cho, M.; Chung, H.; Choi, W.; Yoon, J. Linear correlation between inactivation of E. coli and OH radical concentration in TiO2 photocatalytic disinfection. Water Res. 2004, 38, 1069-1077. (11) Bader, H.; Hoigne´, J. Determination of ozone in water by the indigo method; a submitted standard method. Ozone Sci. Eng. 1982, 4, 169-176. (12) Ayad, N. B.; Johnson, G. R. A.; Nazhat, N. B.; Saadalla-Nazhat, R. A. A simple spectrophotometric determination of hydrogen peroxide at low concentrations in aqueous solution. Anal. Chim. Acta 1988, 204, 349-353. (13) Comninellis, Ch., Electrocatalysis in the electrochemical conversion/combustion of organic pollutants for waste water treatment. Electrochim. Acta 1994, 39 (11/12), 1857-1862. (14) Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. 2000, 52, 662-668. (15) Ko¨tz, E. R.; Stucki, S. Ozone and oxygen evolution on PbO2 electrodes in acid solution. J. Electroanal. Chem. 1987, 228, 407415. (16) USEPA. Ultraviolet Disinfection Guidance Manual; EPA 815D-03-007; United States Environmental Protection Agency Office of Water: Washington, DC, 2003. (17) Matsunaga, T.; Namba, Y.; Nakajima, T. Electrochemical sterilization of microbial cells. Bioelectrochem. Bioenerg. 1984, 174 (13), 393-400.

6122

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 19, 2006

(18) Michaud, R.-A.; Panizza, M.; Ouattara, L.; Diaco, T.; Foti, G.; Comninellis, Ch. Electrochemical oxidation of water on synthetic boron-doped diamond thin film anodes. J. Appl. Electrochem. 2003, 33, 151-154. (19) Kim, J.; von Gunten, U.; Marin ˇ as, B. J. Simultaneous prediction of Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of synthetic waters. Environ. Sci. Technol. 2004, 38, 2232-2241. (20) Pletcher, D.; Walsh, F. C. Industrial Electrochemistry; Chapman and Hall: New York, 1990. (21) Staehelln, J.; Hoigne´, J. Decomposition of ozone in water in the presence of organic solutes acting as promoters and inhibitors of radical chain reactions. Environ. Sci. Technol. 1985, 19, 12061213. (22) Hunt, N. K.; Marin ˇ as, B. J. Inactivation of Escherichia coli with ozone: chemical and inactivation kinetics. Water Res. 1999, 33 (11), 2633-2641. (23) Khadre, M. A.; Yousef, A. E. Sporicidal action of ozone and hydrogen peroxide: a comparative study. Int. J. Food Microbiol. 2001, 71, 131-138. (24) Poortinga, A. T.; Bos, R.; Norde, W.; Busscher, H. J. Electric double layer interactions in bacterial adhesion to surfaces. Surf. Sci. Rep. 2002, 47, 1-32. (25) USEPA, Alternative Disinfectants and Oxidants Guidance Manual; EPA 815-R-99-014; United States Environmental Protection Agency Office of Water: Washington, DC, 1999. (26) Sotelo, J. L.; Beltra´n, F. J.; Benitez, F. J.; Beltra´n-Heredia, J. Henry’s law constant for the ozone-water system. Water Res. 1989, 23 (10), 1239-1246. (27) Driedger, A. M.; Rennecker, J. L.; Marin ˜ as, B. J. Sequential inactivation of Cryptosporidium parvum oocysts with ozone and free chlorine. Water. Res. 2000, 34, 3591-3597.

Received for review February 23, 2006. Revised manuscript received July 25, 2006. Accepted June 21, 2006. ES0604313