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 con-
ducted 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 electrochemi-
cal 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: +82-2-876-
8911; e-mail: jeyong@snu.ac.kr.
10.1021/es0604313 CCC: $33.50  2006 American Chemical Society
Published on Web 08/30/2006
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 Ch 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 mech-
anism 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 micro-
organisms 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 spectro-
photometer (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-dimethyl-
1,10-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 accord-
ingly, 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 transmis-
sion 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 (Cole-
Parmer 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 pos-
sibility 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 inac-
tivation 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 one-
electron 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)
• concentration than that formed by electrolysis as shown in
O + O2 f O3 (4)
•
OH + •OH f H2O2 (5)
•
OH + O3 f HO2• + O2 (6)
• electrolyzed solution was not measurable. The possibility of
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
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
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
h T ) 0.04 mg min/l) (9), the residual disinfecting activity of
C
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 3. Morphological change of E. coli cells resulting from the
electrolysis at 100 mA/cm2 for 5 min ([E. coli]0 ) 108 CFU/mL, [KH2-
PO4]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 inactiva-
tion 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 transmis-
sion 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 inactiva-
tion), 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 morpho-
6120 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 40, NO. 19, 2006
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).
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 dif-
ference 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 character-
istics 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 rate-
limiting 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 decolori-
zation 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] (11)
dt
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 con-
centrations 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 signifi-
cantly reduce the solubility of O3 and accelerate its decom-
position 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 elec-
trochemical 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.
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Received for review February 23, 2006. Revised manuscript
received July 25, 2006. Accepted June 21, 2006.
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