AgandCuloadedonTiO2 graphite as a catalyst for Escherichia coli contaminated water disinfection
Chemical Papers 64 (5) 557–565 (2010)
DOI: 10.2478/s11696-010-0036-4
ORIGINAL PAPER
Ag and Cu loaded on TiO2 /graphite as a catalyst
for
-contaminated water disinfection
y
Fitria Rahmawati*, Triana Kusumaningsih, Anita M. Hapsari, Aris Hastuti
op
Department of Chemistry, The Faculty of Mathematics and Natural Sciences, Sebelas Maret University,
Jl. Ir. Sutami 36 A Kentingan Surakarta, Indonesia
Received 13 October 2009; Revised 11 February 2010; Accepted 12 February 2010
ut
ho
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TiO2 film was synthesized by means of the chemical bath deposition (CBD) method from TiCl4
as a precursor and surfactant cetyl trimethyl ammonium bromide (CTAB) as a linking and assembling agent of the titanium hydroxide network on a graphite substrate. Ag and Cu were loaded
on the TiO2 film by means of electrodeposition at various applied currents. Photoelectrochemical
testing on the composite of Ag–TiO2 /G and Cu–TiO2 /G was used to define the composite for
Escherichia coli-contaminated water disinfection. Disinfection efficiency and the rate of disinfection
of E. coli-contaminated water with Ag–TiO2 /G as a catalyst was higher than that observed for
Cu–TiO2 /G in all disinfection methods including photocatalysis (PC), electrocatalysis (EC), and
photoelectrocatalysis (PEC). The highest rate constant was achieved by the PEC method using
Ag–TiO2 /G, k was 6.49 × 10−2 CFU mL−1 min−1 . Effective disinfection times of 24 h (EDT24 )
and 48 h (EDT48 ) were achieved in all methods except the EC method using Cu–TiO2 /G.
c 2010 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Ag–TiO2 /G, Cu–TiO2 /G, Escherichia coli, disinfection
A
Introduction
Waste produced by humans and animals significantly contributes to the contamination of surface
water and ground water. Contaminants of concern
include nutrients (nitrate-nitrogen and phosphorus)
and bacteria. Escherichia coli is a well-known indicator bacterium (Cho et al., 2004). If the environment
is conducive to the survival of E. coli, it may also
be conducive to supporting other potentially harmful
pathogens.
Chlorine, chlorine dioxide, ozone, and ultraviolet
radiation are used as disinfectants in water treatment
facilities around the world. Among them, chlorine is
widely used to disinfect drinking water because of its
low cost and wide range of available information on it.
However, it can form toxic by-products such as carcinogenic trihalomethanes (THMs). Increasing chlorine dosage and residence time can improve disinfection levels. Yet, this can lead to increased THM for*Corresponding author, e-mail: fitria@uns.ac.id
mation; therefore, the reduction of organic pollutants
is required prior to chlorine disinfection (Kabir et al.,
2003). Ultraviolet radiation studies on E. coli have
been carried out since the mid-20th century (Kantor
& Deering, 1966; Barner & Cohen, 1956). Doubleday
et al. (1977) studied the ultraviolet-induced mutation
in E. coli, and the evaluation of E. coli cells damage
has been studied in recent years (Sinha & Häder, 2002;
Kappke et al., 2005; Friedberg, 2003).
Over the recent years, much attention has been directed to heterogeneous photocatalytic detoxification,
where water pollutants are degraded by irradiation of
an aqueous suspension of pollutants with powdered
semiconductor oxides, primarily focusing on TiO2 as
a highly active and durable photocatalyst (Xu et al.,
2008; Körösi et al., 2007; Chu et al., 2008; Akurati
et al., 2007; Rincón et al., 2001). Escherichia coli inactivation in the photocatalytic treatment using TiO2
has been extensively studied (Wei et al., 1994; Kikuchi
et al., 1997; Ohno et al., 1998; Dunlop et al., 2002).
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
fectiveness of disinfection times of 24 h (EDT24 ), and
48 h (EDT48 ) in E. coli-contaminated water disinfection with Ag and Cu loaded on TiO2 film.
Experimental
op
y
All solutions and reagents were prepared with
deionized distilled water and analytical grade chemicals (Merck Chemicals, Indonesia) were used. All
glassware used was washed with distilled water and
then autoclaved at 121 ◦C for 15 min.
Escherichia coli bacteria were cultured in a sterilized lactose broth in a shaking unit at 37 ◦C for 24 h.
The approximate formulae per litre of lactose broth
medium (pH 6.9 ± 0.2) are beef extract 3.0 g, peptone 5.0 g, and lactose 5.0 g. The model of E. colicontaminated water was prepared by pouring 1 mL of
E. coli suspension into 99 mL of sterilized deionized
water, and was then twice diluted to 100 mL. Triplicate dilution produced Escherichia coli with average
colonies of 261 ± 6, equal to 1.04 × 105 ± 2263 CFU
mL−1 . Bacterial colonies counts were usually between
30 and 300, fewer than 30 are inaccurate because a
single contamination can cause an at least 4 % error
and over 300 the colonies are difficult to count (Christensen et al., 2003). Measurement of the E. coli population in contaminated water was done by pouring 1
mL of contaminated water into a Petri dish and adding
sterilized EMBA (eosin methylene blue lactose sucrose
agar) medium at 45 ◦C. After the medium was solidified, it was incubated at 37 ◦C for 24 h. Colonies were
counted using a colony counter (Stuart Scientific, UK)
and the number of surviving cells in the sample was
calculated according to Eq. (1). In all counts, three
replicate experiments were carried out.
A
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Study on the killing mechanism of bacteria by photocatalytic treatment was carried out by Maness et
al. (1999). However, the quantum efficiency of photocatalytic degradation on bare titania is relatively low
as this process, which involves the transfer of photogenerated electrons and holes created within the
semiconductor particles, competes with the recombination of these charge carriers. A potentially promising route to improve photocatalytic efficiency is to
separate and hence reduce the recombination using
an applied potential. A research conducted by Butterfield et al. (1997) produced photoelectrochemical
disinfection not only for Escherichia coli but also for
Clostridium perfringens and Cryptosporidium parvum
(Christensen et al., 2003). Other studies on photoelectrochemical disinfection were conducted by Harper et
al. (2001), Christensen et al. (2003), and Egerton et
al. (2006). Many studies have been devoted to increasing the photocatalytic activity of TiO2 . In particular,
Ag deposition on thin film of TiO2 on an ITO (indium tin oxide) substrate (TiO2 /ITO) was found to
improve the photocatalytic activity of TiO2 because
migration of photogenerated electrons into metal particles can increase the lifetime of holes and reduce the
possibility of recombination (He et al., 2003).
TiO2 powder is commonly used in the laboratory
in the form of a suspension. This method yields a
high catalyst surface area to volume ratio for pollutant hydroxyl radical interaction. However, the catalyst must be removed by a post-treatment separation
stage, which may not be cost effective in large scale
(Li et al., 2008). An effective way to avoid the need
for a post-treatment separation is to load TiO2 on a
substrate, such as activated carbon (Hammer et al.,
2007; Li et al., 2008), titanium plate (Harper et al.,
2001), titanium mesh cylinder (Egerton et al., 2006),
titanium foil (Dunlop et al., 2002), and titanium metal
mesh (Christensen et al., 2003).
In the research reported in this paper, photocatalytic, electrocatalytic, and photoelectrocatalytic inactivation of Escherichia coli was examined over Ag
and Cu loaded on a TiO2 film on a graphite substrate.
The graphite substrate was used in this research due to
its cheapness, inertness, and good electronic conductor properties. Regarding the conductor properties, it
is possible to improve photo catalytic efficiency by the
application of a small positive potential to a UV irradiated TiO2 /G electrode. Meanwhile, the TiO2 film
was deposited by chemical bath deposition due to the
ease of the process, and the fact that there is no requirement for special equipment and high temperature. Here, surfactant molecules were used as linking
and assembling agents of the titanium hydroxide network on the graphite substrate. The titanium hydroxide network was transformed into titanium oxide films
by an annealing process (Rahmawati et al., 2006).
This research paid particular attention to investigating disinfection effectiveness, rates of inactivation, ef-
N
nd
CFU mL−1 =
v
(1)
where n is the number of colonies per plate, d is the
dilution factor, v is the plated volume (mL), and CFU
is the colony forming unit.
Synthesis of TiO2 /G was carried out by means of
chemical bath deposition (CBD). Synthesis solution
was prepared in a pyrex glass vessel by dissolving 1.1
mL of TiCl4 (99 %) into 100 mL of 1 M HCl, then 0.58
g of cetyl trimethyl ammonium bromide was added
into this solution. The solution was stirred for 5 min
using a magnetic stirrer. A graphite plate was hung on
a wire, dipped into the synthesis solution, and kept at
60 ◦C without stirring for 4 days in an incubator (Selecta, Spain, Hot-Cold M). Then, the plate was washed
with deionized water and heated to 450 ◦C. Deposition of Ag onto TiO2 /graphite was done by electrodeposition of 0.4 M AgNO3 solution at 0.01 A, 0.015
A, 0.020 A, 0.025 A, and 0.030 A for 30 min. An
analytic-analyzer electrolysis (Yanaco, Japan, AES2D ± 1 × 10−2 V) was used for the electrodeposition.
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
op
y
min killed 60 % of bacteria in open circuit conditions
providing 95 % of bacteria were killed at 1.3 V potential and 99.5 % at 3 V. Christensen et al. (2003) also
found that application of 3.0 V on the sol–gel TiO2
electrode with UV irradiation has better results than
the application of 1.3 V. This was the reason for using the potential of 3 V in this research. The cathodic
cell in the EC and PEC methods was a graphite rod
dipped into a 0.1 M KCl solution, and the anodic cell
was a disk of Ag–TiO2 /G or Cu–TiO2 /G dipped into
E. coli-contaminated water. A salt bridge connected
the two cells, and an Ni wire allowed electrons to flow
into an external circuit. Each experiment was repeated
twice and the results were reproducible within the relative error of < 5 %.
Inactivation of Escherichia coli was determined by
measuring the number of surviving cells after a definite time, and presented as % of inactivation by Eq.
(3), where N0 /(CFU mL−1 ) is the initial population
and Nt /(CFU mL−1 ) is the population after time t of
disinfection
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Meanwhile, 0.4 M CuSO4 solution and current of 0.01
A, 0.015 A, 0.02 A, 0.025 A, and 0.03 A, and time of
30 min were used for Cu electrodeposition. XRD (Shimadzu 6000, Japan) analysis was used to identify the
prepared materials, while the microstructure analysis
was carried out using a scanning electron microscope
(Philips XL-20, Japan).
Optimization of the applied current variation
is based on % of incident photon to current efficiency (%IPCE) of Cu–TiO2 /G and Ag–TiO2 /G.
The %IPCE measurement was carried out in the 200–
700 nm wavelength range. The Cu–TiO2 /G or Ag–
TiO2 /G electrode was dipped into 0.1 M tetraethylammonium iodide and 0.1 M I2 in acetonitrile, and
connected to a microamperemeter, with a standard
calomel electrode (SCE) as the reference electrode.
The samples were illuminated with monochromatic
light (diffraction grating C-T monochromator of UVVIS spectrophotometer Seiki Ogawa, Japan, was used)
from a deuterium lamp (100 mA, 10 mV) as a UV
light source, and a wolfram lamp (100 mA 10 mV)
as a visible light source. The incident photon to current efficiency (%IPCE) was calculated according to
the following equation
Isc A cm−2 1240
%IPCE =
× 100
(2)
Pinc (W cm−2 ) λ/nm
A
ut
where Isc is the measured current under short circuit
conditions, Pinc is the incident light power, and λ is
the incident photon wavelength.
Inactivation of E. coli was done by means of photocatalysis (PC), electrocatalysis (EC), and photoelectrocatalysis (PEC) methods in a pyrex glass vessel.
Photolysis (Lys), photon irradiation without a semiconductor material as a catalyst, was carried out for
control purposes. An ultraviolet lamp, 254 nm, 6 W
(Cole–Palmer 9815-series, France), located 12 cm from
the Escherichia coli suspension, was used as a photon
source. The light intensity was 0.3 mW cm−2 , which
was determined by a UVC-254 short wave UV meter
254 nm, ± 2 % F.S. The UVC lamp was used based on
the research result of Zelle and Hollaender (1954) who
found that the highest quantum yield for 37 % survival
of E. coli in quanta incident per cm2 was obtained by
UVC with 265 nm. The most conventional UV lamps
used for disinfection are low-pressure UV lamps. Their
monochromatic emission at the wavelength of 254 nm
is most efficiently absorbed by DNA bases and therefore has one of the greatest germicidal effects among
UV wavelengths (Harm, 1980). In addition, the use
of low power aiming to minimize the heat released by
the lamp into the disinfection cell while keeping the
cell at room temperature (25–28 ◦C). Meanwhile, a 3 V
and 0.5 A power source was used as an electron source
in the EC and PEC processes. A study conducted by
Egerton et al. (2006) showed that UV irradiation on
0.1 atom % Fe-doped TiO2 sol–gel electrode for 25
% inactivation =
N0 − Nt
× 100
N0
(3)
EDT24 and EDT48 were investigated by storing the
samples after disinfection in dark conditions for 24 h
and 48 h under rest condition; then, 1 mL of the sample was poured into 12 mL of sterilized EMBA, solidified, and incubated at 37 ◦C for 24 h. The Escherichia
coli population was then counted by the plate count
method. In all counts, triplicate experiments were used
and the results were reproducible within the relative
error of 5 %. Bars representing typical error were included in Figs. 6 and 7. EDT24 or EDT48 was achieved
if the E. coli population was lower than or the same
as the population at the time after disinfection. If the
population was greater, the % of re-growth were determined by the following equation
% of re-growth =
Nt∗ − Nt
× 100
Nt
(4)
where Nt∗ is the E. coli population after storage for
24 h or 48 h.
A disinfection kinetic model was used to compare
the results obtained under different experimental conditions. In this study, the Chick–Watson model was
used as a reference. This model assumes that the photocatalytic reaction of microorganisms using a semiconductor follows the first order reaction kinetics. In
this research, the Chick–Watson model was tested
by plotting the logarithm of the number of surviving
colonies after disinfection vs. the disinfection time. If
the linear regression coefficient R2 ≥ 0.9, we preferred
the Chick–Watson model with the rate law presented
by the following equation
ln Nt = −kt + ln N0 + c1
(5)
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
16
14
Ag
12
%IPCE · 10
Cu
Cu
4
d
c
b
2
a
6
200
R A
40
50
R
R
60
2 θ /°
ho
Results and discussion
A
ut
A difractogram of synthesized TiO2 /G is presented
in Fig. 1. The difractogram shows that TiO2 deposited
on a graphite substrate by the CBD method is present
in rutile and anatase phases. A study of the influence of the sintering temperature on the TiO2 film
is needed due to the effect of sintering temperature
on the phase transformation to anatase, which is well
known as the most effective phase for catalysis. The
peaks of Ag are present at 2θ of 38.15◦, which matches
the ICSD database #44387, and at 2θ of 52.45◦ , which
matches ICSD #56269. Meanwhile, the peaks of Cu
are present as new peaks found at 2θ of 43.38◦ and
50.53◦ , which matches ICSD # 43493. The difractogram of Ag–TiO2 /G and Cu–TiO2 /G is presented
in Fig. 1.
The %IPCE curves of Cu–TiO2 /G are shown in
Fig. 2. Cu loaded on the TiO2 film enhances the %
conversion of incident photons to current efficiency.
This parallels the effect of metal deposition on the
enhancement of the surface recombination reduction
due to the actions of photogenerated electron collection by metal. Migration of photogenerated electrons into metal particles can increase the lifetime
of holes (He et al., 2003) and activate the oxidation
zone. The general pattern is that there is a %IPCE
rise with the increasing applied current of electrodeposition. Gravimetric analysis found that Cu loaded
on TiO2 /G was 6.3 mass % at 0.03 A of the applied
current, and also for Cu loaded at 0.01 A. However,
600
700
Table 1. Amount of metal deposited on TiO2 film
Applied current
where k/min−1 is the rate constant, t/min is time, and
c1 is a constant.
500
Fig. 2. %IPCE of TiO2 /G (a), Cu–TiO2 /G obtained by electrodeposition of CuSO4 at 0.01 A (b), 0.015 A (c), 0.02
A (d), 0.025 A (e), and 0.03 A (f).
rc
Fig. 1. XRD pattern of TiO2 /G (R: rutile, A: anatase), Ag–
TiO2 /G, and Cu–TiO2 /G.
400
λ/nm
R
70
300
y
R
30
8
R
R
20
e
0
R
A
10
op
Intensity/a.u.
2
Ag
R
f
Metal load/mass %
A
0.010
0.015
0.020
0.025
0.030
Cu
6.3
6.7
8.4
8.3
6.3
±
±
±
±
±
0.60
0.79
0.29
1.66
0.80
Ag
4.1
6.1
5.7
4.1
4.1
±
±
±
±
±
0.37
0.25
0.31
0.30
0.06
their %IPCE are significantly different. Data on metal
loaded (mass %) are listed in Table 1. SEM analysis
showed that the cluster size of Cu loaded at 0.03 A
was 1.18 µm, smaller than that of Cu loaded at 0.01
A which was in the range of 2.56–3.2 µm. Clusters
produced at 0.03 A have a more regular form than
those produced at 0.01 A, Therefore, it can be concluded that a small percentage of loaded metal with
small cluster size and more regular form provides a
better performance in the photogenerated electron collection. However, this explanation may not be applicable to Ag loaded on TiO2 film. SEM micrographs
of TiO2 /graphite and TiO2 /graphite-supported metal
surfaces are given in Fig. 3. Fig. 3a shows that TiO2
coating of the graphite plate is created by a porous
film. The pores are the result of a release of surfactant
molecules during the heating of the graphite plate with
chemically deposited titanium salt at 450 ◦C. Photoelectrochemical testing on Ag–TiO2 /G found that
the %IPCE values of this composite produced at 0.025
A and 0.03 A are similar in the measurement wavelength range (Fig. 4). Table 1 shows that the amount
of Ag loaded at these applied currents is 4.1 %, the
same as that observed applying the current of 0.01 A.
However, the %IPCE value of Ag–TiO2 /G deposited
561
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
12
e
d
10
8
6
c
b
4
2
rc
f
ho
14
a
0
300
400
A
200
ut
%IPCE · 10
2
Fig. 3. Micrograph of TiO2 /G (a) (insert: the disc of TiO2 /G), Cu–TiO2 /G obtained by electrodeposition of CuSO4 at 0.01 A
(b) and at 0.03 A (c), and Ag–TiO2 /G obtained by electrodeposition of AgNO3 at 0.01 A (d), and at 0.03 A (e).
500
600
700
λ/nm
Fig. 4. %IPCE of TiO2 /G (a), Ag–TiO2 /G produced by electrodeposition of 0.4 M AgNO3 at 0.01 A (b), 0.015 A
(c), 0.02 A (d), 0.025 A (e), and 0.03 A (f).
at 0.01 A is significantly lower. The SEM micrograph
in Fig. 4 shows that the cluster size of Ag produced at
0.01 A is 7.19 µm. This value is lower than the cluster size produced at 0.03 A, which is 26.52 µm. The
pattern of the changes in % of metal loaded, listed in
Table 1, can be explained by the fact that the percentage of metal loaded is not linearly dependent on
the applied current as declared by the Faraday’s law.
This subject is discussed in Rahmawati et al. (2008).
Photoelectrochemical data were used as reference
for the material selection for disinfection. In this research, the disinfection experiment was carried out
with a disk of composite TiO2 which has an average
mass of 2.55 × 10−3 mg. The composite was dipped
into 100 mL of an Escherichia coli solution with an average of colonies of 261 ± 6, equal to (1.04 × 105 ±
2263) CFU mL−1 . Cu was loaded using the 0.03 A applied current while Ag was loaded using the 0.025 A
applied current since it had almost the same %IPCE
value as when loaded at 0.03 A.
Escherichia coli inactivation can occur in an irradiated photon without a catalyst (photolysis process,
Lys). The data on surviving cells in CFU mL−1 after
the treatment are presented in Table 2. The data in
Table 2 were plotted in Eq. (2) to calculate the % inactivation with the initial colony number of 1 × 105
CFU mL−1 . The calculation shows that in 30 min,
40.6 % of E. coli are inactivated by the UV light (254
nm). DNA is certainly one of the key targets of UVinduced damage in a variety of organisms ranging from
bacteria (Peak & Peak, 1982; Peak et al., 1984) to humans (Stein et al., 1989; Kripke et al., 1992). UV radiation induces two of the most abundant mutagenic
and cytotoxic DNA lesions: cyclobutane–pyrimidine
dimmers (CPDs), and 6-4 photoproducts (6-4 PPs)
and their Dewar valence isomers. After the UV irradiation, the CPDs are the most abundant and probably
most cytotoxic lesions but the 6-4 PPs may have more
serious, potentially lethal, mutagenic effects (Sinha &
Häder, 2002). The lesions inhibit normal replication of
the genome and result in the inactivation of the microorganisms. Besides genomes, proteins and enzymes
with unsaturated bonds are known to absorb UVC
and UVB radiation which may also result in significant damage to the organisms (Oguma et al., 2002).
Escherichia coli inactivation by means of the PC
method has higher success rate than photolysis. In
30 min, Ag–TiO2 /G application on PC under UV ir-
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
Table 2. Number of surviving cells after treatment and % of inactivation after 30 min of disinfectiona
Surviving cells/(CFU mL−1 )
%inactivation
Method
Lys (without catalyst)
PC
EC
PEC
Cu–TiO2 /G
Ag–TiO2 /G
Cu–TiO2 /G
Ag–TiO2 /G
62000
59600
43600
33600
62000
21200
17600
10800
40.6
42.9
58.2
67.8
40.6
79.7
83.1
89.7
±
±
±
±
2000
400
1600
400
±
±
±
±
2000
400
400
400
±
±
±
±
2.00
0.38
1.53
0.38
±
±
±
±
2.00
0.38
0.38
0.38
a) Initial concentration of cells is (1.0 × 105 ± 2263) CFU mL−1 .
Cu–TiO2 /G
R2
k/min−1
R2
0.0156
0.0467
0.0510
0.0649
0.896
0.958
0.954
0.942
0.0156
0.0220
0.0272
0.0320
0.896
0.932
0.998
0.986
rc
Photolysis
Photocatalysis
Electrocatalysis
Photoelectrocatalysis
k/min−1
y
Ag–TiO2 /G
Method
conductor interface which is called the depletion layer.
Every electron promoted by photon excitation or external potential application in the depletion layer is
accelerated into the bulk of the semiconductor and released to the external circuit through graphite as a
conductive substrate. Conversely, the holes produced
near the depletion layer by photon illumination are
accelerated into the semiconductor surface and move
freely before being recombined with electrons in the
bulk phase of the semiconductor. It is clear that the
electric field significantly increases the charge separation and thus enhances the hydroxyl radical formation.
This is called the electric field enhancement effect.
The PEC method is the combination of PC and
EC. Based on the reduction of the colony number (Table 2) and the comparison of the rate constant value
(Table 3), it can be concluded that the PEC method
has the best performance in Escherichia coli inactivation. The E. coli population decreases up to 89.7 %
(equal to about 1-log reduction) by PEC under UV
irradiation on Ag–TiO2 /G in 30 min, and 67.8 % on
Cu–TiO2 /G. In this study, inactivation of E. coli using
the Ag–TiO2 /G has reached the lag stage in 30 min.
This is the reason the 30 min disinfection time was
used as the purpose of this research was to compare
disinfection efficiency of different methods and different materials. This is in accordance with the correct
application of the Chick–Watson equation requiring
that the disinfection rate does not vary at all during
the process. Consequently, this model is only valid for
the description of the second region (log-linear). However, the model is capable to compare the efficiency of
different disinfection methods through the values of
the kinetic constant k (Marugán et al., 2008).
Christensen et al. (2003) found that applying the
potential of 3 V to the sol–gel electrode of TiO2 , 95 %
(equal to 1.3-log reduction) of E. coli were destroyed
in 25 min. However, there are differences in the type of
the lamp and the electrode surface area. Christensen
et al. (2003) used a tubular UV source (two 8 W black
lamps) located within the electrode cassette of a cylindrical TiO2 diamond mesh with the 100 mm length
and 22 mm radius. Meanwhile, Fe doping on the Christensen’s sol–gel electrode was done by Egerton et al.
(2006). UV irradiation of 0.1 atom % Fe-doped sol–
gel heated at 600 ◦C for 25 min killed 95 % of E. coli
op
Table 3. Rate constants of Escherichia coli inactivation
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ho
radiation is capable of inactivating 79.7 % of the E.
coli population. Meanwhile, Cu–TiO2 /G only achieves
42.9 %, see Table 2. The value of the rate constant
calculated by the first order kinetics, compares the reaction rate of different processes and using different
catalyst materials. The rate constant values are listed
in Table 3. In a photocatalytic reaction, the energy
of photons is absorbed by a semiconductor to generate an electron-hole pair in the TiO2 matrix. The hole
in the valence band can react with H2 O or hydroxide
ions adsorbed on the surface to produce hydroxyl radicals (OH.), and the electron in the conduction band
can reduce O2 to produce superoxide ions (O−
2 ). Both
holes and OH. are extremely reactive in contact with
organic compounds. Detailed mechanism of the TiO2
photochemical reaction and the various reactive oxygen species (ROS) such as OH., O−
2 , and H2 O2 produced are well-documented (Blake et al., 1999; Hoffmann et al., 1995; Mills & Le Hunte, 1997). Maness
et al. (1999) found the ROS generated on the irradiated TiO2 surface to attack polyunsaturated phospholipids in Escherichia coli. The lipid peroxidation reaction that subsequently causes a breakdown of the cell
membrane structure and therefore its associated function is the mechanism underlying cell death. Sunada et
al. (1998) reported that endotoxin, an integral component of the outer membrane of E. coli, was destroyed
under photocatalytic conditions when TiO2 was used.
The calculation of %inactivation by plotting the
data in Table 2 into Eq. (2) shows that the EC method
has a better performance than the PC one. This conclusion is supported by the rate constant data (Table 3). The potential application to TiO2 reduces the
Fermi level and forms an electric field near the semi-
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
a
b
c
d
60000
60000
1
40000
30000
10000
Lys
PC
EC
Method
PEC
ut
).
ho
Fig. 6. Variation of E. coli population with the disinfection
method used in the presence of Ag–TiO2 /G: after dis), after storage for 24 h (
), and
infection (
for 48 h (
(equal to 1.3-log reduction) at 1.3 V and 99.5 % of E.
coli (equal to 2.3-log reduction) at 3 V. The electrode
used by Egerton et al. (2006) had the same dimensions
as the one used by Christensen et al. (2003) with the
surface area of 22 cm2 . In this study, the surface area
of the electrode was only 0.504 cm2 , however, highenergy UVC lamp was used.
Fast reduction of the E. coli population by the
PEC disinfection is promoted by the formation of excited electron-hole pairs by photon irradiation and
separation of excited electron-holes applying an external potential. This allows the holes to move freely
in the semiconductor surface and to oxidize the species
on the interface of the semiconductor, which results in
the formation of hydroxyl radicals that are the main
contributors to cell degradation. Reduction of the E.
coli population is shown in Fig. 5.
Although it is well known that both Ag and Cu
have antimicrobial properties, the antimicrobial activity of these two metals is still far below the antimicrobial activity of the PEC method using composite
materials. Yoon et al. (2007) found that for the disinfection of water contaminated with E. coli (initial concentration 200 CFU), the 90 % antimicrobial efficiency
was obtained using 58.41 µg mL−1 of Ag nanoparti-
A
40000
30000
20000
rc
20000
0
50000
op
50000
y
70000
E.coli population/(CFU mL¯ )
70000
1
E.coli population/(CFU mL¯ )
Fig. 5. Escherichia coli population during PEC degradation using Ag–TiO2 /G: initial population (a), and population after 10 min
(b), 20 min (c), and 30 min (d) of treatment.
10000
0
Lys
PC
EC
PEC
Method
Fig. 7. Variation of E. coli population with the disinfection
method used in the presence of Cu–TiO2 /G: after dis), after storage for 24 h (
), and
infection (
for 48 h (
).
cles (particle size of 40 nm) or 33.49 µg mL−1 of Cu
nanoparticles (100 nm). Calculations using the data
in Table 1 show that 1.046 µg mL−1 of Ag or 1.607
µg mL−1 of Cu is sufficient to reduce the E. coli population by 89.7 % or 67.8 %, respectively. The initial
E. coli concentration used in this study was 1.0 × 105
CFU mL−1 . However, this is only a rough comparison,
given the differences in some parameters of the experiment including the particle size of metal and the time
of disinfection.
EDT24 and EDT48 were achieved when applying
Ag–TiO2 /G in all disinfection methods. It is shown in
Fig. 6 that the Escherichia coli population after the
storage for 24 h and 48 h decreased compared to the
population at the time after the disinfection. However,
when applying Cu–TiO2 /G, EDT24 and EDT48 were
not achieved using the EC method (Fig. 7). The % of
re-growth of E. coli calculated by Eq. (4) was 36.17 %
for EDT24 and 110.64 % for EDT48 . This confirms
that the catalysis by electrons using Cu–TiO2 /G as
the electrode only inactivates E. coli but does not kill
them as E. coli bacteria have the capability of selfprotection under extreme conditions and may reactivate when the conditions return to normal. This result
also shows that the distilled water medium was not the
564
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
rc
Deposition of Ag and Cu on TiO2 /G is capable
of enhancing the effectiveness of Escherichia coli inactivation. Ag deposition has a better performance
than Cu. EDT24 and EDT48 can be achieved in
all catalytic methods using Ag–TiO2 /G and photonassisted catalytic methods using a Cu–TiO2 /G electrode, however, they cannot be achieved in electrocatalytic methods using a Cu–TiO2 /G electrode.
y
Conclusions
Doubleday, O. P., Green, M. H. L., & Bridges, B. A.
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436–440. DOI: 10.1038/nature01408.
Hammer, N., Kvande, I., Xu, X., Gunnarsson, V., Tøtdal, B.,
Chen, D., & Rønning, M. (2007). Au–TiO2 catalysts on
carbon nanofibres prepared by deposition-precipitation and
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10.1016/j.cattod.2007.03.001.
Harm, W. (1980). Biological effects of ultraviolet radiation (pp.
31–39). New York, NY, USA: Cambridge University Press.
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kinetics of the photoelectrochemical disinfection of water inoculated with E. coli. Journal of Applied Electrochemistry,
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He, C., Xiong, Y., Zha, C., Wang, X., & Zhu, X. (2003). Approach to a pulse photoelectrocatalytic process for the degradation of organic pollutants. Journal of Chemical Technology
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D. W. (1995). Environmental application of semiconductor photocatalysis. Chemical Reviews, 95, 69–96. DOI:
10.1021/cr00033a004.
Kabir, M. F., Haque, F., Vaisman, E., Langford, C. H., &
Kantzas, A. (2003). Disinfecting E. coli bacteria in drinking water using a novel fluidized bed reactor. International
Journal of Chemical Reactor Engineering, 1, 1–10.
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studies of filamentous Escherichia coli B. Journal of Bacteriology, 92, 1062–1070.
Kappke, J., da Silva, E. R., Schelin, H. R., Paschuk, S. A.,
Pashchuk, A., de Oliveira, A., Filho, N. C., Szanto, E. M.,
Takahashi, J., & Calvacante de Souza, J. (2005). Evaluation of Escherichia coli cells damages induced by ultraviolet
and proton beam radiation. Brazilian Journal of Physics, 35,
805–807. DOI: 10.1590/S0103-97332005000500022.
Kikuchi, Y., Sunada, K., Iyoda, T., Hashimoto, K., & Fujishima,
A. (1997). Photocatalytic bactericidal effect of TiO2 thin
films: dynamic view of the active oxygen species responsible for the effect. Journal of Photochemistry and Photobiology A: Chemistry, 106, 51–56. DOI: 10.1016/S10106030(97)00038-5.
Körösi, L., Papp, S., Bertóti, I., & Dékány, I. (2007). Surface and
bulk composition, structure, and photocatalytic activity of
phosphate-modified TiO2 . Chemistry of Materials, 19, 4811–
4819. DOI: 10.1021/cm070692r.
Kripke, M. L., Cox, P. A., Alas, L. G., & Yarosh, D. B. (1992).
Pyrimidine dimmers in DNA initiate systemic suppression in
UV-irradiated mice. Proceedings of the National Academy of
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op
main factor driving the decline in E. coli survival after
their storage for 24 h or 48 h; instead, the result of the
EC disinfection showed an increase in the number of
colonies of E. coli.
These results clearly show that the deposition of
Ag on the TiO2 /G film has better performance in enhancing catalytic activity of TiO2 compared to the
Cu deposition. Based on the data in Table 2, it can be
concluded that Ag–TiO2 /G as a catalyst for the PEC
disinfection of Escherichia coli-contaminated water is
by 21.9 % more efficient than Cu–TiO2 /G.
ho
Acknowledgements. This research was partially supported
by the DIPA project program of the Sebelas Maret University
and the HIBAH Bersaing project program of the Directorate
General for Higher Education. The support was greatly appreciated.
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565
DOI: 10.2478/s11696-010-0036-4
ORIGINAL PAPER
Ag and Cu loaded on TiO2 /graphite as a catalyst
for
-contaminated water disinfection
y
Fitria Rahmawati*, Triana Kusumaningsih, Anita M. Hapsari, Aris Hastuti
op
Department of Chemistry, The Faculty of Mathematics and Natural Sciences, Sebelas Maret University,
Jl. Ir. Sutami 36 A Kentingan Surakarta, Indonesia
Received 13 October 2009; Revised 11 February 2010; Accepted 12 February 2010
ut
ho
rc
TiO2 film was synthesized by means of the chemical bath deposition (CBD) method from TiCl4
as a precursor and surfactant cetyl trimethyl ammonium bromide (CTAB) as a linking and assembling agent of the titanium hydroxide network on a graphite substrate. Ag and Cu were loaded
on the TiO2 film by means of electrodeposition at various applied currents. Photoelectrochemical
testing on the composite of Ag–TiO2 /G and Cu–TiO2 /G was used to define the composite for
Escherichia coli-contaminated water disinfection. Disinfection efficiency and the rate of disinfection
of E. coli-contaminated water with Ag–TiO2 /G as a catalyst was higher than that observed for
Cu–TiO2 /G in all disinfection methods including photocatalysis (PC), electrocatalysis (EC), and
photoelectrocatalysis (PEC). The highest rate constant was achieved by the PEC method using
Ag–TiO2 /G, k was 6.49 × 10−2 CFU mL−1 min−1 . Effective disinfection times of 24 h (EDT24 )
and 48 h (EDT48 ) were achieved in all methods except the EC method using Cu–TiO2 /G.
c 2010 Institute of Chemistry, Slovak Academy of Sciences
Keywords: Ag–TiO2 /G, Cu–TiO2 /G, Escherichia coli, disinfection
A
Introduction
Waste produced by humans and animals significantly contributes to the contamination of surface
water and ground water. Contaminants of concern
include nutrients (nitrate-nitrogen and phosphorus)
and bacteria. Escherichia coli is a well-known indicator bacterium (Cho et al., 2004). If the environment
is conducive to the survival of E. coli, it may also
be conducive to supporting other potentially harmful
pathogens.
Chlorine, chlorine dioxide, ozone, and ultraviolet
radiation are used as disinfectants in water treatment
facilities around the world. Among them, chlorine is
widely used to disinfect drinking water because of its
low cost and wide range of available information on it.
However, it can form toxic by-products such as carcinogenic trihalomethanes (THMs). Increasing chlorine dosage and residence time can improve disinfection levels. Yet, this can lead to increased THM for*Corresponding author, e-mail: fitria@uns.ac.id
mation; therefore, the reduction of organic pollutants
is required prior to chlorine disinfection (Kabir et al.,
2003). Ultraviolet radiation studies on E. coli have
been carried out since the mid-20th century (Kantor
& Deering, 1966; Barner & Cohen, 1956). Doubleday
et al. (1977) studied the ultraviolet-induced mutation
in E. coli, and the evaluation of E. coli cells damage
has been studied in recent years (Sinha & Häder, 2002;
Kappke et al., 2005; Friedberg, 2003).
Over the recent years, much attention has been directed to heterogeneous photocatalytic detoxification,
where water pollutants are degraded by irradiation of
an aqueous suspension of pollutants with powdered
semiconductor oxides, primarily focusing on TiO2 as
a highly active and durable photocatalyst (Xu et al.,
2008; Körösi et al., 2007; Chu et al., 2008; Akurati
et al., 2007; Rincón et al., 2001). Escherichia coli inactivation in the photocatalytic treatment using TiO2
has been extensively studied (Wei et al., 1994; Kikuchi
et al., 1997; Ohno et al., 1998; Dunlop et al., 2002).
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
fectiveness of disinfection times of 24 h (EDT24 ), and
48 h (EDT48 ) in E. coli-contaminated water disinfection with Ag and Cu loaded on TiO2 film.
Experimental
op
y
All solutions and reagents were prepared with
deionized distilled water and analytical grade chemicals (Merck Chemicals, Indonesia) were used. All
glassware used was washed with distilled water and
then autoclaved at 121 ◦C for 15 min.
Escherichia coli bacteria were cultured in a sterilized lactose broth in a shaking unit at 37 ◦C for 24 h.
The approximate formulae per litre of lactose broth
medium (pH 6.9 ± 0.2) are beef extract 3.0 g, peptone 5.0 g, and lactose 5.0 g. The model of E. colicontaminated water was prepared by pouring 1 mL of
E. coli suspension into 99 mL of sterilized deionized
water, and was then twice diluted to 100 mL. Triplicate dilution produced Escherichia coli with average
colonies of 261 ± 6, equal to 1.04 × 105 ± 2263 CFU
mL−1 . Bacterial colonies counts were usually between
30 and 300, fewer than 30 are inaccurate because a
single contamination can cause an at least 4 % error
and over 300 the colonies are difficult to count (Christensen et al., 2003). Measurement of the E. coli population in contaminated water was done by pouring 1
mL of contaminated water into a Petri dish and adding
sterilized EMBA (eosin methylene blue lactose sucrose
agar) medium at 45 ◦C. After the medium was solidified, it was incubated at 37 ◦C for 24 h. Colonies were
counted using a colony counter (Stuart Scientific, UK)
and the number of surviving cells in the sample was
calculated according to Eq. (1). In all counts, three
replicate experiments were carried out.
A
ut
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Study on the killing mechanism of bacteria by photocatalytic treatment was carried out by Maness et
al. (1999). However, the quantum efficiency of photocatalytic degradation on bare titania is relatively low
as this process, which involves the transfer of photogenerated electrons and holes created within the
semiconductor particles, competes with the recombination of these charge carriers. A potentially promising route to improve photocatalytic efficiency is to
separate and hence reduce the recombination using
an applied potential. A research conducted by Butterfield et al. (1997) produced photoelectrochemical
disinfection not only for Escherichia coli but also for
Clostridium perfringens and Cryptosporidium parvum
(Christensen et al., 2003). Other studies on photoelectrochemical disinfection were conducted by Harper et
al. (2001), Christensen et al. (2003), and Egerton et
al. (2006). Many studies have been devoted to increasing the photocatalytic activity of TiO2 . In particular,
Ag deposition on thin film of TiO2 on an ITO (indium tin oxide) substrate (TiO2 /ITO) was found to
improve the photocatalytic activity of TiO2 because
migration of photogenerated electrons into metal particles can increase the lifetime of holes and reduce the
possibility of recombination (He et al., 2003).
TiO2 powder is commonly used in the laboratory
in the form of a suspension. This method yields a
high catalyst surface area to volume ratio for pollutant hydroxyl radical interaction. However, the catalyst must be removed by a post-treatment separation
stage, which may not be cost effective in large scale
(Li et al., 2008). An effective way to avoid the need
for a post-treatment separation is to load TiO2 on a
substrate, such as activated carbon (Hammer et al.,
2007; Li et al., 2008), titanium plate (Harper et al.,
2001), titanium mesh cylinder (Egerton et al., 2006),
titanium foil (Dunlop et al., 2002), and titanium metal
mesh (Christensen et al., 2003).
In the research reported in this paper, photocatalytic, electrocatalytic, and photoelectrocatalytic inactivation of Escherichia coli was examined over Ag
and Cu loaded on a TiO2 film on a graphite substrate.
The graphite substrate was used in this research due to
its cheapness, inertness, and good electronic conductor properties. Regarding the conductor properties, it
is possible to improve photo catalytic efficiency by the
application of a small positive potential to a UV irradiated TiO2 /G electrode. Meanwhile, the TiO2 film
was deposited by chemical bath deposition due to the
ease of the process, and the fact that there is no requirement for special equipment and high temperature. Here, surfactant molecules were used as linking
and assembling agents of the titanium hydroxide network on the graphite substrate. The titanium hydroxide network was transformed into titanium oxide films
by an annealing process (Rahmawati et al., 2006).
This research paid particular attention to investigating disinfection effectiveness, rates of inactivation, ef-
N
nd
CFU mL−1 =
v
(1)
where n is the number of colonies per plate, d is the
dilution factor, v is the plated volume (mL), and CFU
is the colony forming unit.
Synthesis of TiO2 /G was carried out by means of
chemical bath deposition (CBD). Synthesis solution
was prepared in a pyrex glass vessel by dissolving 1.1
mL of TiCl4 (99 %) into 100 mL of 1 M HCl, then 0.58
g of cetyl trimethyl ammonium bromide was added
into this solution. The solution was stirred for 5 min
using a magnetic stirrer. A graphite plate was hung on
a wire, dipped into the synthesis solution, and kept at
60 ◦C without stirring for 4 days in an incubator (Selecta, Spain, Hot-Cold M). Then, the plate was washed
with deionized water and heated to 450 ◦C. Deposition of Ag onto TiO2 /graphite was done by electrodeposition of 0.4 M AgNO3 solution at 0.01 A, 0.015
A, 0.020 A, 0.025 A, and 0.030 A for 30 min. An
analytic-analyzer electrolysis (Yanaco, Japan, AES2D ± 1 × 10−2 V) was used for the electrodeposition.
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F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
op
y
min killed 60 % of bacteria in open circuit conditions
providing 95 % of bacteria were killed at 1.3 V potential and 99.5 % at 3 V. Christensen et al. (2003) also
found that application of 3.0 V on the sol–gel TiO2
electrode with UV irradiation has better results than
the application of 1.3 V. This was the reason for using the potential of 3 V in this research. The cathodic
cell in the EC and PEC methods was a graphite rod
dipped into a 0.1 M KCl solution, and the anodic cell
was a disk of Ag–TiO2 /G or Cu–TiO2 /G dipped into
E. coli-contaminated water. A salt bridge connected
the two cells, and an Ni wire allowed electrons to flow
into an external circuit. Each experiment was repeated
twice and the results were reproducible within the relative error of < 5 %.
Inactivation of Escherichia coli was determined by
measuring the number of surviving cells after a definite time, and presented as % of inactivation by Eq.
(3), where N0 /(CFU mL−1 ) is the initial population
and Nt /(CFU mL−1 ) is the population after time t of
disinfection
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Meanwhile, 0.4 M CuSO4 solution and current of 0.01
A, 0.015 A, 0.02 A, 0.025 A, and 0.03 A, and time of
30 min were used for Cu electrodeposition. XRD (Shimadzu 6000, Japan) analysis was used to identify the
prepared materials, while the microstructure analysis
was carried out using a scanning electron microscope
(Philips XL-20, Japan).
Optimization of the applied current variation
is based on % of incident photon to current efficiency (%IPCE) of Cu–TiO2 /G and Ag–TiO2 /G.
The %IPCE measurement was carried out in the 200–
700 nm wavelength range. The Cu–TiO2 /G or Ag–
TiO2 /G electrode was dipped into 0.1 M tetraethylammonium iodide and 0.1 M I2 in acetonitrile, and
connected to a microamperemeter, with a standard
calomel electrode (SCE) as the reference electrode.
The samples were illuminated with monochromatic
light (diffraction grating C-T monochromator of UVVIS spectrophotometer Seiki Ogawa, Japan, was used)
from a deuterium lamp (100 mA, 10 mV) as a UV
light source, and a wolfram lamp (100 mA 10 mV)
as a visible light source. The incident photon to current efficiency (%IPCE) was calculated according to
the following equation
Isc A cm−2 1240
%IPCE =
× 100
(2)
Pinc (W cm−2 ) λ/nm
A
ut
where Isc is the measured current under short circuit
conditions, Pinc is the incident light power, and λ is
the incident photon wavelength.
Inactivation of E. coli was done by means of photocatalysis (PC), electrocatalysis (EC), and photoelectrocatalysis (PEC) methods in a pyrex glass vessel.
Photolysis (Lys), photon irradiation without a semiconductor material as a catalyst, was carried out for
control purposes. An ultraviolet lamp, 254 nm, 6 W
(Cole–Palmer 9815-series, France), located 12 cm from
the Escherichia coli suspension, was used as a photon
source. The light intensity was 0.3 mW cm−2 , which
was determined by a UVC-254 short wave UV meter
254 nm, ± 2 % F.S. The UVC lamp was used based on
the research result of Zelle and Hollaender (1954) who
found that the highest quantum yield for 37 % survival
of E. coli in quanta incident per cm2 was obtained by
UVC with 265 nm. The most conventional UV lamps
used for disinfection are low-pressure UV lamps. Their
monochromatic emission at the wavelength of 254 nm
is most efficiently absorbed by DNA bases and therefore has one of the greatest germicidal effects among
UV wavelengths (Harm, 1980). In addition, the use
of low power aiming to minimize the heat released by
the lamp into the disinfection cell while keeping the
cell at room temperature (25–28 ◦C). Meanwhile, a 3 V
and 0.5 A power source was used as an electron source
in the EC and PEC processes. A study conducted by
Egerton et al. (2006) showed that UV irradiation on
0.1 atom % Fe-doped TiO2 sol–gel electrode for 25
% inactivation =
N0 − Nt
× 100
N0
(3)
EDT24 and EDT48 were investigated by storing the
samples after disinfection in dark conditions for 24 h
and 48 h under rest condition; then, 1 mL of the sample was poured into 12 mL of sterilized EMBA, solidified, and incubated at 37 ◦C for 24 h. The Escherichia
coli population was then counted by the plate count
method. In all counts, triplicate experiments were used
and the results were reproducible within the relative
error of 5 %. Bars representing typical error were included in Figs. 6 and 7. EDT24 or EDT48 was achieved
if the E. coli population was lower than or the same
as the population at the time after disinfection. If the
population was greater, the % of re-growth were determined by the following equation
% of re-growth =
Nt∗ − Nt
× 100
Nt
(4)
where Nt∗ is the E. coli population after storage for
24 h or 48 h.
A disinfection kinetic model was used to compare
the results obtained under different experimental conditions. In this study, the Chick–Watson model was
used as a reference. This model assumes that the photocatalytic reaction of microorganisms using a semiconductor follows the first order reaction kinetics. In
this research, the Chick–Watson model was tested
by plotting the logarithm of the number of surviving
colonies after disinfection vs. the disinfection time. If
the linear regression coefficient R2 ≥ 0.9, we preferred
the Chick–Watson model with the rate law presented
by the following equation
ln Nt = −kt + ln N0 + c1
(5)
560
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
16
14
Ag
12
%IPCE · 10
Cu
Cu
4
d
c
b
2
a
6
200
R A
40
50
R
R
60
2 θ /°
ho
Results and discussion
A
ut
A difractogram of synthesized TiO2 /G is presented
in Fig. 1. The difractogram shows that TiO2 deposited
on a graphite substrate by the CBD method is present
in rutile and anatase phases. A study of the influence of the sintering temperature on the TiO2 film
is needed due to the effect of sintering temperature
on the phase transformation to anatase, which is well
known as the most effective phase for catalysis. The
peaks of Ag are present at 2θ of 38.15◦, which matches
the ICSD database #44387, and at 2θ of 52.45◦ , which
matches ICSD #56269. Meanwhile, the peaks of Cu
are present as new peaks found at 2θ of 43.38◦ and
50.53◦ , which matches ICSD # 43493. The difractogram of Ag–TiO2 /G and Cu–TiO2 /G is presented
in Fig. 1.
The %IPCE curves of Cu–TiO2 /G are shown in
Fig. 2. Cu loaded on the TiO2 film enhances the %
conversion of incident photons to current efficiency.
This parallels the effect of metal deposition on the
enhancement of the surface recombination reduction
due to the actions of photogenerated electron collection by metal. Migration of photogenerated electrons into metal particles can increase the lifetime
of holes (He et al., 2003) and activate the oxidation
zone. The general pattern is that there is a %IPCE
rise with the increasing applied current of electrodeposition. Gravimetric analysis found that Cu loaded
on TiO2 /G was 6.3 mass % at 0.03 A of the applied
current, and also for Cu loaded at 0.01 A. However,
600
700
Table 1. Amount of metal deposited on TiO2 film
Applied current
where k/min−1 is the rate constant, t/min is time, and
c1 is a constant.
500
Fig. 2. %IPCE of TiO2 /G (a), Cu–TiO2 /G obtained by electrodeposition of CuSO4 at 0.01 A (b), 0.015 A (c), 0.02
A (d), 0.025 A (e), and 0.03 A (f).
rc
Fig. 1. XRD pattern of TiO2 /G (R: rutile, A: anatase), Ag–
TiO2 /G, and Cu–TiO2 /G.
400
λ/nm
R
70
300
y
R
30
8
R
R
20
e
0
R
A
10
op
Intensity/a.u.
2
Ag
R
f
Metal load/mass %
A
0.010
0.015
0.020
0.025
0.030
Cu
6.3
6.7
8.4
8.3
6.3
±
±
±
±
±
0.60
0.79
0.29
1.66
0.80
Ag
4.1
6.1
5.7
4.1
4.1
±
±
±
±
±
0.37
0.25
0.31
0.30
0.06
their %IPCE are significantly different. Data on metal
loaded (mass %) are listed in Table 1. SEM analysis
showed that the cluster size of Cu loaded at 0.03 A
was 1.18 µm, smaller than that of Cu loaded at 0.01
A which was in the range of 2.56–3.2 µm. Clusters
produced at 0.03 A have a more regular form than
those produced at 0.01 A, Therefore, it can be concluded that a small percentage of loaded metal with
small cluster size and more regular form provides a
better performance in the photogenerated electron collection. However, this explanation may not be applicable to Ag loaded on TiO2 film. SEM micrographs
of TiO2 /graphite and TiO2 /graphite-supported metal
surfaces are given in Fig. 3. Fig. 3a shows that TiO2
coating of the graphite plate is created by a porous
film. The pores are the result of a release of surfactant
molecules during the heating of the graphite plate with
chemically deposited titanium salt at 450 ◦C. Photoelectrochemical testing on Ag–TiO2 /G found that
the %IPCE values of this composite produced at 0.025
A and 0.03 A are similar in the measurement wavelength range (Fig. 4). Table 1 shows that the amount
of Ag loaded at these applied currents is 4.1 %, the
same as that observed applying the current of 0.01 A.
However, the %IPCE value of Ag–TiO2 /G deposited
561
op
y
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
12
e
d
10
8
6
c
b
4
2
rc
f
ho
14
a
0
300
400
A
200
ut
%IPCE · 10
2
Fig. 3. Micrograph of TiO2 /G (a) (insert: the disc of TiO2 /G), Cu–TiO2 /G obtained by electrodeposition of CuSO4 at 0.01 A
(b) and at 0.03 A (c), and Ag–TiO2 /G obtained by electrodeposition of AgNO3 at 0.01 A (d), and at 0.03 A (e).
500
600
700
λ/nm
Fig. 4. %IPCE of TiO2 /G (a), Ag–TiO2 /G produced by electrodeposition of 0.4 M AgNO3 at 0.01 A (b), 0.015 A
(c), 0.02 A (d), 0.025 A (e), and 0.03 A (f).
at 0.01 A is significantly lower. The SEM micrograph
in Fig. 4 shows that the cluster size of Ag produced at
0.01 A is 7.19 µm. This value is lower than the cluster size produced at 0.03 A, which is 26.52 µm. The
pattern of the changes in % of metal loaded, listed in
Table 1, can be explained by the fact that the percentage of metal loaded is not linearly dependent on
the applied current as declared by the Faraday’s law.
This subject is discussed in Rahmawati et al. (2008).
Photoelectrochemical data were used as reference
for the material selection for disinfection. In this research, the disinfection experiment was carried out
with a disk of composite TiO2 which has an average
mass of 2.55 × 10−3 mg. The composite was dipped
into 100 mL of an Escherichia coli solution with an average of colonies of 261 ± 6, equal to (1.04 × 105 ±
2263) CFU mL−1 . Cu was loaded using the 0.03 A applied current while Ag was loaded using the 0.025 A
applied current since it had almost the same %IPCE
value as when loaded at 0.03 A.
Escherichia coli inactivation can occur in an irradiated photon without a catalyst (photolysis process,
Lys). The data on surviving cells in CFU mL−1 after
the treatment are presented in Table 2. The data in
Table 2 were plotted in Eq. (2) to calculate the % inactivation with the initial colony number of 1 × 105
CFU mL−1 . The calculation shows that in 30 min,
40.6 % of E. coli are inactivated by the UV light (254
nm). DNA is certainly one of the key targets of UVinduced damage in a variety of organisms ranging from
bacteria (Peak & Peak, 1982; Peak et al., 1984) to humans (Stein et al., 1989; Kripke et al., 1992). UV radiation induces two of the most abundant mutagenic
and cytotoxic DNA lesions: cyclobutane–pyrimidine
dimmers (CPDs), and 6-4 photoproducts (6-4 PPs)
and their Dewar valence isomers. After the UV irradiation, the CPDs are the most abundant and probably
most cytotoxic lesions but the 6-4 PPs may have more
serious, potentially lethal, mutagenic effects (Sinha &
Häder, 2002). The lesions inhibit normal replication of
the genome and result in the inactivation of the microorganisms. Besides genomes, proteins and enzymes
with unsaturated bonds are known to absorb UVC
and UVB radiation which may also result in significant damage to the organisms (Oguma et al., 2002).
Escherichia coli inactivation by means of the PC
method has higher success rate than photolysis. In
30 min, Ag–TiO2 /G application on PC under UV ir-
562
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
Table 2. Number of surviving cells after treatment and % of inactivation after 30 min of disinfectiona
Surviving cells/(CFU mL−1 )
%inactivation
Method
Lys (without catalyst)
PC
EC
PEC
Cu–TiO2 /G
Ag–TiO2 /G
Cu–TiO2 /G
Ag–TiO2 /G
62000
59600
43600
33600
62000
21200
17600
10800
40.6
42.9
58.2
67.8
40.6
79.7
83.1
89.7
±
±
±
±
2000
400
1600
400
±
±
±
±
2000
400
400
400
±
±
±
±
2.00
0.38
1.53
0.38
±
±
±
±
2.00
0.38
0.38
0.38
a) Initial concentration of cells is (1.0 × 105 ± 2263) CFU mL−1 .
Cu–TiO2 /G
R2
k/min−1
R2
0.0156
0.0467
0.0510
0.0649
0.896
0.958
0.954
0.942
0.0156
0.0220
0.0272
0.0320
0.896
0.932
0.998
0.986
rc
Photolysis
Photocatalysis
Electrocatalysis
Photoelectrocatalysis
k/min−1
y
Ag–TiO2 /G
Method
conductor interface which is called the depletion layer.
Every electron promoted by photon excitation or external potential application in the depletion layer is
accelerated into the bulk of the semiconductor and released to the external circuit through graphite as a
conductive substrate. Conversely, the holes produced
near the depletion layer by photon illumination are
accelerated into the semiconductor surface and move
freely before being recombined with electrons in the
bulk phase of the semiconductor. It is clear that the
electric field significantly increases the charge separation and thus enhances the hydroxyl radical formation.
This is called the electric field enhancement effect.
The PEC method is the combination of PC and
EC. Based on the reduction of the colony number (Table 2) and the comparison of the rate constant value
(Table 3), it can be concluded that the PEC method
has the best performance in Escherichia coli inactivation. The E. coli population decreases up to 89.7 %
(equal to about 1-log reduction) by PEC under UV
irradiation on Ag–TiO2 /G in 30 min, and 67.8 % on
Cu–TiO2 /G. In this study, inactivation of E. coli using
the Ag–TiO2 /G has reached the lag stage in 30 min.
This is the reason the 30 min disinfection time was
used as the purpose of this research was to compare
disinfection efficiency of different methods and different materials. This is in accordance with the correct
application of the Chick–Watson equation requiring
that the disinfection rate does not vary at all during
the process. Consequently, this model is only valid for
the description of the second region (log-linear). However, the model is capable to compare the efficiency of
different disinfection methods through the values of
the kinetic constant k (Marugán et al., 2008).
Christensen et al. (2003) found that applying the
potential of 3 V to the sol–gel electrode of TiO2 , 95 %
(equal to 1.3-log reduction) of E. coli were destroyed
in 25 min. However, there are differences in the type of
the lamp and the electrode surface area. Christensen
et al. (2003) used a tubular UV source (two 8 W black
lamps) located within the electrode cassette of a cylindrical TiO2 diamond mesh with the 100 mm length
and 22 mm radius. Meanwhile, Fe doping on the Christensen’s sol–gel electrode was done by Egerton et al.
(2006). UV irradiation of 0.1 atom % Fe-doped sol–
gel heated at 600 ◦C for 25 min killed 95 % of E. coli
op
Table 3. Rate constants of Escherichia coli inactivation
A
ut
ho
radiation is capable of inactivating 79.7 % of the E.
coli population. Meanwhile, Cu–TiO2 /G only achieves
42.9 %, see Table 2. The value of the rate constant
calculated by the first order kinetics, compares the reaction rate of different processes and using different
catalyst materials. The rate constant values are listed
in Table 3. In a photocatalytic reaction, the energy
of photons is absorbed by a semiconductor to generate an electron-hole pair in the TiO2 matrix. The hole
in the valence band can react with H2 O or hydroxide
ions adsorbed on the surface to produce hydroxyl radicals (OH.), and the electron in the conduction band
can reduce O2 to produce superoxide ions (O−
2 ). Both
holes and OH. are extremely reactive in contact with
organic compounds. Detailed mechanism of the TiO2
photochemical reaction and the various reactive oxygen species (ROS) such as OH., O−
2 , and H2 O2 produced are well-documented (Blake et al., 1999; Hoffmann et al., 1995; Mills & Le Hunte, 1997). Maness
et al. (1999) found the ROS generated on the irradiated TiO2 surface to attack polyunsaturated phospholipids in Escherichia coli. The lipid peroxidation reaction that subsequently causes a breakdown of the cell
membrane structure and therefore its associated function is the mechanism underlying cell death. Sunada et
al. (1998) reported that endotoxin, an integral component of the outer membrane of E. coli, was destroyed
under photocatalytic conditions when TiO2 was used.
The calculation of %inactivation by plotting the
data in Table 2 into Eq. (2) shows that the EC method
has a better performance than the PC one. This conclusion is supported by the rate constant data (Table 3). The potential application to TiO2 reduces the
Fermi level and forms an electric field near the semi-
563
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
a
b
c
d
60000
60000
1
40000
30000
10000
Lys
PC
EC
Method
PEC
ut
).
ho
Fig. 6. Variation of E. coli population with the disinfection
method used in the presence of Ag–TiO2 /G: after dis), after storage for 24 h (
), and
infection (
for 48 h (
(equal to 1.3-log reduction) at 1.3 V and 99.5 % of E.
coli (equal to 2.3-log reduction) at 3 V. The electrode
used by Egerton et al. (2006) had the same dimensions
as the one used by Christensen et al. (2003) with the
surface area of 22 cm2 . In this study, the surface area
of the electrode was only 0.504 cm2 , however, highenergy UVC lamp was used.
Fast reduction of the E. coli population by the
PEC disinfection is promoted by the formation of excited electron-hole pairs by photon irradiation and
separation of excited electron-holes applying an external potential. This allows the holes to move freely
in the semiconductor surface and to oxidize the species
on the interface of the semiconductor, which results in
the formation of hydroxyl radicals that are the main
contributors to cell degradation. Reduction of the E.
coli population is shown in Fig. 5.
Although it is well known that both Ag and Cu
have antimicrobial properties, the antimicrobial activity of these two metals is still far below the antimicrobial activity of the PEC method using composite
materials. Yoon et al. (2007) found that for the disinfection of water contaminated with E. coli (initial concentration 200 CFU), the 90 % antimicrobial efficiency
was obtained using 58.41 µg mL−1 of Ag nanoparti-
A
40000
30000
20000
rc
20000
0
50000
op
50000
y
70000
E.coli population/(CFU mL¯ )
70000
1
E.coli population/(CFU mL¯ )
Fig. 5. Escherichia coli population during PEC degradation using Ag–TiO2 /G: initial population (a), and population after 10 min
(b), 20 min (c), and 30 min (d) of treatment.
10000
0
Lys
PC
EC
PEC
Method
Fig. 7. Variation of E. coli population with the disinfection
method used in the presence of Cu–TiO2 /G: after dis), after storage for 24 h (
), and
infection (
for 48 h (
).
cles (particle size of 40 nm) or 33.49 µg mL−1 of Cu
nanoparticles (100 nm). Calculations using the data
in Table 1 show that 1.046 µg mL−1 of Ag or 1.607
µg mL−1 of Cu is sufficient to reduce the E. coli population by 89.7 % or 67.8 %, respectively. The initial
E. coli concentration used in this study was 1.0 × 105
CFU mL−1 . However, this is only a rough comparison,
given the differences in some parameters of the experiment including the particle size of metal and the time
of disinfection.
EDT24 and EDT48 were achieved when applying
Ag–TiO2 /G in all disinfection methods. It is shown in
Fig. 6 that the Escherichia coli population after the
storage for 24 h and 48 h decreased compared to the
population at the time after the disinfection. However,
when applying Cu–TiO2 /G, EDT24 and EDT48 were
not achieved using the EC method (Fig. 7). The % of
re-growth of E. coli calculated by Eq. (4) was 36.17 %
for EDT24 and 110.64 % for EDT48 . This confirms
that the catalysis by electrons using Cu–TiO2 /G as
the electrode only inactivates E. coli but does not kill
them as E. coli bacteria have the capability of selfprotection under extreme conditions and may reactivate when the conditions return to normal. This result
also shows that the distilled water medium was not the
564
F. Rahmawati et al./Chemical Papers 64 (5) 557–565 (2010)
rc
Deposition of Ag and Cu on TiO2 /G is capable
of enhancing the effectiveness of Escherichia coli inactivation. Ag deposition has a better performance
than Cu. EDT24 and EDT48 can be achieved in
all catalytic methods using Ag–TiO2 /G and photonassisted catalytic methods using a Cu–TiO2 /G electrode, however, they cannot be achieved in electrocatalytic methods using a Cu–TiO2 /G electrode.
y
Conclusions
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op
main factor driving the decline in E. coli survival after
their storage for 24 h or 48 h; instead, the result of the
EC disinfection showed an increase in the number of
colonies of E. coli.
These results clearly show that the deposition of
Ag on the TiO2 /G film has better performance in enhancing catalytic activity of TiO2 compared to the
Cu deposition. Based on the data in Table 2, it can be
concluded that Ag–TiO2 /G as a catalyst for the PEC
disinfection of Escherichia coli-contaminated water is
by 21.9 % more efficient than Cu–TiO2 /G.
ho
Acknowledgements. This research was partially supported
by the DIPA project program of the Sebelas Maret University
and the HIBAH Bersaing project program of the Directorate
General for Higher Education. The support was greatly appreciated.
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A
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