Environmental Pollution 247 (2019) 847e856

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Superior disinfection effect of Escherichia coli by hydrothermal

synthesized TiO2-based composite photocatalyst under LED
irradiation: Influence of environmental factors and disinfection
mechanism*
Na Liu a, Qi Zhu b, Nan Zhang a, Cheng Zhang a, Naoki Kawazoe c, Guoping Chen c,
Nobuaki Negishi b, Yingnan Yang a, *
a
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8577, Japan
b
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba,
Ibaraki, 305-8569, Japan
c
Research Center of Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The photocatalytic inactivation of Escherichia coli (E. coli) under light-emitting diode (LED) light irradi-
Received 3 October 2018 ation was performed with P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst to investigate the photocatalytic
Received in revised form bactericidal activity. Our work showed that this composite photocatalyst possessed remarkable bacterial
7 January 2019
disinfection ability and could completely inactivate 108 cfu/mL of E. coli within just 40 min under the
Accepted 19 January 2019
optimum catalyst loading of 0.5 g/L. The effects of different environmental factors, including light
Available online 28 January 2019
wavelength, light intensity, temperature, solution pH and inorganic ions, on the inactivation efficiency
were evaluated. The results showed that bacteria inactivation by P/Ag/Ag2O/Ag3PO4/TiO2 was more
Keywords:
P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst
favorable with blue colored LED irradiation, light intensity at 750 W/m2, temperature in the range of 30
Escherichia coli inactivation e37
C and pH values at natural or slightly alkaline condition. The existence of different inorganic ions
Environmental factors under normal environmental level had no significant impact on the bactericidal performance. In addi-
Disinfection mechanism tion, during the inactivation process, the morphology changes of E. coli cells were directly observed by
LED light irradiation scanning electron microscope (SEM) and further proved by the measurement of Kþ leakage from the
inactivated E. coli. The results demonstrated that the photocatalytic inactivation caused drastic damage
on bacterial cells membrane. Furthermore, the mechanisms of photocatalytic bacterial inactivation were
also systemically studied and the results confirmed that the excellent disinfection activity of P/Ag/Ag2O/
Ag3PO4/TiO2 resulted from the major reactive species: hþ and $O 2 from photocatalytic process instead of
the leakage of Agþ (0.085 ± 0.005 mg/L) from photocatalyst. These results indicate that P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst has promising potential for real water sterilization application.
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction Usually, the conventional water disinfection methods including

Annually, waterborne diseases can cause up to 1.8 million
deaths around the world (World Health Organization (WHO),
2008). Many drinking water sources are not only contaminated
by various hazardous chemicals, but also polluted by many path-
ogenic microganisms, therefore, need to be sterilized before use.
*
This paper has been recommended for acceptance by Prof. Dr. Klaus Kümmerer.
* Corresponding author.
E-mail address: yo.innan.fu@u.tsukuba.ac.jp (Y. Yang).
chlorination, ozonation and ultraviolet (UV) radiation, are effective
against most pathogens (Pablos et al., 2013; Carbajo et al., 2016).
Unfortunately, all of these methods have several disadvantages,
such as the formation of over 600 kinds of potentially hazardous
disinfection byproducts (DBPs) or the emergence of chlorine-
resistant microbial (Sharma et al., 2014; Park et al., 2016).
Furthermore, these treatment methods are often chemically,
energetically or operationally expensive (Shannon et al., 2008).
Therefore, it is of great importance to develop a versatile new
technology to remove pathogenic contaminants from water.
As a “green” advanced oxidation technology, semiconductor

https://doi.org/10.1016/j.envpol.2019.01.082
0269-7491/© 2019 Elsevier Ltd. All rights reserved.
848 N. Liu et al. / Environmental Pollution 247 (2019) 847e856
titanium dioxide (TiO2) photocatalysis has attracted tremendous
attention since the first photocatalytic inactivation was reported
(Matsunaga et al., 1985). It has been shown to be superior over the
conventional methods for water disinfection, due to its relatively
high photocatalytic oxidation ability, long-term stability and non-
toxicity (Zheng et al., 2018; Santaella et al., 2014). Nevertheless, the
low effective utilization of solar light limits the practical application
of pure TiO2. Due to its large band gap, TiO2 is only activated under
UV light which only accounts for 4% of the total solar light spec-
trum. As such, in order to develop highly efficient visible-light-
driven photocatalysts, considerable efforts have been made to
modify TiO2 by metal deposition (Ag and Au) (Chen et al., 2010), ion
doping (S, C, P and N) (Asahi et al., 2001) and combination with
narrow-band gap semiconductors (AgBr and Ag3PO4) (Cao et al.,
2012). Based on abovementioned modification methods, in our
previous study, a visible-light-driven P/Ag/Ag2O/Ag3PO4/TiO2
photocatalyst has been successfully synthesized by sol-gel method,
exhibiting a small band gap and a strong absorption band in the
visible region (Hu et al., 2015). It has been demonstrated with
remarkable photocatalytic efficiency in decomposing the organic
matters and sterilization of bacteria (Zhu et al., 2017). However,
some reports have pointed out that the specific surface area of sol-
gel prepared photocatalyst is relatively small, which is not benefit
for high efficiency of photocatalytic wastewater treatment
(Schwarz et al., 1995). Additionally, the high temperature calcina-
tion during material synthesis may break the sensitive structure of
Ag salts. On the other hand, hydrothermal method has been an
interesting technique to prepare materials with the advantages of
facile synthesis at low temperature (Ohshima et al., 2004). There-
fore, recently with further research, a novel hydrothermal synthe-
sized P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst has been successfully
developed by our lab for the first time and showed bigger specific
area, smaller crystal size and higher organic degradation ability
than the composite synthesized by sol-gel method. However,
regarding its photocatalytic disinfection ability, there is no research
being conducted. In addition, as the next generation indoor lighting
source, commercial visible light-emitting diode (LED) lamps have
drawn a lot of attention due to its long lifetime, high efficiency, low
cost and energy saving (Bergh, 2004). Moreover, compared to other
light sources, the wavelength of the LED lamp is more specific (Ng
et al., 2016). Therefore, by using a suitable LED lamp as light source,
the light emitted form LED lamp can be better utilized by the
photocatalyst, further reducing the operating cost of water sterili-
zation. However, to date, there are few studies available for
combining the photocatalytic disinfection with LED irradiation (Ng
et al., 2016; Wu et al., 2016).
Actually, in the real water environment, photocatalytic disin-
fection reactions are affected by various factors such as intensity of
the light source, temperature of the reaction, pH of the solution, etc.
(Bhatkhande et al., 2002; Sontakke et al., 2011). Besides, natural
water resources contain many different kinds of inorganic ions
(such as Cl, SO2 
4 and NO3 ) (Sontakke et al., 2011). And it has been
reported that the presence of these ions has a great influence on
photocatalytic degradation of organic substances (Bhatkhande
et al., 2002). However, there are quite few studies which reported
the effects of different environmental factors on the photocatalytic
inactivation of microorganisms. Moreover, for the photocatalytic
mechanism, it is largely different in each system based on the
particle size, electronic potential of band structure, surface states
and morphology of the photocatalysts (Alrousan et al., 2009). As
such, the photocatalytic mechanism needs to be considered indi-
vidually. Surprisingly, by far, there are few researches on the
investigation of photocatalytic disinfection mechanism by different
photocatalysts. To our knowledge, it is the first time to study the
effects of environmental factors on bacterial inactivation, especially
the inactivation mechanism by the novel hydrothermal synthesized
P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst under LED light irradiation.
Hence, the objective of this work was to study the effects of
different environmental factors (such as light intensity, light
wavelength, temperature, pH, and existence of ions) on the inac-
tivation efficiency of Escherichia coli (E. coli) by using P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst under LED irradiation. Furthermore, the
possible bacterial inactivation mechanism of P/Ag/Ag2O/Ag3PO4/
TiO2 photocatalyst was systematically investigated. In order to get a
result closer to the actual water environment, the E. coli used in the
experiments was isolated from the natural environment.
2. Materials and methods
2.1. Synthesis of P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst
The P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst with visible light
response was prepared by a hydrothermal method. At first, 6 mL
tetrabutyl titanate was dissolved in 46 mL ethanol. After stirring for
20 min at ambient temperature, the required amount of AgNO3,
Ag3PO4 and 11 mL 1 mol/L HNO3 solution was added drop wise into
the compound under vigorous stirring. The final sol-gel solution
obtained was transparent, homogeneous and stable after stirring
12 h at room temperature (Hu et al., 2015). Then the obtained sol
was transferred into a Teflon-lined autoclave and heated at
110e130
C for 3 h, followed by cooling to room temperature
naturally. After the hydrothermal reaction, the resultant pre-
cipitates were collected by three times centrifuge at 9390  g
(TOMY, Multipurpose Refrigerated Centrifuge LX-120, Japan),
washed thoroughly with ethanol and dried at 60
C for 12 h. Finally,
the P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst powder was successfully
synthesized and stored in desiccator. In order to compare the
antibacterial activity with the novel photocatalyst, pure TiO2 was
also prepared as control by the same method. All of the agents used
during this study were obtained from FUJIFILM Wako Pure Chem-
ical Corporation, Japan.
2.2. Bacteria preparation
In this study, E. coli, a Gram-negative bacterium, was chosen as
model bacteria. It was isolated from Matsumi Lake (University of
Tsukuba, Japan) by using XM-G agar medium. At first, the collected
water sample was streaked on the XM-G agar selective medium by
streak plate method and incubated for 18 h at 35
C. Then, the E. coli
colony with blue color can be easily isolated from the environment.
After isolation, E. coli was incubated in Luria Bertani broth medium
at 37
C for 24 h to reach a stationary phase. It is usually observed
that the resistance of stationary phase bacteria is greater than that
of exponential phase bacteria (Cherchi and Gu, 2011). After incu-
bation, cells were harvested from overnight culture by centrifuga-
tion at 2960  g for 10 min and then washed twice with Phosphate-
buffered saline (PBS). Finally, the prepared cells were resuspended
and diluted to 108 colony-forming units (cfu/mL) with PBS by
measuring the optical density at 600 nm by a spectrophotometer
(Shimadzu, UV-1600, Japan). The accurate initial cell concentration
was determined by the spread plate method with a serial dilution.
All the materials used in the experiments were autoclaved at 121
C
for 15 min before use to ensure sterility.
2.3. Photocatalytic bacterial inactivation
The photocatalytic disinfection reactor used in this study con-
sisted of three parts: a reaction vessel, visible lamps and a voltage
regulator used to adjust light intensity (Fig. S1). Specially, the
visible light source of our photocatalytic disinfection experiments
 N. Liu et al. / Environmental Pollution 247 (2019) 847e856
was provided by LED lamps (Nichia, NSSW-064, Japan) with the
light intensity around 750 W/m2 for 40 min irradiation and the
output power of this kind LED lamp was less than 0.3 W. At first, a
20 mL mixture suspension including P/Ag/Ag2O/Ag3PO4/TiO2 pho-
tocatalyst and E. coli of 108 cfu/mL was put into the reactor and
vigorously dispersed by a magnetic stirrer. The photocatalyst con-
centration was adjusted to 0.25, 0.50, 0.75, 1.00 g/L, respectively.
Then, at different time intervals, 1 mL of reaction mixture was
collected and immediately diluted with PBS solution for a serial
dilution. Following that, 1 mL of the diluted sample was spread
uniformly on Luria Bertani nutrient agar plates in triplicate and
incubated at 37
C for 24 h. After incubation, the number of viable
cells in each sample were then determined by counting the bac-
terial colonies on the plates. Control experiment (bacterial cells
suspension without catalyst) was also conducted for comparison at
the beginning while investigating the photocatalytic disinfection
activity of the novel P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst. For the
bacterial cell suspension used in controls, they derived from the
same stem suspension with that used in photocatalytic disinfection
experiment. Each set of the experiments was performed in tripli-
cate and the average value was given.
For practical application of P/Ag/Ag2O/Ag3PO4/TiO2 photo-
catalyst, the potential effects of different parameters on the pho-
tocatalytic disinfection ability were evaluated according to the
possible conditions in real environment, including light intensity
(250, 500, 750 W/m2), light wavelength (465e470, 515e521 and
620e625 nm), temperature (20, 25, 30 and 37
C), solution pH (5,
6,7 and 8) and different inorganic ions (NO 2 
3 , SO4 and Cl ). At the
beginning of photocatalytic reaction, the solution pH was adjusted
with HCl or NaOH (0.1 mM). The concentration of all inorganic ions
was adjusted to 0.2 mM based upon the reported values in surface
water (Alrousan et al., 2009).
2.4. Preparation procedure for bacterial SEM observation
Before and after inactivation, the destruction process of E. coli
cells at different time intervals (0, 20, 40, 90 min) was observed by
scanning electron microscope (SEM) (Hitachi, FE-SEM S-4800 EDX,
Japan). The mixture of E. coli and photocatalyst was collected and
centrifuged at 2960  g for 10 min, and the cells were prefixed by
4% glutaraldehyde for 4 h, and then washed with PBS 3 times
(10 min each). Subsequently, the specimens were dehydrated by a
graded series of ethanol (25%, 50%, 80% and then 100%) and then
100% butyl alcohol for 3 times (15 min each), respectively. The
resulting cells were finally critical point dried, gold sputter coated
on the substrates and visualized using SEM at a magnification of
10000 times.
2.5. Detection of released Agþ and Kþ
During the photocatalytic inactivation process with P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst, the inactivation solution samples were
collected at different time intervals and centrifuged at 11860  g for
3 times (10 min each). Then, the concentration of released Kþ from
bacteria cells and Agþ ions from photocatalyst were measured by
inductively coupled plasma optical emission spectrometry (ICP-
OES) (Hitachi, SPS3520UV-DD, Japan), respectively (Fassel and
Kniseley, 1974).
2.6. Scavenger study
To clearly identify the effects of different active species gener-
ated by P/Ag/Ag2O/Ag3PO4/TiO2 (0.5 g/L), different scavengers were
applied to remove the corresponding active species in the photo-
catalytic process. In brief, sodium oxalate (Na2C2O4) was utilized to
849
remove hþ (Shi et al., 2014), isopropanol was utilized to remove
diffusing ·OH in the solution bulk (Zhang et al., 2010) and TEMPOL
was employed to remove ·O 2 (Chen et al., 2011a). The applied
concentration of Na2C2O4, isopropanol and TEMPOL was optimized
as 0.5, 5 and 2 mM, respectively (Shi et al., 2014; Zhang et al., 2010;
Chen et al., 2011a). Each of scavenger was added into the reactor
simultaneously with the photocatalyst. Following that, the bacteria
suspension was added into the reactor to start the photocatalytic
disinfection reaction. The contributions of different reactive oxygen
species (ROSs) during the photocatalytic disinfection process were
determined by comparing the inactivation efficiency with and
without the addition of different scavengers. In addition, the blank
control experiments were also performed by only adding scaven-
gers into bacterial solutions.
3. Results and discussion
3.1. Photocatalytic inactivation
The photocatalytic disinfection activity of novel P/Ag/Ag2O/
Ag3PO4/TiO2 composite was investigated by the inactivation of
E. coli (initial cell concentration: 107 cfu/mL) under LED irradiation.
As shown in Fig. 1, the results demonstrated that 107 cfu/mL of
E. coli was 100% sterilized by the novel composite only within
20 min of photocatalytic treatment. Even under dark condition, an
approximately 2-log reduction of E. coli was achieved after 20 min.
For the disinfecting ability of dark control, it can be attributed to the
tiny amount of nanometer metal Ag loaded on the composite (Sun
et al., 2008). However, the bacterial population remained almost
unchanged by pure TiO2 under dark condition or even after 20 min
treatment of LED irradiation. In addition, when the experiment was
conducted under light alone without catalyst (No catalyst), there
was no decrease in the concentration of viable bacteria observed,
indicating that light irradiation alone could not photolyze the
bacterial cells. Compared with other reported photocatalysts, such
as g-C3N4/TiO2 which need 180 min for complete inactivation of
107 cfu/mL E. coli (Li et al., 2015), the as-prepared composite
demonstrated significantly enhanced photocatalytic disinfection
ability. The results suggest that the novel composite P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst possesses superior disinfection ability
under LED irradiation and can be used efficiently for the inactiva-
tion studies.
Fig. 1. Photocatalytic inactivation of E. coli by pure TiO2 and P/Ag/Ag2O/Ag3PO4/TiO2
composite under LED irradiation. Initial bacterial concentration ¼ 107 cfu/ml; Catalyst
concentration ¼ 0.5 g/L; Temperature ¼ 25
C; Lamp intensity ¼ 750 W/m2; LED
wavelength: 465e470 nm; pH ¼ 7.
850 N. Liu et al. / Environmental Pollution 247 (2019) 847e856
To further investigate the photocatalytic disinfection perfor-
mance under extremely high bacterial concentration, the effect of
different initial bacterial concentrations on E. coli removal was
examined with varying initial cell number from 107 to 109 cfu/mL
by P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst under LED irradiation
(Fig. 2). Until now, there were almost no other reported studies
conducted under such high bacterial concentration. From the
figure, it could be clearly observed that E. coli cell number showed a
rapid reduction at all concentrations. 107 cfu/mL and 108 cfu/mL of
E. coli were completely inactivated within 20e40 min, and even the
initial cell number was increased to 109 cfu/mL the complete ster-
ilization time was just 150 min. In order to obtain the kinetic in-
formation, the first-order rate constants (k) at three different
concentrations were calculated and the plot of constant (k) versus
initial bacterial concentration (cfu/mL) has been represented as
inset in the figure. With the initial concentration of E. coli increasing
from 107 to 109 cfu/mL, the rate constants correspondingly
decreased from 0.0364 to 0.0106 min1. The presumed reason is
that as the initial E. coli concentration increasing, the number of
E. coli cells also increases which will block the light to reach to the
surface of catalyst. Similar observation also has been reported by
Alikhani et al. (2013) that with increasing the initial E. coli con-
centration from 1.5  104 to 1.5  108 cfu/mL, the first-order rate
constants correspondingly decreased from 0.001 to 0.0008 min1
(Alikhani et al., 2012). Compared to the reported result, it was
apprent that our material showed higher rate constant over dozens
of times even at higher initial bacterial concentration which further
confirmed the superior disinfection ability of the novel composite
P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst. From the results obtained,
in order to clearly reveal the effects of different factors on the
inactivation efficiency of E. coli and observe the changes under
different conditions, the initial bacterial concentration was set to
108 cfu/mL and used during the following experiments.
In photocatalytic experiments, catalyst loading is an important
factor that can highly influence the efficiency of photocatalytic
treatment. To investigate the optimum catalyst loading, experi-
ments on photocatalytic inactivation of E. coli (initial bacterial
concentration: 108 cfu/mL) by P/Ag/Ag2O/Ag3PO4/TiO2 catalyst
were performed under catalyst concentration ranging from 0.25 to
1.00 g/L. The results are shown in Fig. 3. With increasing the
amount of P/Ag/Ag2O/Ag3PO4/TiO2 in a low range, the activity
Fig. 2. Effect on inactivation with different initial E. coli concentrations (cfu/ml) by P/
Ag/Ag2O/Ag3PO4/TiO2 composite under LED irradiation. The inset represents the plot of
rate constant at various E. coli initial concentrations. Catalyst concentration ¼ 0.5 g/L;
Temperature ¼ 25
C; Lamp intensity ¼ 750 W/m2; LED wavelength: 465e470 nm;
pH ¼ 7.
Fig. 3. Effect of P/Ag/Ag2O/Ag3PO4/TiO2 loading on the inactivation of E. coli. Initial
bacterial concentration ¼ 108 cfu/ml; Temperature ¼ 25
C; Lamp intensity ¼ 750 W/
m2; LED wavelength: 465e470 nm; pH ¼ 7.
increased significantly and a maximum inactivation was observed
when the concentration went up to 0.50 g/L. 108 cfu/mL of the E. coli
was 100% sterilized only within 40 min of photocatalytic treatment.
However, further increment in catalyst loading beyond 0.50 g/L
lead to decrease in photocatalytic disinfection efficiency. The re-
sults indicated 0.50 g/L was the optimum catalyst loading for this
system.
During this photocatalytic disinfection process, at low catalyst
concentration (0.25 g/L), the observed inactivation was not signif-
icant which can be attributed to the less availability of ROSs to
target the large number of bacteria. The improved inactivation at
the concentration of 0.50 g/L is due to the increase of the total
number of active sites on the photocatalyst surface thus causing an
increase in the number of ROSs which are sufficient enough to
target the present cells number. At catalyst loading beyond the
optimum, the activity reduction can be ascribed to the light
screening effect. A high amount of catalyst in the solution causes
the increase of the turbidity in the suspension and thus prevents
the radiation from reaching to cells and other catalyst particles
which leads to the lower rate of inactivation (Benabbou et al., 2007;
Sontakke et al., 2012). Therefore, the optimum concentration of
0.50 g/L which was also a common loading amount during photo-
catalytic degradation process, was used for following disinfection
experiments.
3.2. Influence of different environmental factors
In general, photocatalytic reactions are influenced by various
parameters in the real water environment. In this regard the effects
of different environmental factors which could influence the pho-
tocatalytic disinfection of microorganisms (such as wavelength and
intensity of light source, temperature of reaction, solution pH,
presence of inorganic ions) were discussed.
At first, the influence of light wavelengths on the photocatalytic
inactivation of E. coli by P/Ag/Ag2O/Ag3PO4/TiO2 was examined
under different colored LED lamps (blue, green and red LED) as
shown in Fig. 4A. The red colored LED possesses the longest
wavelength range of approximately 620e625 nm, green LED
515e521 nm, and blue LED 465e470 nm. It can be found that the
photocatalytic disinfection efficiency under blue LED was much
higher than that under green LED and red LED. 108 cfu/mL of E. coli
could be totally disinfected within 40 min photocatalytic treatment
by using blue LED. The inactivation process of green LED was
 N. Liu et al. / Environmental Pollution 247 (2019) 847e856 851

Fig. 4. Effect of different environmental factors: (A) light wavelength, (B) light intensity, (C) temperature, (D) pH and (E) inorganic ions on the inactivation of E. coli by P/Ag/Ag2O/
Ag3PO4/TiO2 composite under LED irradiation. Initial bacterial concentration ¼ 108 cfu/mL; Catalyst concentration ¼ 0.5 g/L.

similar to red LED, and both of them caused 5-log reduction in cell
density after 40 min treatment. This result suggested the most
effective wavelength of LED was in the range of 465e470 nm for the
P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst. This is because the photon
energy of blue LED (465e470 nm) is higher than those of other
colored LED lamps (Bergh, 2004). Therefore, under shorter light
wavelength the photocatalyst showed higher disinfection ability
and the most effective wavelength ranges of LED light for photo-
catalytic disinfection by this novel catalyst was 465e470 nm.
For the photocatalytic inactivation reactions, they are highly
dependent on the illumination intensity of the light source (Bergh,
2004). According to the above research result, blue LED
(465e470 nm) has been demonstrated as the optimum wavelength
range. Hence, experiments to investigate the effect of light intensity
on bacterial disinfection efficiency by the as-prepared composite
photocatalyst were performed using blue LED as light source.
Although the maximum intensity of natural sunlight reaching
the surface of the earth is about 1000 W/m2, while the actual in-
tensity will vary according to atmospheric conditions, geographical
location, daytime and season (Rinco
n and Pulgarin, 2003). For the
light intensity effect of photo-disinfection study, a series of exper-
iments were conducted under the light intensity in the range of
250e750 W/m2. As expected, Fig. 4B shows that the photocatalytic
disinfection efficiency increased with increasing the light intensity
from 250 to 750 W/m2 in the presence of P/Ag/Ag2O/Ag3PO4/TiO2.
At the lower light intensity (250 W/m2), there was a less decrease in
bacterial survival number than higher light intensity, which sug-
gests that low photon incidence limits the photocatalytic reaction.
852 N. Liu et al. / Environmental Pollution 247 (2019) 847e856
At the high intensity (750 W/m2), a high flow of photons is available
which can promote generation of more oxidative species on the
surface of catalyst that can subsequently attack bacterial cells
(Rinco
n and Pulgarin, 2003). Therefore, an increased bacterial
inactivation rate was observed under high light intensity. In addi-
tion, when the experiment was conducted under environmental
average light intensity level (500 W/m2), an approximately 6-log
reduction of E. coli was achieved within 40 min treatment which
suggests the P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst holds enor-
mous potential for actual water sterilization by using solar light as
irradiation source.
The influence of temperature on the inactivation of E. coli was
also examined in this research as shown in Fig. 4C. The reaction
time necessary to achieve 100% of E. coli inactivation was 40 min at
20 and 25
C, while a complete inactivation was achieved within
just 30 min when the temperature was at 30 and 37
C. It was
verified that the photocatalytic disinfection efficiency can be
significantly improved when the temperature was increased.
Generally, it is believed that the production of ROSs is not affected
by the experimental temperature (Herrmann, 2005), thus the
observed temperature-induced effect may be due to the changes in
the fatty acid composition of the cell membrane at different tem-
peratures. It is well known that the temperature can cause the
change of unsaturated and saturated fatty acids amount in bacterial
cell membrane (Sinensky, 1974) and the susceptibility of bacterial
cells towards photocatalytic disinfection is influenced by the pro-
portion of unsaturated to saturated fatty acid in cell membrane
(Cheng et al., 2007; Gao et al., 2012). When the relative amount of
unsaturated fatty acid increases, the membrane of cells will become
less rigid (Tra€uble and Overath, 1973). Then, the bacterial cells
become easier to be attacked by the ROSs (Chu et al., 2016). The
results indicate that bacteria inactivation by P/Ag/Ag2O/Ag3PO4/
TiO2 can perform well at environmental temperature range
(20e37
C) and is more favorable with temperature in the range of
30e37
C.
Fig. 4D shows pH effect on the photocatalytic disinfection of
E. coli by the P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst under blue LED
irradiation with the light intensity of 750 W/m2. The results showed
that an increase of pH would lead to a faster inactivation of bacterial
cells by P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst. Total inactivation of
108 cfu/mL E. coli was achieved within 40 min at pH 7 and 8. Even at
acidic pH value of 5 and 6, the inactivation was still around 5-log.
The results in this study indicate that bacterial inactivation by P/Ag/
Ag2O/Ag3PO4/TiO2 is more favorable under natural or slightly
alkaline conditions, which is probably due to more oxidative spe-
cies produced at more alkaline pH so that it exhibits higher disin-
fection efficiency at natural or slightly alkaline conditions (Chen
et al., 2011b). The common pH of surface water and wastewater
are in the range of 6.5e8.5 (Watts et al., 1995). Therefore, P/Ag/
Ag2O/Ag3PO4/TiO2 would perform well in the common surface
water environment and is suitable for future actual water sterili-
zation application.
In order to match with the realistic treatment process, it is of
great importance to study the effect of common inorganic ions on
the photocatalytic inactivation of microorganisms. Several re-
searchers have reported that the presence of some inorganic ions
has a significant effect on the photocatalytic degradation of organic
compounds, such as Ag/reduced graphene oxide nanocomposite
photodegradation of azo dyes (Borthakur et al., 2017), 1% PteTiO2
photodegradation of trichloroethylene (Burns et al., 1999) and P-25
TiO2 photodegradation of humic acids (Wiszniowski et al., 2004).
Therefore, the effect of presence of various ions (Cl, SO2 
4 and NO3 )
on the photocatalytic inactivation by P/Ag/Ag2O/Ag3PO4/TiO2 was
studied. Based on the results shown in Fig. 4E, the presence of SO2 4
and NO 3 exhibited a negligible effect on the photocatalytic
inactivation of E. coli. On the other hand, Cl had a very little
negative effect on the bacterial inactivation. This is mainly because
Cl can act as scavengers of hydroxyl radicals or some oxidizing
radical species ($Ox) by the following reaction and react invariably
together to decrease the photocatalytic efficiency (Bhatkhande
et al., 2002).
$Ox þ Cl / Cl$ þ Ox (1)
Although the presence of chloride ions showed a very little
negative effect on the bacterial inactivation, there was still around
6-log E. coli inactivated. Therefore, the results suggest that the ex-
istence of different inorganic ions under normal environmental
level has no significant impact on the bactericidal performance of P/
Ag/Ag2O/Ag3PO4/TiO2.
3.3. The cell damage process
To confirm the destruction of bacterial cells by P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst, the morphology changes of E. coli cells
during the inactivation process was examined by SEM. As shown in
Fig. 5A, before photocatalytic inactivation, the bacterial cells
exhibited a rod shape and intact cell structure. After 20 min pho-
tocatalytic treatment, the bacterial cell became abnormal and
showed a rough surface with the formation of pits and holes in
their cell wall. Further, the cell was deeply damaged, forming bigger
pits and holes in their cell walls with much rougher surface after
40 min photocatalytic inactivation. Prolonging the inactivation time
to 90 min, resulted in disorganized membrane structures, which
demonstrated that the cells were completely decomposed. This
observation confirmed that the destruction process of bacterial
cells progresses from the cell membrane to the internal cellular
components, causing the bacterial cells to eventually collapse.
To further confirm the destruction of cell membrane, the
leakage of Kþ from the inactive E. coli was also measured as Kþ is an
important component virtually existing in bacteria (Chen et al.,
2013). Any damage in the cell membrane structure during the
photocatalytic inactivation will cause the Kþ leakage and accumu-
lation in the solution. As the result shown in Fig. 5B, in the exper-
iment without catalyst, the leakage of Kþ from cells was nearly the
same when the time prolonged. Contrarily, Kþ leakage increased
notably in the presence of P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst
under LED light irradiation. Considering the Kþ measurements
combined with SEM images, the photocatalytic inactivation caused
cell membrane to be destroyed and brought a resultant leakage of
intracellular substances.
3.4. Disinfection mechanism of P/Ag/Ag2O/Ag3PO4/TiO2
For Ag-based composite photocatalysts, it is generally accepted
that the released Agþ ions at high concentration can exhibit
bactericidal activity. Furthermore, a high release of Agþ will shorten
the effective lifetime of catalysts and harm the environment and
human health (Akhavan, 2009). As such, the release of Agþ ions
from P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst was examined by ICP-
OES, as shown in Table 1. It can be found that Agþ leakage from
P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst were kept in the range from
0.046 ± 0.004 to 0.085 ± 0.005 mg/L for 12 h treatment, which was
much lower than the reported Minimum Inhibitory Concentration
(MIC) value of Agþ (0.78 mg/L) (Takayama, 1994). According to
previous report, Agþ concentration below 0.6 mg/L does not show
obvious inactivation effect to E. coli (Zhang et al., 2010). These facts
suggest that the quick inactivation of E. coli should result from
photocatalytic performance instead of the released Agþ, which also
further confirms that the results of previous dark control
 N. Liu et al. / Environmental Pollution 247 (2019) 847e856 853

Fig. 5. SEM images of E. coli (A) and leakage of Kþ from E. coli (B) within 90 min photocatalytic treatment by P/Ag/Ag2O/Ag3PO4/TiO2. Initial bacterial concentration ¼ 108 cfu/mL;
Catalyst concentration ¼ 0.5 g/L; Temperature ¼ 25
C; Light intensity ¼ 750 W/m2; LED wavelength: 465e470 nm; pH ¼ 7.

Table 1
Agþ leakage from P/Ag/Ag2O/Ag3PO4/TiO2 photocatalyst in 12 h.

Time 0 min 10 min 20 min 30 min 40 min 12 h

Agþ leakage (mg/L) 0.085 ± 0.005 0.072 ± 0.003 0.074 ± 0.006 0.068 ± 0.002 0.046 ± 0.004 0.079 ± 0.006

destruction should be attributed to the disinfecting ability of
nanometer metal Ag loading on the composite rather than the Agþ
release. Moreover, when the experimental time was extended to
12 h, the concentration of released Agþ was just 0.079 ± 0.006 mg/L
and there was no increased release of Agþ ions observed. The result
demonstrates that the bactericidal activity of P/Ag/Ag2O/Ag3PO4/
TiO2 photocatalyst exhibits high stability under the LED lamp
irradiation. In addition, based on the safety standard value (less
than 0.1 mg/L) of Agþ (World Health Organization (WHO), 2008),
this composite photocatalyst meets the safety standard and could
be an appropriate candidate for practical application.
To further obtain insight into the contribution of various ROSs
during the photocatalytic disinfection process by P/Ag/Ag2O/
Ag3PO4/TiO2, the scavengers study was carried out. During the
854 N. Liu et al. / Environmental Pollution 247 (2019) 847e856

blank control experiments, the addition of each scavenger as
mentioned concentration had no toxic on E. coli within 40 min (see
Fig. S2 in the Supplementary Material). As shown in Fig. 6,
compared with the bacterial inactivation without scavenger, the
addition of TEMPOL and Na2C2O4 significantly inhibited the inac-
tivation of E. coli, proving that ·O þ
2 and h played important role in
the inactivation process. However, when isopropanol was added
into the solution, only a slight decrease in the inactivation efficiency
was observed as compared with that of no scavenger added,
implying that ·OH was not critical in the whole reaction process. In
brief, the above results indicate that hþ and ·O 2 are the major
reactive species contributing to the inactivation process in the
present photocatalytic disinfection system.
Based on aforementioned results and analyses, a possible
mechanism of E. coli inactivation by P/Ag/Ag2O/Ag3PO4/TiO2 pho-
tocatalyst is proposed as shown in Fig. 7. The complete inactivation
of bacteria from the solution may be due to two factors: The main
factor is due to the photocatalytic property exhibited by P/Ag/Ag2O/
Ag3PO4/TiO2 composite under LED irradiation where ·O þ
2 and h are
formed on the surface of catalyst leading to the death of bacteria
and the other factor seems to be attributed to the action of nano-
meter metallic Ag of composite to kill the cells. According to the
results of scavenger study, a detail photocatalytic disinfection
pathway is explained as follows: Firstly, under LED light irradiation,
the photogenerated electron-hole pairs are excited from Ag2O,
Ag3PO4 and TiO2. The possible pathways for generating active
species has been proposed based on the previous report (Hu et al.,
2015). Then the hþ accumulated on Ag2O could directly react with
E. coli. Meanwhile, the photogenerated e at the surface of the TiO2
could react with the adsorbed molecular oxygen to yield ·O 2 . The
reactive species of ·O þ
2 and h could attack bacterial cells, caused
the complete destroy of cell membrane and finally brought massive
cell lyses and inactivation.

3.5. Comparison of different photocatalysts on microbicidal

efficiency

In order to further investigate the photocatalytic disinfection

Fig. 6. Photocatalytic inactivation of E. coli with different scavengers by P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst (Inactivation time ¼ 40 min; Control: with catalyst and
without any scavengers).
ability of this novel photocatalyst, the inactivation efficiency of
E. coli by P/Ag/Ag2O/Ag3PO4/TiO2 was also calculated in comparison
of other reported photocatalysts, such as Cu/TiO2 (Khraisheh et al.,
2015), g-C3N4/TiO2 (Li et al., 2015) and Ag/AgCl-activated carbon
(McEvoy et al., 2013). All of their experimental parameters are lis-
ted in Table 2. It could be found that P/Ag/Ag2O/Ag3PO4/TiO2
composite showed the highest ability even at the highest initial
E. coli concentration, and its photocatalytic inactivation efficiency
reached to 6.67  109 cfu/(g$min) under LED light irradiation.
However, the reported Cu/TiO2, g-C3N4/TiO2 and Ag/AgCl-activated
carbon just exhibited 1.67  107, 9.26  107 and 3.33  106 cfu/
(g$min), respectively. Therefore, the novel composite photocatalyst
showed higher inactivation efficiency than reported Ag/AgCl-
activated carbon, over 2000 times. In addition, the disinfection
experiments on Enterococcus species (a Gram-positive bacterium)
were also conducted. Although the inactivation of Enterococcus
species was more difficult than that of Gram-negative E. coli, the
novel P/Ag/Ag2O/Ag3PO4/TiO2 composite still showed remarkable
disinfection ability against the inactivation of Gram-positive bac-
teria. These results indicate that the as-prepared P/Ag/Ag2O/
Ag3PO4/TiO2 composite photocatalyst holds great potential for
actual water sterilization application in the future solar light driven
photocatalytic disinfection system due to its high efficiency, energy
saving and environment friendly.

4. Conclusions

Hydrothermal synthesized P/Ag/Ag2O/Ag3PO4/TiO2 composite

Fig. 7. Schematic diagram of proposed mechanism of bacterial disinfection by P/Ag/
Ag2O/Ag3PO4/TiO2.
photocatalyst was found to be a promising bactericidal catalyst
because of its notable photocatalytic antibacterial capability under
LED light irradiation. Better bactericidal activity of P/Ag/Ag2O/
Ag3PO4/TiO2 was achieved under high light intensity, shorter light
wavelength, ambient temperature and natural or slightly alkaline

Table 2
Comparison of inactivation efficiency of E. coli by P/Ag/Ag2O/Ag3PO4/TiO2 with different photocatalysts.

Photocatalyst Initial concentration Photocatalyst Concentration Inactivation time Inactivation efficiency Reference
(cfu/mL) (g/L) (min) (cfu/(g$min))

P/Ag/Ag2O/Ag3PO4/TiO2 (LED light) 108 0.50 30 6.67  109 This study

Cu/TiO2 (UVA) 106 1.00 60 1.67  107 Khraisheh et al., 2015
g-C3N4/TiO2 (visible light) 107 0.60 180 9.26  107 Li et al., 2015
Ag/AgCl-activated carbon (visible light) 106 5.00 60 3.33  106 McEvoy et al., 2013
 N. Liu et al. / Environmental Pollution 247 (2019) 847e856
condition. The existence of different inorganic ions under normal
environmental level had no significant impact on the bactericidal
performance, suggesting that this photocatalyst can be applied for
actual water environment. The results of SEM observation and Kþ
leakage measurement verified that the destruction process of the
bacterial cell is progressive from the cell membrane to the inner
cellular components, leading to the final collapse of bacterial cells.
Especially, this work also systemically revealed the mechanisms of
photocatalytic bacterial inactivation. The excellent disinfection ac-
tivity of P/Ag/Ag2O/Ag3PO4/TiO2 resulted from the major reactive
species: hþ and $O 2 from photocatalytic process instead of the
leakage of Agþ from photocatalyst. In comparison with other re-
ported photocatalysts, P/Ag/Ag2O/Ag3PO4/TiO2 composite exhibi-
ted extremely high disinfection efficiency under LED light
irradiation. Therefore, on the basis of its remarkable disinfection
ability, energy saving and environment friendly nature, P/Ag/Ag2O/
Ag3PO4/TiO2 photocatalyst is of great potential for the actual water
sterilization and could greatly facilitate water disinfection system.
Acknowledgement
This work was supported by Scientific Research (B) 15H02859
from Japan Society for the Promotion of Science and JSPS KAKENHI
JP17J00358 and JP18J20620. The authors also thank Takenouchi
Satoshi of Materials Analysis Station, Chemical Analysis and X-ray
Diffraction Group, Research Network and Facility Services Division,
NIMS for part of the technical supporting.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.envpol.2019.01.082.
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