w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

The properties of ZnO nanofluids and the

role of H2O2 in the disinfection activity
against Escherichia coli

Lingling Zhang a,e,*, Yu Li b, Xiaoming Liu b, Lihua Zhao b,

Yulong Ding c, Malcolm Povey d, Daqiang Cang b
a
School of Civil and Environmental Engineering, University of Science and
Technology Beijing, No. 30 Xueyuan Road, Beijing 100083, China
b
School of Metallurgical and Ecological Engineering, University of Science and Technology Beijing,
Beijing 100083, China
c
Institute of Particle Science & Engineering, University of Leeds, Leeds LS2 9JT, UK
d
Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK
e
Key Laboratory of Educational Ministry for High Efficient Mining and Safety in Metal Mine, University of Science
and Technology Beijing, Beijng 100083, China

article info abstract

Article history: This work investigates the disinfection property of ZnO nanofluids, focusing on H2O2
Received 31 May 2012 production and the disinfection activities of ZnO suspensions with different particles/ag-
Received in revised form gregates. The possible disinfection mechanisms of ZnO suspensions are analysed. In this
2 October 2012 work, a medium mill was used to produce ZnO suspensions with different sizes of parti-
Accepted 22 October 2012 cles/aggregates. During the milling process, five ZnO suspension samples (AeE) were
Available online 29 March 2013 produced. X-ray Diffraction (XRD) and Dynamic Light Scattering (DLS) analyses revealed
that after milling, the size of ZnO particles/aggregates in the suspensions decreased.
Keywords: Disinfection tests, H2O2 detection assays and fluorescent analyses were used to explore the
ZnO disinfection activities and mechanism of ZnO suspensions. Disinfection tests results
Nanofluid showed that all the produced ZnO suspension exhibited disinfection activity against
Disinfection Escherichia coli. ZnO suspensions with smaller particles/aggregates showed better disin-
H2O2 fection activities. The presence of H2O2 in ZnO suspension was analysed. The H2O2
Bacteria detection assay suggested that there is 1 mM H2O2 in 0.2 g/l ZnO Sample A, while there was
Active oxygen no H2O2 present in ZnO Sample E. Though results showed that there was no H2O2 present
in ZnO Sample E, Sample E with a size of 93 nm showed the best disinfection activities.
Fluorescence tests detected that the interaction between E. coli lipid vesicles and ZnO
Sample E was much faster and more efficient. This study firstly demonstrated that ZnO
suspensions with different particles/aggregates produced different amount of H2O2. Re-
sults suggested that H2O2 is responsible for the disinfection activity of larger ZnO particles/
aggregates while the interaction between smaller ZnO particles/aggregates and vesicle
lipids is responsible for the disinfection activity of smaller ZnO particles/aggregates.
Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.

* Corresponding author. School of Civil and Environmental Engineering, University of Science and Technology Beijing, No. 30 Xueyuan
Road, Beijing 100083, China. Tel.: 86 13401120996.
0043-1354/$ e see front matter Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.watres.2012.10.054
4014 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1
1. Introduction
In wastewater treatment procedures, disinfection is an
important stage. Disinfection will minimize contamination
and protect public health. There are different disinfection
technologies, each possessing unique advantages and draw-
backs. Though disinfection methods currently used in waste-
water treatment can effectively control microbial pathogens,
research in the past few decades found there are harmful
disinfection byproducts (DBPs). More than 600 DBPs have been
reported (Krasner et al., 2006). In order to overcome the limi-
tations of traditional disinfection methods, research has focus
on developing alternative disinfection methods (Li et al., 2008).
Nanoscale materials are defined as substances with at least
one critical dimension less than w100 nm. These materials, if
properly engineered, have unique electrical, thermal, me-
chanical, and chemical properties that are highly desirable for
applications in electronics, energy, biotechnology, medical,
and environmental sectors (Ball, 2001; Belloc et al., 2003; Kong
et al., 2000; Wasan and Nikolov, 2003; Mallin, 2006; Brayner
et al., 2006). Previous work investigated the disinfection and
toxicity activities of metal oxide nanoparticles, such as ZnO,
SiO2, Al2O3, Fe3O4, TiO2, MgO and CaO. Among these metal
oxide nanoparticles, ZnO is of particular interest due to
extensive application of the material in personal care and
home care products (Axtell et al., 2005; Li et al., 2005;
Schumacher et al., 2004). ZnO is a metal oxide, which is much
more stable and has a longer life than organic-based disin-
fection agents. This is particularly important for harsh con-
ditions such as high temperatures and/or pressures occurring
during product manufacturing, storage and transportation
(Brayner et al., 2006; Utamapanya et al., 1991; Wang et al.,
1998; Hewitt et al., 2001; Stoimenov et al., 2002; Sawai, 2003;
Fu et al., 2005; Makhluf et al., 2005).
Previous studies show that ZnO particles are effective for
inhibiting both Gram-positive and Gram-negative bacteria,
and even spores that are high-temperature and high-pressure
resistant (Sawai et al., 1995a,b, 1996a,b; Li et al., 2011). A higher
concentration of smaller particles with a higher surface area
gives a better disinfection behaviour (Makhluf et al., 2005;
Sawai et al., 1996a; Yamamoto, 2001; Zhang et al., 2007),
whereas crystalline structure and particle shape have a very
small effect (Yamamoto et al., 1998). A number of mechanisms
have also been proposed to interpret these experimental ob-
servations (Sawai et al., 1996b, 1998; Zhang et al., 2010). These
include production of active oxygen species due to the pres-
ence of nanoparticles, damage of membrane cell wall through
adhesion onto the cell membrane, interaction between mem-
brane lipid and nanoparticles, penetration through the mem-
brane cell wall, and cellular internalisation of nanoparticles.
Research on the disinfection activities of ZnO suspensions
with different sizes of particles/aggregates is limited. As is
known, ZnO cannot dissolve in water. Once ZnO is added to
distilled water, aggregates of ZnO will form in the suspension
(Hyung and Kim, 2009). It has been mentioned by Makhluf that
the microwave-assisted synthesis of MgO with a particle size
of 11  1 nm became an agglomerate with a size of 350 nm after
being dispersed in water, using analysis by dynamic light
scattering measurements (Makhluf et al., 2005). Work related
to the size of ZnO in suspensions was done by Adams et al.
(2006), where they clarified the difference between the size of
the advertised particle by supplier and actual particle size in
the suspension. The results showed that the size of the ag-
gregation was important with reference to the disinfection
properties. The information of the size of the aggregates is
so vital, because most industrial uses and experimental tests
are in aqueous systems. Particle solubility was proposed
to contribute to the disinfection property of nanoparticles
(Franklin et al., 2007). However, our previous work (Zhang et al.,
2010) used ZnCl2 to investigate the contribution caused by free
Zn2 and suggested that free Zn2 contributed little in disin-
fection property. Sawai et al. detected the production of H2O2 in
the ZnO slurry and proposed that the production of H2O2 in the
ZnO slurry was one of the primary factors in the disinfection
mechanism (Sawai et al., 1998). One of the objective of this
work focuses on the role of H2O2 in ZnO suspensions.
In this work, series of aqueous suspensions of ZnO nano-
particles are produced, characterised, and tested for their
disinfection activity against Escherichia coli bacteria. Fluores-
cent analyses are used to investigate interactions between
ZnO particles and E. coli cells. This work mainly aims at
exploring the disinfection mechanism differences among ZnO
suspensions with different particles/aggregates. The produc-
tion of H2O2 in ZnO suspensions with different particles/ag-
gregates is analysed in this work.
2. Materials and methods
2.1. Materials
Dry zinc oxide nanoparticles (90e200 nm) from Nano-
structured & Amorphous Materials (USA), was used in this
work. LuriaeBertani (LB) agar and broth medium used for
growing and maintaining bacterial cultures, Catalase from
Corynebacterium glutamicum and H2O2 used for the mechanistic
investigations were purchased from SigmaeAldrich (UK). E.
coli DH5a was provided by the Department of Biological Sci-
ence of the University of Leeds. For the fluorescent analysis,
the natural E. coli polar lipid was purchased from Avanti Polar
lipid, Inc. Chemicals 8-hydroxypyrene-1,3,6-trisulfonic acid
trisodium (Pyranine), 2-(cyclohexylamino) ethanesulfonic
acid (CHES), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid (HEPES) and 2-(N-morpholino)ethanesulfonic acid (MES)
used in the buffer solution were purchased from Sigma-
eAldrich (UK). An Amplex Red Hydrogen Peroxide/Peroxi-
dase assay kit purchased from Invitrogen Detection
Technologies (UK) was used to detect the production of H2O2
in ZnO suspensions.
2.2. Formulation and characterization of ZnO
suspensions
To gain more information of the shape, size distribution and
morphology of the as-received nanoparticles, scanning elec-
tron microscopy (SEM) was run under LEO Gemini 1530 field
emission SEM at a voltage of 5 kV. Fig. 1A shows the SEM
images of the particles from the supplier. It can be seen that
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1
Fig. 1 e SEM images of ZnO nanofluids.
ZnO particles are in the form of agglomerates. Fig. 1A also
shows the existence of particles that are much larger than
200 nm and some are even micron sized.
As is known, once the powder is dissolved in water, ag-
gregates will form, as seen in Fig. 1A. To get an ideal ZnO
suspension with better size distribution, an ultrasonicator
(Clifton, UK) and a Dyno-Mill (Willy A. Bachofen, Switzerland)
were used. A preset amount of dry ZnO nanoparticles was
mixed with distilled water in a glass beaker with the aid of a
magnetic stirrer. Once particles were dispersed in water, the
beaker was placed into the ultrasonicator. The pH value of the
suspension was adjusted to 7.2. After 1 h of sonication, ZnO
Sample A was produced. To get further differently sized dis-
tribution samples, the Dyno-Mill was used to process different
samples of ZnO suspensions at room temperature. During the
milling process, pH value of the nanofluid was monitored and
kept at 7.2 all the time. ZnO Samples BeE were taken out after
the milling treatment and put in the ultrasonicator for an
4015
extra 30 min sonication. The size distribution of these nano-
fluids were analysed using a Nano-Sizer from Malvern In-
strument. For each nanofluids samples, the average particles
sizes were measured for three times. Also, to obtain the
properties of these ZnO nanofluid samples, SEM investigation
was used. Besides, these ZnO nanofluid samples were then
diluted with distilled water to the concentrations of 0.5 g/l and
1 g/l for the disinfection activity analysis.
In this work, X-ray diffraction (XRD) analyses were used to
confirm the phase and purity of ZnO before and after milling.
The patterns of original ZnO nanoparticles, ZnO nanofluids
Samples A and E were tested. For the XRD analyses, ZnO
suspensions were dried at 60  C for 24 h, and were analysed
with a Siemens D500 XRD, scanning over 2q 30e70 with Cu
Ka radiation (l 1.54056).
ZnO is an n type semiconductor with a wide band gap
(3.37 eV) and a large excitation binding energy (60 meV) (Chen
et al., 1997). Similar to TiO2, ZnO is photocatalytic. As a
4016 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1
consequence, H2O2 and possibly other species may be pro-
duced in ZnO suspensions. The production of H2O2 in the
ZnO suspension was measured by using an Amplex Red
Hydrogen Peroxide/Peroxidase assay kit. The experiments
were performed on a fluorescence microplate, supplied by
Fisher, UK. This microplate had a volume of 100 ml per
microplate well and allowed 96 samples to be run at one
time. H2O2 concentrations were detected following the in-
structions for the Amplex Red Hydrogen Peroxide/Peroxi-
dase assay kit.
2.3. Disinfection tests
The reduction ratios of bacteria and growth curves of bacteria
with and without the presence of different ZnO nanofluids
were investigated to estimate the disinfection activities of the
produced ZnO nanofluids. All the disinfection tests were
performed in the dark. The microorganism strain used in this
study was E. coli DH5a.
The reduction ratio of the bacteria is estimated by the plate
counting method. The test process is described as follows:
50 ml of the E. coli culture with an approximate concentration
of 106e107 colony forming units per millilitre (CFU/ml) were
inoculated in a 5 ml LB broth medium as a control and solution
mixer, which contained autoclaved LB broth medium and
0.5 g/l or 1 g/l autoclaved ZnO nanofluid. Cultures were grown
at 37  C under an agitation condition for 24 h. After 24 h, a
500 ml culture from the control sample and 500 ml cultures with
the presence of ZnO were taken out and diluted in 10-fold
increments in LB medium. After the dilution, 100 ml of the
proper dilution is transferred directly onto the LB agar plate.
The solution is then spread over the surface of the agar with a
sterilized bent glass rod. Three agar plates were used for each
dilution. After incubating at 37  C for 48 h, the plates, which
have an ideal number of colonies between 30 and 300, were
counted. By knowing how much the sample was diluted prior
to being plated, along with the amount of the dilution used in
the plating, the concentration of viable cells per millilitre in
the original sample can be calculated. The reduction ratio of
bacteria can be evaluated by the following equation (Wang
et al., 2006): R(%) A  B/A  100%, where R is the percent-
age reduction ratio, A the number of bacterial colonies from
the untreated bacteria suspension (without ZnO nanofluid)
and B is the number of bacterial colonies from the bacteria
culture treated by the ZnO nanofluid.
Growth curves of bacteria treated with and without ZnO
suspension and catalase were investigated to further under-
stand the role of H2O2 produced in ZnO suspensions. Catalase,
as a H2O2 quencher, was used in the disinfection tests. Cata-
lase is a common enzyme that is able to decompose hydrogen
peroxide into water and oxygen: 2H2O2 / 2H2O O2. In the
tests, 50 ml of the E. coli culture with an approximately con-
centration of 106e107 colony forming units per millilitre (CFU/
ml) were inoculated in a 5 ml solution mixer, which contained
autoclaved LB broth medium, ZnO nanofluid and catalase.
Cultures were grown at 37  C under agitation. The growth
curve was determined by measuring the time evolution of the
optical density (OD) of the sample contained in a 10 mm op-
tical path length quartz cuvette. The measurements were
done at 600 nm with a spectrophotometer (Thermo Electron
Corporation, USA) at a frequency of once an hour. A blank LB
broth medium cultured under the same conditions was used
as a control.
2.4. Fluorescence test
To understand the mechanisms of the disinfection effect, the
fluorescence tests were used, where pyranine was used as a
sensitive pH probe to detect the changes of the pH values in
the lipid vesicles. Lipid vesicles were produced as described
in previous work (Zhang et al., 2010). In a typical fluorescence
measurement, 1.3 ml purified lipid vesicle solution was
transferred to a precision cell (HELLMA). The fluorescence
intensity was analysed online using a fluorescence spec-
trometer (RF-5300, Shimadzu, UK) at an emission wavelength
of 510 nm, and two excitation wavelengths of 405 nm and
455 nm. During the measurement, a certain amount of ma-
terials to be tested, such as ZnO nanofluids and H2O2, was
injected into the solution. The injection was done every
15 min. During the injection, the excitation spectra of the
pyranine at the emission wavelength of 510 nm in the buffer
solution and the pH values of the solution (external pH) were
measured. For all the measurements, the fluorescence in-
tensity was tested every 3 s. The equivalent internal pH value
of vesicles can be determined by using the ratio of the
emission intensity at the wavelengths of 405 and 455 nm
(Kano and Fendler, 1978; Clement and Gould, 1981; Damiano
et al., 1984).
3. Results and discussions
3.1. Formulation of ZnO suspension
In this work, ZnO nanofluids were produced via sonication
and milling processes. With these treatments, the size of ZnO
particles decreased gradually. Fig. 2 depicts the decreasing
trend of the particle size of ZnO in suspension. The first size
result was measured when ZnO powder was dispersed in
water and placed in the ultrasonicator for 1 h. The size of ZnO
particles was measured by the Nano-Sizer (Malvern instru-
ment). The average size of the particles in this suspension was
2000 Average sizes of ZnO particle
1000
Average size, nm
600
500
400
300
200
100
0 20 40 60 80 100 120 140 160 180 200 220 240 260
Time, minute
Fig. 2 e The trend of the size change using Dyno-Mill.
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1
approximately 2115 nm. During the first 10 min of the milling
process, the size of the ZnO particles dropped sharply, when
the average size of ZnO particles decreased to around 400 nm.
Followed by a gradual size decrease, the size of the ZnO in the
suspension finally reached about 93 nm. During the milling
process, five samples (AeE) were taken for further investiga-
tion. These five samples of different size distributions were
named A, B, C, D and E.
3.2. Characterization of ZnO suspension
ZnO Samples AeE were characterized using SEM and the
Nano-Sizer. The SEM images of these five samples are shown
in Fig. 1, where the magnification is 100 K. For Samples D and
E, the enlarged SEM images with a magnification of 250 K are
also provided in the insets. It is clear that particles in these
samples have an irregular shape and some are in the form of
aggregates. A comparison between the SEM images of ZnO
Samples A and E reveals that the combination of milling and
ultrasonication leads to significant changes to the morphology
of ZnO particles. Note that the aggregates of ZnO particles
may also be due to the sample preparation for the SEM ana-
lyses. The size distributions by volume of these 5 samples are
Fig. 3 e Size distributions by volume for Samples AeE.
4017
shown in Fig. 3. It can be seen that Sample A has a single peak
and fairly narrow size distribution. Milling of the sample for
about 10 min leads to ZnO Sample B, which has a bimodal and
wide size distribution. Further milling results in ZnO Samples
CeE, and the following observations can be made from the
size distribution data:
 With increased milling duration, a second peak of smaller
particle size appears.
 The smaller size peak becomes sharper and taller, whereas
the larger size peak becomes smaller with increasing milling
duration.
 The small size peak moves toward an even smaller size
range with increasing milling time.
 After about 4 h milling, the large size peak disappears.
It is interesting to note that the large size peaks for ZnO
Samples B and C occur at sizes that are bigger than that for
ZnO Sample A. This may be associated with the principle of
DLS measurement. In the DLS measurement, particles are
assumed to be spherical and the size measured is the hydro-
dynamic diameter. However, milling process seems to lead to
the particles in ZnO Samples B and C being flat in shape,
4018 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1

(101) significantly and peak widths are broadened for Sample E

indicating significant size reduction due to breakage of crys-
(100) tals. This is in agreement with the SEM and Nano-Sizer ana-
(110)
(002) (103)
(112)
lyses as presented in Figs. 1 and 3.
(102)
(200)
(201) The concentrations of H2O2 in ZnO Samples A and E were
converted and reported in Fig. 5. For ZnO Sample A, the con-
Intensity

Original ZnO centration of H2O2 increases with increasing concentration of

30 40 50 60
2-Theta (degree)
Fig. 4 e X-ray diffraction spectrums of ZnO nanoparticles.
which may give a larger hydrodynamic diameter. This can be
seen from the SEM images shown in Fig. 1.
X-ray diffraction (XRD) was taken in order to further
confirm the ZnO phase of the nanoparticles and the purity of
the ZnO before and after milling. The top pattern in Fig. 4 is for
the original ZnO powder, the middle one is for ZnO Sample A,
and the bottom one is for ZnO Sample E. One can see from the
figure that the positions of the peaks for all the three samples
are in good agreement with those reported in the JCPDS 36-
1451 data and the crystalline structure of ZnO used in this
work can be indexed to hexagonal wurtzite structure. The
XRD patterns also suggest little impurities in the sample and
virtually no changes in the crystalline structure during sus-
pension formulation. The peaks at 2q values of 31.9 , 34.5 ,
36.4 , 47.6 , 56.7 , 62.9 , 66.4 , 68.0 and 69.2 correspond to the
crystal planes of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0),
(1 1 2), (2 0 1), respectively, of the crystalline zinc oxide. A
comparison between the three patterns shows that the
heights and widths of ZnO Sample A are almost the same as
that of the original Sample, indicating little breakage of the
ZnO crystals and size reduction by sonication is mainly due to
de-aggregation. However, the peak heights are reduced
ZnO, and over 1 mM H2O2 is produced in the 0.2 g/l suspension.
However, almost no H2O2 is produced in Sample E. The for-
mation of H2O2 in Sample A may be attributed to excess hy-
Sample A droxyl group on the surfaces of ZnO particles. For ZnO Sample
E, however, there are no excess hydroxyl groups due to very
Sample E high surface/volume ratio of ZnO particles. The existence of
70 H2O2 in ZnO slurries was also reported by a Japanese group
(Sawai et al., 1996b). They found that the bulk concentration of
H2O2 in 100 mg/ml ZnO slurry was about 50 mg/ml. This is
equivalent to w1.5 mM, which is much higher than that
measured in this work. The reason for the difference may be
because the size of ZnO used by Sawai et al. (1996b) is larger
than that used in this work.
3.3. Disinfection tests
Fig. 6 and Table 1 present the number of the E. coli colonies
after 24 h treatment by 1 g/l different ZnO samples. Fig. 6
shows that after 24 h of culturing, the control sample con-
tains approximately 109 CFU/ml, whereas samples treated by
the milled Samples AeE give a much lower number of bacte-
ria, and samples with smaller particle sizes give an increased
bacteria number reduction. Based on the plate counting re-
sults, the reduction ratios can be calculated and these are
shown in Table 1. It is found that in Sample E, which has an
average particle size of 93 nm, 99.998% E. coli bacteria were
dead. In contrast, the same amount of ZnO in Sample A only
killed 98.936% E. coli bacteria within 24 h. The reduction ratios
of the bacteria in Sample BeD are 99.866%, 99.978% and
99.992% respectively. It is very clear that the reduction ratio of
the bacteria is increasing from Sample A to Sample E. The
possible reason is that smaller particles have a higher specific
surface area, and hence a higher extent of interaction between

1.4 10
10
ZnO sample A
1.2 ZnO sample E
9
10
Number of bacterial colonies,

1.0
8
0.8
10
H 2O 2, M

7
CFU/ml

0.6 10

0.4 6
10
0.2
5
10
0.0
4
10
-0.2 Control A B C D E
0.00 0.05 0.10 0.15 0.20 Samples
Concentration of ZnO suspension, g/l
Fig. 6 e Number of bacteria colonies with and without the
Fig. 5 e Concentrations of H2O2 in ZnO suspensions. presence of 1 g/l different ZnO samples for 24 h.
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1 4019

Table 1 e The reduction ratios of the bacteria treated by 1 g/l different ZnO samples for 24 h.
Samples A B C D E Negative control
7 6 5 5 4
Average number of colonies (CFU/ml) 1.82  10 2.29  10 3.73  10 1.35  10 3.07  10 1.71  109
Reduction ratio (100%) 98.936 99.866 99.978 99.992 99.998 0

the particles and bacterial cells. Another possible reason is production of H2O2 is the major mechanism but not the only
that smaller particles may have higher surface oxygen va- disinfection mechanism. The same conclusion was also
cancies and defects. Such oxygen vacancies and defects can drawn by Sawai et al. (1998), who used 2.6 mm ZnO slurries
help to effectively inhibit the recombination of photo-induced and tested their disinfection behaviour against E. coli. The
electrons and holes by becoming centres to capture photo- results for the ZnO Sample E show a significant difference e
induced electrons (Jing et al., 2004). In addition, the oxygen the use of catalase does not significantly affect the disin-
vacancies favour adsorption of O2, capture photo-induced fection activity of the suspension. This indicates that H2O2 is
electrons, and simultaneously produce O2 radical groups not a dominant mechanism for the nano-sized ZnO
(Jing et al., 2001). suspension.
To further understand the role of H2O2 produced in ZnO
suspensions, a H2O2 quencher, catalase, was used in the
3.4. Fluorescence test
disinfection tests. The underlying thoughts were: if H2O2
played a major role in the disinfection activity of ZnO sus-
The interaction between ZnO particles and E. coli lipid vesicles
pensions, the addition of catalase would remove such an
was investigated by fluorescence analysis on pyranine, where
effect. As suggested by Sigma, the supplier, 1 ml catalase
the equivalent internal pH values of the lipid vesicle can be
solution contains 5000 catalase working units (U), and 1 U
determined based on the recorded ratio of fluorescence in-
of catalase can convert 1 mM H2O2 to water and oxygen at pH
tensity at two excitation wavelengths, 405 nm and 455 nm at
7 and 25  C. In this work, 1 ml catalase was used for a 5 ml
the emission wavelength of 510 nm (Kano and Fendler, 1978).
0.1 g/l ZnO suspension, which was enough to decompose all
ZnO Samples A and E, were used in the fluorescence tests.
H2O2 in the suspension according to the measurement data
Fig. 8 shows the time evolution of the DpHi. It can be seen that
of H2O2, shown in Fig. 5. Fig. 7 shows the growth curves of E.
after injection of the milled ZnO Sample E, DpHi increases
coli in the presence of different additives. One can see that
sharply initially, and then tends to level off at a time
the E. coli bacteria grow very well in the absence of any ad-
depending on the ZnO concentration. Addition of ZnO Sample
ditions. The addition of 0.1 g/l ZnO suspensions (ZnO Sam-
A 0.01 g/l leads to a DpHi of about 0.35 units, whereas a DpHi of
ples A and E) shows good disinfection activity with Sample E
around 0.58 units is seen when 0.01 g/l ZnO Sample E is
showing better activity. These agree with the results shown
injected. These results suggest possible leakage of the vesicles
in the previous work (Zhang et al., 2007). With the addition of
and/or increased mass transfer between the interior and
the catalase in the ZnO suspensions, the disinfection activity
exterior of the vesicles. Leakage of E. coli cells has actually
of ZnO Sample A drops significantly, indicating decomposi-
been observed by our previous study (Zhang et al., 2010). The
tion of H2O2 in the suspension with ZnO Sample A. It is noted
DpHi changes more rapidly in ZnO Sample E compared to ZnO
that, despite the addition of catalase, ZnO Sample A still
Sample A. For ZnO particles of smaller size, more surface
exhibits some disinfection activity, suggesting that
active sites were exposed to the lipid vesicle and cause more
and quicker leakage of pyranine inside the vesicles. This result
2.5
Control
0.1g/l ZnO sample A
2.0 0.1g/l ZnO sample A + catalase 0.6
0.1g/l ZnO sample E
0.1g/l ZnO sample E + catalase
1.5 0.5
OD at 600nm

0.4
pH changes

1.0

0.3
0.5

0.2

0.0 Sample E (93nm), 0.01g/l

0.1 Sample A (2115nm), 0.01g/l
0 2 4 6 8
0.0
Time, hour 0 200 400 600 800 1000
Time /S
Fig. 7 e Growth curves of E. coli bacterial cells with the
presence of different sizes ZnO nanoparticles and Fig. 8 e The DpHi after inducing 0.01 g/l ZnO suspensions
supplementary with catalase. into the lipid vesicle solutions.
4020 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 4 0 1 3 e4 0 2 1
0.9 H2O2 310 g/l, 620 g/l
ZnO Sample E 0.01 g/l, 0.02 g/l, 0.05 g/l
0.8
0.7
0.6
0.5
pH i
0.4
0.3
0.2
0.1
0.0
0 500 1000 1500 2000 2500
Time , second
Fig. 9 e DpHi as a function of time upon injection of H2O2
solutions and ZnO suspensions (Sample E).
is in agreement with the one in the disinfection tests (see
Fig. 6), where a larger number of bacteria was killed in the
presence of ZnO Sample E.
Because previous work done by Sawai et al. suggested that
H2O2 is the main disinfection factor for ZnO slurry, the effect
of H2O2 on E. coli lipid vesicles was also investigated by
applying a H2O2 solution in the fluorescence analyses. In our
work, the concentrations of H2O2 solution applied in the
fluorescence analyses are 310 and 620 mg/ml, which are much
higher than the actual concentration of H2O2 present in the
ZnO suspension. This is because with a higher concentration
of H2O2, there would be a substantially better observation
opportunity of the interactions between H2O2 and E. coli lipid
vesicles. The DpHi is as a function of time, caused by the in-
duction of the H2O2 solution and ZnO Sample E are both
illustrated in Fig. 9. The data displays that with the induction
of higher concentrations of the ZnO suspension and the H2O2
solution, DpHi increases. At the end of the test (45 min), the
DpHi caused by the ZnO nanoparticle suspension (w0.7) is
found to be far higher than that by the H2O2 solution (w0.025).
The negligible effect of H2O2 is somewhat surprising as H2O2 is
a traditional and highly effective disinfection agent. A possible
explanation is that the disinfection activity of the H2O2 solu-
tion is not due to the damage of the lipid-rich membrane of
the biological cell, but rather due to the interaction of H2O2
with membrane proteins. Alternatively, H2O2 might pass the
membrane and induce oxidative damage inside the bacteria.
The result indicated that the behaviour of H2O2 and ZnO
nanoparticles against E. coli lipid vesicles is quite different.
Therefore, it is suggested that in ZnO Sample E, interaction
between ZnO nanoparticles and lipid vesicles is responsible
for the disinfection activities.
4. Conclusions
In this work, ZnO suspensions with different sizes of particles/
aggregates were produced. H2O2 measurement revealed that
there were different concentrations of H2O2 present in the
ZnO suspensions with different sizes of particles/aggregates.
There was H2O2 in ZnO suspension with large particles/ag-
gregates, while no H2O2 was present in ZnO suspension with
small particles/aggregates. The fluorescence tests indicate
that there is interaction between ZnO particles and E. coli lipid
vesicles. The interaction between ZnO and lipid is more effi-
cient and rapid in the ZnO suspension with a smaller particle
size. Disinfection tests showed that ZnO suspensions had
disinfection activities against E. coli DH5a. ZnO suspension
with smaller ZnO particles/aggregates exhibited a better
disinfection effect. Mechanism investigation suggested that
ZnO suspensions with different sizes of particles/aggregates
had different disinfection mechanisms. ZnO suspensions
with a large particles/aggregates size (2115 nm) showed the
3000
disinfection activities mainly because of H2O2 was present in
the ZnO suspension, while ZnO suspensions with a small
particles/aggregates size (93 nm) showed strong interaction
between ZnO particles and the lipids.
Acknowledgements
This work is partially supported by the fundamental research
funds for the central universities, National Natural Science
Foundation of China (Grant No. 41203073) and Chinese 863
programme (Grant No. 2011AA06A105). The authors would
like to extend their thanks to Dr Lars Jeuken and Dr Nikolaos
Daskalakis of the School of Physics and Astronomy at the
University of Leeds for helping with fluorescence analysis.
Sincere thanks are due to Dr Alexei ONeil of the School of
Biological Sciences at the University of Leeds for advices on
performing H2O2 measurement experiment.
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