Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology B: Biology

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

Controllable synthesis of ZnO nanoparticles and their morphology-dependent

antibacterial and optical properties
Nasrin Talebian a,⇑, Seyedeh Matin Amininezhad a, Monir Doudi b
a
Science Faculty, Department of Chemistry, Shahreza Branch, Islamic Azad University, Razi Chemistry Research Centre, Women Research Council, 86145-311 Shahreza, Isfahan, Iran
b
Department of Microbiology, Falavarjan Branch, Islamic Azad University, Falavarjan, Isfahan, Iran

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

Article history: ZnO materials with different morphologies have been synthesized via a simple solvothermal method
Received 6 October 2012 using different solvents without any catalysts, templates or surfactants. The ZnO samples are employed
Received in revised form 2 December 2012 in the inactivation of gram-negative Escherichia coli and gram-positive Staphylococcus aureus in MilliQ
Accepted 7 January 2013
water. The photocatalytic activities of samples to degrade an azo dye, Acid Orange 74 (CI 18745), were
Available online 1 February 2013
also tested. XRD data showed that single-phase ZnO with the wurtzite crystal structure but different
growth habits were obtained in the different solvents. SEM imaging illustrated that ZnO with flower-like,
Keywords:
rod-like, and spherical shape were produced when water, 1-hexanol, and ethylene glycol were used as
ZnO nanostructures
Solvothermal
the solvent, respectively. The optical properties of the as-prepared ZnO materials were investigated by
Optical property UV–vis absorption and photoluminescence spectra. The antibacterial efficiencies were affected by the
Bacteria inactivation physiological status of the bacterial cells, different morphologies and crystal growth habits, particle size
and optical properties of ZnO samples. Results indicate that ZnO flower-like showed significantly higher
photocatalytic inactivation than ZnO rod- and sphere-like against E. coli compared with S. aureus. It was
found that the antibacterial activity of ZnO increased with decreasing crystallite size. The inactivation
efficiencies for both organisms under light conditions were higher than under dark conditions. The
obtained results were discussed according to the morphologies, optical and structural properties of
ZnO powders as key parameters in photocatalytic and antibacterial activity.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction
Nanostructured materials offer promising opportunities for im-
proved and tailored properties for application in environmental
catalysis due to their unique physicochemical properties, caused
by their nanosized dimensions and large surface/volume ratios
[1]. More recently, several natural and engineered nanomaterials
have also been shown to have strong antimicrobial properties,
including silver nanoparticles [2], TiO2 [3], ZnO [4] and SiO2 [5].
Unlike conventional chemical disinfectants, the antimicrobial
nanomaterials are not expected to produce harmful disinfection
byproducts (DBPs). Since the properties of materials at the nano-
scale can be profoundly affected by the shape and size of the nano-
structures what must follow is the ‘‘design’’ rather than the
‘‘preparation’’ of nanocatalysts. Therefore, control of the morphol-
ogy and size of particulate inorganic materials has received in-
creased attention. Besides, controlling the crystal planes of
nanomaterials is a paramount target in catalyst preparation. The
ideal way is to first fully understand the relationship between crys-
⇑ Corresponding author. Tel.: +98 321 329 2260; fax: +98 321 3232701.
E-mail addresses: nasrin_talebian@yahoo.com, talebian@iaush.ac.ir (N. Tale-
bian).
tal structure and corresponding catalytic properties, and then syn-
thesize nanocatalysts with well-defined reactive crystal planes
[6,7].
Among metal oxide semiconductors, ZnO is increasingly recog-
nized as a suitable alternative due to its richest range of morphol-
ogies among the wide band gap semiconductors (3.37 eV) and
relatively lower cost of production [8,9]. ZnO was also reported
to exhibit higher quantum efficiency and photocatalytic activity
than TiO2 in certain cases [10].
It has been demonstrated that ZnO nanostructures exhibit anti-
microbial activity against on a broad spectrum of bacteria includ-
ing Staphylococcus auerus [11] and Escherichia coli [12–14]. ZnO
nanoparticles seem to have relative toxicity to bacteria but exhibit
minimal effect on human cells [12]. The disruption of the cell
membrane is attributed to peroxidation of the unsaturated phos-
pholipids due to photocatalytically induced hydrogen peroxide
[15]. Cell membrane and wall damage upon the contact with ZnO
nanoparticles occur to inhibit bacterial growth [4,12]. In addition,
binding Zn2+ ion to the membranes of microorganisms results in
prolong the lag phase of the microbial growth cycle [16]. A recent
toxicology study by Poynton et al. [17] suggested that both ZnO
nanoparticles and Zn2+ are toxic, but have different modes of
action.

1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2013.01.004
 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73
There are several individual reports on the morphology-depen-
dent photocatalytic inactivation of ZnO catalysts against some bac-
teria [18–20]. However, to our knowledge, no work has been
reported about the comparative study of ZnO nanoparticles anti-
bacterial properties with different morphologies particularly re-
sulted from solvent effect.
The wet-chemical method offers finer tailoring of the size and
shape of the nanocrystals. However, among solution-based meth-
ods, solvothermal process has many advantages such as an easier
composition control, better homogeneity, low processing tempera-
ture, narrow grain size-distribution, easier fabrication of large scale
samples and possibility of using high purity starting materials [21].
Besides, the solvothermal synthesis is one of the most powerful
tools for providing distinct morphologies of nanomaterials [22].
Since the solvent physico-chemical properties such as polarity,
viscosity, and softness will strongly influence the solubility and
transport behavior of the precursors, the solvent in a solvothermal
process could be used as shape-controller of nanoparticles. It was
found that the crystallite growth habit and particle sizes of ZnO
nanocrystalline could be controlled by changing the solvent during
the solvothermal synthesis [23–25].
In this study, the solvothermal method was employed to syn-
thesize ZnO materials with different morphologies in the absence
of templates or structure-directing agents under mild conditions.
The desired characteristics were attained through the change and
control of the solvents with the aim of evaluation in the inactiva-
tion efficiencies for two microorganisms under light and dark con-
ditions. Results indicate that ZnO nanoflowers showed significantly
higher photocatalytic activity than ZnO nanorods and nanospher-
ers. Regarding the photocatalytic activities of the prepared ZnO
samples to degrade azo dye, similar to some reports of organic pol-
lutant degradation efficiency [26], the present results are showed
morphology-dependent photocatalytic activity of samples. The ob-
tained results were discussed according to the morphologies, opti-
cal and structural properties of ZnO nanoparticles as key
parameters in photocatalytic and antibacterial activity.
2. Experimental
2.1. Materials and synthesis methods
Table 1 shows some physical features of used solvents including
water, 1-hexanol and ethylene glycol. In a typical synthesis, 5.60 g
Zn(CH3COO)22H2O was dissolved in 50 ml each used solvent un-
der vigorous stirring at 60 °C. In the case of water as solvent,
10 ml NaOH 0.5 M was also added drop by drop into zinc acetate
dihydrate aqueous solution until a transparent solution was ob-
tained at pH = 9. The pH solution was about 10–11 when using
1-hexanol and ethylene glycol as solvent. After stirring at 60 °C
for 30 min, the mixture was then transferred into a Teflon-lined
autoclave and was maintained at 170 °C for 18 h. Subsequently,
after the autoclave was cooled, the as-formed white precipitate
Table 1
Some physical features of used solvents [27].
Solvent Water 1- Ethylene
Hexanol glycol
Formula H2O C6H14O C2H6O2
Boiling point (°C) 100 155–159 197.3
Relative polarity index 10.2 0.88 6.9
Dielectric constant 80 13.3 41
Saturated vapor pressure (hPa) 2.338 0.418 0.0075
(20 °C)
M.W. (g mol1) 18.02 102.17 62.07
Viscosity (mPa s) 1.0 0.494 16.1
67
was filtered, washed with ethanol and distilled water, and dried
in the air at 80 °C.
2.2. Antibacterial experiment of ZnO nanoparticles
The antibacterial activity of the synthesized ZnO nanoparticles
was evaluated using bacterium as per the colony count method.
To examine the bacterial growth rate, cultures (bacterial strain)
were grown in the nutrient broth medium (NB) supplemented
with ZnO nanoparticle colloidal suspension at concentration of
20 ppm. The ZnO nanoparticles were added into 10 ml NB medium
which was added with 200 ll bacteria at a concentration of
1.5  105 CFU (colony forming unit). The culture of nanoparticle
free medium as blank was grown under the same reaction condi-
tions and was used as a control. The samples and blank were illu-
minated with four 8 W lamps (Philips UV-A, kmax = 365 nm), at
room temperature for 20, 40, 60 and 80 min, respectively. The tem-
perature of the culture medium was controlled using the water cir-
culation through the reactor. During each time period, the sample
containers were placed horizontally onto a shaker platform and
agitated at a speed of 225 rpm. After catalyst centrifugation and
separation, 100 ll of each bacteria suspension was dispersed on
the Nutrient Agar medium. The number of colonies forming units
(CFUs) was counted taking into account the dilution factor after
24 h of incubation at 37 °C. The respective data are the average
of values obtained from triplicate runs. The catalysts under consid-
eration have not shown a decrease in their photoinduced microbi-
ocide efficiency when the antimicrobial experiments were
repeated three times using the same samples. No significant de-
crease in CFU in nanoparticle free medium observed after UV light
exposure. This indicates that the photochemical inactivation was
negligible under UV-A irradiation with low energy.
To evaluate both catalytic and photoinduced biocide activities,
two parameters were calculated: (i) the survival ratio and (ii) the
discriminative factor. The survival ratio (S = N/N0  100%, where
N0 and N are the numbers of CFUs at initial (1.5  105 CFU/ml)
and at 20 min after UV light exposure for photocatalytic condi-
tion/or at the same time after contact with catalytic powder under
dark for catalytic condition, respectively) provides an information
on the overall bactericidal efficiency of the catalytic systems under
investigation. The discriminative factor (DF = Nc/N, where N and Nc
are the numbers of CFUs remaining in suspension at 20 min under
dark condition in contact with catalyst powder and at the same
time after UV light exposure in contact with catalyst powder,
respectively) permits one to compare the efficiency of photocata-
lytic inactivation with that of catalytic one.
2.3. Photocatalytic experiment of ZnO nanoparticles
A photoreactor fitted with four 8 W lamps of wavelength
365 nm and highly polished anodized aluminum reflector was em-
ployed for the photodegradation experiments. The reaction vessel
was placed at the center of the reactor. The reactor was fitted with
cooling fans at the bottom to dissipate the generated heat. Solu-
tions of the dye of 10 ppm concentration were prepared afresh at
pH = 4 and used. It should mention that the photodegradation
reactions were performed at different pH ranging from 2 to 10,
and the highest efficiency was obtained at pH = 4. After the addi-
tion of the catalyst to the dye solution and given illumination
times, the oxide was removed by centrifugation and the unde-
graded dye was spectrophotometrically estimated at the maxi-
mum absorption wavelength of dye.
68
2.4. Characterization
To characterize the ZnO powders, X-ray power diffraction (XRD)
experiments were performed on a Bruker, D8 ADVANCE XRD dif-
fraction spectrometer with a Cu Ka line at 1.5406 Å and a Ni filter
for an angle range of 2h = 20–80°. The morphologies of samples
were also investigated using a Philips, XL30 scanning electron
microscope (SEM) with an accelerating voltage of 17 kV. The opti-
cal properties were performed on a Shimadzu, MPC-2200 UV–vis
spectrophotometer operated over the range of 350–600 nm at a
resolution of 2.0 nm. The optical emission studies on ZnO samples
were made by PL spectroscopy using 325 nm beam of He–Cd laser
as the excitation light source at room temperature over the range
of 300–700 nm at a resolution of 1.0 nm. The BET method was em-
ployed to measure the surface area of ZnO samples with a Microm-
eritics Gemini 2360 surface area analyzer.
3. Results and discussion
3.1. Structure
As shown in Fig. 1, all of the diffraction peaks from the samples
corresponded to those of (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3),
(2 0 1), (1 1 2) and (2 0 0) plane of reflections for a single phase
wurzite structure of ZnO [28]. Afterward, ZnO powders refer as
sample A, B and C for synthesis in 1-hexanol, ethylene glycol and
water, respectively. The crystallite size was calculated from wurz-
ite (0 0 2) reflection, using the Scherrer equation [29]. The average
crystallite size is 76, 65 and 45 nm for sample A, B and C, respec-
tively (see Table 2). The XRD intensity ratios provide information
about the degree of relatively preferred orientations and crystal
structure of synthesized ZnO samples using the various solvents.
Table 2 shows the (0 0 2) peak relative intensity from XRD patterns
of synthesized ZnO powders. The solvents used in this study had
widely varying polarities. The intensity ratios of synthesized sam-
ples decrease approximately in consistence with increase in sol-
vent polarities in the following order sample C < sample
B < sample A.
In general, the structure of the solvent led to the morphology
control depending on its interaction with the growing crystal
[30]. ZnO is a polar crystal consisting polar zinc-terminated plane
as (0 0 1), the polar oxygen-terminated plane as (0 0 1)  and non-po-
lar plane as (1 0 0). Then, the relative growth rates of the various
crystal planes are different in solvents with different polarities.
Our results illustrated that the growth along the (0 0 2) direction
(c-axis) was affected by solvent polarity as well as hydroxyl groups
[25]. In ethylene glycol with two hydroxyl groups in short chain,
the solvent-interface interaction would be stronger than 1-hexanol
containing one hydroxyl group in long chain but weaker than
(101)
(002)
(100)
(102)
Intensity
25 30 35 40 45 50
2-Theta ( o )
Fig. 1. XRD patterns of ZnO powders.
N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73
Table 2
Some parameters from XRD analysis of ZnO samples.
Sample Solvent I(0 0 2)/ Crystallite size (nm)
[I(1 0 0) + I(0 0 2) + I(1 0 1)]
A 1-Hexanol 0.262 76
B Ethylene 0.236 65
glycol
C Water 0.209 45
water with highest polarity. For 1-hexanol, the weaker interaction
leads to more growth rate along the (0 0 2) direction. When water
used as solvent, there is a strong interaction between the polar
(0 0 2) surface and solvent. Besides, there are two kinds of precur-
sors, e.g., Zn(OH)2 and [ZnðOHÞ2 4 ] that might exist or coexist in
the reaction aqueous medium and the concentrations of each spe-
cies depend on the pH values. For the hydrothermal crystal growth
process of ZnO (water as solvent), as the solution appears weak
alkaline, small quantity of growth units [ZnðOHÞ2 4 ] are also ob-
tained in the solution [31,32]. It has been reported that the (0 0 2)
facet of the crystal, which is the slightly positively charged Zn sur-
face, can be adsorbed by negatively charged chemical species [33].
The adsorbed molecules, therefore, retard the growth of the crystal
in that direction.
3.2. Morphology
The surface morphology of the ZnO samples was studied by
using SEM, as shown in Fig. 2. It was clearly illustrated that mor-
phology of particles synthesized in 1-hexanol, ethylene glycol
and water, was rod-, sphere- and flower-like, respectively.
There are two main factors that contribute to the morphology of
the final products obtained under solvothermal conditions: these
are the initial nucleation of the crystals, and the solubility of the
precursor in the solvents with different saturated vapor pressures
[24]. The lower saturated vapor pressures allow for more rapid
growth of the ZnO nuclei, resulting in larger particles for 1-hexanol
and ethylene glycol.
The chain length of the solvent molecule also affected the crys-
tal growth through interface-solvent interactions. For 1-hexanol, in
which the polarity decreases with the chain length of the molecule,
ZnO particles with branch rod-like shape were obtained due to
ineffectively solvent attachment to the (0 0 2) surface of ZnO crys-
tals. Hexagonal ZnO rod-like could be formed (Fig. 2A) when the
particles preferably grow along normal of the (0 0 2) planes. This
phenomenon has been observed in previous reports [34,35].
The ZnO product has spherical morphology, possibly owing to
three reasons. One may arise from the wurtzite structure of ZnO it-
self. Namely, ZnO with wurtzite structure has a basal polar oxygen
 face and a top tetrahedron corner-exposed polar Zn (0 0 1)
(0 0 1)
face, and they have different charge and reactivity, favoring forma-
tion of spherical morphology [36]. Another, it could be closely re-
lated to the coordination between ethylene glycol molecules and
Zn2+ or selective anchoring of ethylene glycol molecules on ZnO
(110)
(103)
(201)
(200)
(112)
crystals, favoring generation of spherical morphology. Further, eth-
ylene glycol which consists of two hydroxyl groups in relatively
high polar molecule, could effectively attach to the (0 0 2) surface
of ZnO crystals. This interaction results in inhibit the preferential
growth of ZnO crystals in the c-direction and causes the spherical
Sample A morphology.
Sample B In weak alkaline aqueous solution, small quantities of growth
Sample C
unit [ZnðOHÞ24 ] decompose to ZnO (low number of nuclei), conse-
55 60 65 70 75 80 quently, ZnO crystalline particles are self assembled one by one
along the [0 0 1] crystal direction and, by this in situ ‘‘soft-tem-
plate’’ function, the flower-like ZnO morphology is formed [31],
as shown in Fig. 2C in the case of water as solvent.
 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73 69

Fig. 3. The change in the optical transmittance spectra of ZnO samples prepared in
different solvents. The inset of the figure shows magnified spectra.
Sample A

linearity was observed for different n values at n = 1 (direct allowed

transition) which was found to give the best fit for ZnO samples (not
shown here). The band gap value increases in the order of sample C
(3.27) < sample B (3.31) < sample A (3.34) which is in consistency
with crystallite size.
PL spectroscopy is a powerful tool in characterizing optical
quality of semiconductor materials. Fig. 4 shows photolumines-
cence spectra of as synthesized ZnO samples with the excited
wavelength of 325 nm. From this figure, one sharp and strong blue
emission band at about 422 nm was observed, which is called near
band edge emission generated by the free-exciton recombination
[38]. ZnO commonly exhibits luminescence in the visible spectral
range due to different intrinsic or extrinsic defects. The blue emis-
sion in ZnO nanobelts at 422 nm was attributed to interstitial
zinc [39]. While other reports indicated the photoluminescence
Sample B
emission of ZnO in the blue region (420 nm) was due to Zn vacancy
[40]. Moreover, the blue emission band (442 nm) in ZnO nano-
wires was also attributed to oxygen vacancies [41]. However, PL
intensity correlates directly with defect densities in the material.
The peak position of the emission band was blue-shifted from sam-
ple C compared to sample A coincides well with the blue shift of
the absorption edge from transmittance spectra in Fig. 3. The pres-
ence of excess surface defects leads to strong blue emission for
sample C corresponding to the formation of hydroxyl radical on
the surface of ZnO leaving oxygen vacancies.

3.4. Antibacterial activity

The antimicrobial activity of the samples was determined by

colony count method using gram-negative E. coli and gram-positive
Sample C
Fig. 2. SEM photographs of ZnO powders.
16000
Sample A
14000
Sample B
3.3. Optical properties 12000 Sample C

10000
Intensity

A shift in absorption edge to a higher wavelength is observed

with increasing solvent polarity as shown in Fig. 3. The optical 8000
band gap, Eg, of the ZnO samples was determined by extrapolation 6000
of the linear portion of (aht)2 versus ht plots using the following
equation [37]: 4000

2000
ðahtÞ2 ¼ Aðht  Eg Þn
0
300 350 400 450 500 550 600 650 700
where a is absorption coefficient, ht is the photon energy, Eg is the
Wavelength (nm)
optical band-gap energy (eV) and A has a value between 105 and
106 cm1 eV1 depending on the electron–hole mobility. A good Fig. 4. Photoluminescence spectra of ZnO samples prepared in different solvents.
70 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73

Table 3
The effect of photocatalytic and catalytic inactivation on the survival of Escherichia 100
coli after 20 min under UV-illumination.
80

Photocatalytic
efficiency (%)
Condition Sample A Sample B Sample C
60
Number of viable cells Irradiation 0.75  102 0.37  102 0.02  102
(CFU/ml) Dark 1.76  102 1.05  102 0.42  102 40
Parameters for evaluation of biocide activity
20
S (%) Irradiation 0.050 0.025 0.001
Dark 0.117 0.070 0.028 0
DF 2.35 2.83 21.0 C
20 B
40
60 A
80
Time (min)

Fig. 5. The photocatalytic results comparing the ZnO nanoparticles: samples A, B

Table 4
and C.
The effect of photocatalytic and catalytic inactivation on the survival of Staphylococcus
aureus after 20 min under UV-illumination.

Condition Sample A Sample B Sample C (0 0 2), are very important for catalytic reactions, because they fa-
Number of viable cells Irradiation 1.25  10 2
0.72  10 2
0.21  102 vor formation of more oxygen vacancies. The formation of nano-
(CFU/ml) Dark 2.49  102 1.82  102 0.65  102 particles with smooth (0 0 2) facet possessing higher energy for
Parameters for evaluation of biocide activity sample C results in higher activity.
S (%) Irradiation 0.083 0.048 0.014
Dark 0.166 0.121 0.043
DF 1.99 2.53 3.09 4. Influence of the morphologies on the catalytic performance
of the ZnO materials

The morphologies of ZnO catalysts play an important role in the

S. aureus exposed to different samples for up to 20 min to assess
their bactericidal effect under UV light and dark conditions as
the test cultures. The results of the bactericidal activity perfor-
mance studies are collected in Tables 3 and 4. Sample C results
in the highest photoinduced microbiocide efficiency: the DF value
increases ca 10-fold in the case of E. coli and ca 1.5-fold in the case
of S. aureus compared with sample A, indicating the photoinduced
microbiocide efficiency being especially pronounced for E. coli.
Drastic decrease of the number of viable cells was observed for
the illuminated ZnO samples as compared to the dark condition
that points to the prominent photokilling ability of the nanostruc-
tured ZnO powder. It is seen from Tables 2 and 3 that inactivation
efficiency order was sample C > sample B > sample A under both
UV light and dark conditions.
3.5. Photocatalytic activity
The degradation of the dye was estimated from the reduction in
absorption intensity of Acid Orange 74 at a fixed wavelength k
max  490 nm. The degradation efficiency (DE) was then calculated
as given in DE = [(C0  C)/C0]  100, where C0 is the initial concen-
tration of the dye and C is the concentration after irradiation at gi-
ven time intervals.
The morphologies and crystal faces of ZnO samples had a signif-
icant effect on the photocatalytic processes. As in Fig. 5, the same
order was observed for ZnO samples photocatalytic activity which
indicates the same parameters affect both activities toward dye
degradation and bacteria inactivation. The flower-like ZnO demon-
strated maximum photocatalytic activity as expected owing to: (1)
higher surface exposed to the contaminant molecules with smaller
particles; (2) according to PL spectra, an increase in the density of
vacancies and interstitial defects can act as the active centers to
capture photo-induced electrons, and the recombination of
photo-induced electrons and holes can be effectively inhibited
[42]; (3) a decrease in band-gap energy for sample C results in
the higher spectral response and higher photocatalytic activity
and (4) catalysts with higher surface energy show better catalytic
performance [43] The growth of crystals with some preferred
structures or planes was controlled by the surface energy of the
planes. For ZnO, the higher-surface-energy polar planes such as
photocatalytic activity; this arises from differences in particle
sizes, polar planes, optical properties and oxygen vacancies as dis-
cussed below.
4.1. Influence of ZnO particle size
Usually, a high specific surface area has a beneficial effect on the
activity for catalysts. Therefore, the crystallite size may play a crit-
ical role in the biocide activity of ZnO nanoparticles. However, con-
tradictory results about the impact of particle size on the
antibacterial activity of ZnO have been reported. Jones et al. [44]
and Yamamoto [11] observed that smaller ZnO particles were more
toxic than bigger particles, but no size related effect was found in
another study by Franklin et al. [45].
At present work, it was found that the antibacterial activity of
ZnO increased with decreasing particle size or increasing surface
area (m2/g) in order of sample C (28.8) > sample B (19.2) > sample
A (15.5). The smaller the average size or higher surface area of the
sample C, the greater the antibacterial effect observed demonstrat-
ing that inactivation process occurs through catalyst surface. It was
also found that the particle sizes of ZnO nanocrystalline could be
controlled by changing the solvent during the solvothermal
synthesis.
4.2. Effects of the crystal habits
The Zn-terminated (0 0 1) and O-terminated (0 0 1)  polar faces
are more active surfaces than the nonpolar surfaces perpendicular
to them. Then, polar faces are facile to adsorb oxygen molecules
and OH ions, resulting in a greater production rate of H2O2 and

OH radicals, hence promoting the photocatalytic reaction
[46,47]. Therefore, it is very desirable to synthesize nanocatalysts
with high ratios of more reactive crystal planes. Unfortunately, sur-
faces with high reactivity usually diminish rapidly during the crys-
tal growth process as a result of the minimization of surface
energy. ZnO prepared when water was used as the solvent showed
the highest inactivation activity. This is due to the formation of
nanoparticles with smooth (0 0 2) facet, which have higher energy,
resulting in privileged activity.
 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73 71

Fig. 6. Photographs demonstrating the effect of three catalysts on the number of CFU following exposure to UV light for 20, 40, 60 min and without UV after 80 min.

4.3. Effects of optical properties
There was an inverse relationship between band gap energy and
biocide activity of ZnO samples. A decrease in band-gap energy for
sample C results in the higher spectral response. The photocatalytic
activity could be affected by a change in photon harvesting
depending on the band gap energy.
4.4. Effects of oxygen vacancies
If the photo-generated electrons recombined with the photo-in-
duced holes, the photocatalytic activity would be decreased. In
general, surface defects in crystalline ZnO can act as the active cen-
ters to capture photo-induced electrons, and the recombination of
photo-induced electrons and holes can be effectively inhibited
[42], so that the photocatalytic activity can be greatly improved.
The higher photocatalytic activity of the ZnO flower-like (sample
C) may result from the larger the content of oxygen vacancy on
the surface.

The presence of excess surface defects leading to strong blue
emission has also been correlated to higher antibacterial response
of sample C. The formation of hydroxyl radical on the surface of
ZnO samples in aqueous dispersion would leave oxygen vacancies
on the surface, according to the mechanism proposed by Hewett
et al. [48]. So one probable mechanism may be the attack of the
reactive hydroxyl radical on the bacteria cell membrane leads to
membrane damage.
5. Influence of the microorganisms on the inactivation
efficiency
All ZnO photocatalysts show much higher activity against E. coli
than against S. aureus. The higher deactivation efficiency in the case
of gram-negative bacteria as compared to gram-positive ones was
reported earlier [49,50]. This difference can be attributed to the dif-
ferences in the cell wall structure inherent in gram-negative and
72 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73

gram-positive bacteria. gram-positive and gram-negative bacteria
have similar internal, but very different external structures. A
gram-positive bacterium has a thick peptidoglycan layer that con-
tains teichoic and lipoteichoic acids. A gram-negative bacterium
has a thin peptidoglycan layer and an outer membrane that con-
tains lipopolysaccharide, phospholipids, and proteins. This permits
a conclusion that the bacteria photoinactivation rate is governed
not only by cell wall thickness but also by the morphology of cell
envelop and resistance of outer membrane to the reactive oxygen
species produced at the photocatalyst surface. Fig. 6 photographi-
cally shows the difference between bacteria photoinactivation
and inactivation efficiencies for all samples against E. coli and S.
aureus.
6. Catalyst recovery and reuse
Catalyst deactivation is one of the drawbacks on the application
of photocatalytic degradation processes, deposition of photoinsen-
sitive species on the photocatalyst surface blocking its active sites.
The economy of the photocatalytic process depends upon how
many times a catalyst can be reused without sacrificing its effi-
ciency and the type of regeneration it requires. The ZnO used cat-
alysts was regenerated by treating with boiling distilled water and
then by drying it in a hot air oven at a temperature of 100 °C.
The catalysts under consideration have not shown a significant
decrease in their photoinduced and dark microbiocide efficiency
when the antimicrobial experiments were repeated three times
350
300
250
Residual servival
Sample A, E.coli
Sample B, E. coli
Sample C, E.coli
Sample A, S. aureus
using the same samples. The reproducibility of experimental data
is shown in Figs. 7 and 8.
7. Photocatalytic inactivation mechanisms
The nanoparticle morphology, surface area, surface charge, its
material solubility and degree of aggregation in a particular system
being studied will strongly influence the availability of nanoparti-
cles for uptake into cells and resulting toxicity in aquatic systems
[51,52]. The following factors may affect the antibacterial activity
of ZnO powders: (1) the cations eluted from the powder [53];
Brunner et al. [54] observed that material solubility strongly influ-
enced the cytotoxic response of a range of oxide nanoparticles,
with more soluble compounds like ZnO and FeO showing greater
acute toxicity than nanoparticles of extremely low solubility such
as CeO2 and TiO2. However, the authors did not consider the influ-
ence of dissolved zinc species on toxicity and attributed the effects
solely to the nanoparticle suspensions. Provided that the ZnO con-
centration is well below the solubility limit obtained in our study,
cells will be exposed to nondissolved nanoparticles. However, it is
reasonable to suggest that toxicity may, at least in part, be related
to zinc solubility. The released metal ions may penetrate bacterial
cell membranes and combine with the intracellular protease of
bacteria to inactive it. (2) Highly reactive oxygen species (ROS)
generated including hydroxyl groups (OH), superoxide anions
ðO
2 Þ, and hydrogen peroxide (H2O2) [15,55,56]; Oxygen dissolved
in the solution can generate superoxide anions O 2 by single-elec-
tron reduction which does not require UV irradiation [57,58]. How-
ever, photocatalytically induced oxygen species improve the
bactericide effect of UV-illuminated samples. (3) pH; In our work,
pH of the powder samples dispersed in physiological saline was
7.5, irrespective of the sample. (4) Membrane dysfunction
[14,53]. (5) Nanoparticle internalization [12].

Sample B, S. aureus
200 Sample C, S. aureus
8. Conclusion
150
Nanocrystalline ZnO powders with different morphologies were
100 successfully prepared by the solvothermal method. Physicochemi-
cal properties of the solvent, which was employed as the reaction
50 medium for ZnO synthesis, have significant impact on both the for-
mation of ZnO nuclei and the growth of the crystals. The dielectric
0
10 20 30 40 constant of the solvent was proposed to be an index that was well-
Time (min) correlated with the morphology of the synthesized products. SEM
images showed that flower-like, hexagonal rod-like and spheri-
Fig. 7. Plot of the survival number of Escherichia coli and Staphylococcus aureus with cal-like ZnO were synthesized in water, 1-hexanol and ethylene
different catalysts under UV illumination.
glycol, respectively. Antibacterial tests indicated that the flower-
like ZnO nanoparticles exhibited excellent antibacterial activities
after UV illumination for a short time. For flower-like ZnO nanopar-
5e+2
ticles, the enhanced intensity of the photoluminescence band in
Sample A, E.coli visible luminescence range resulted from the higher surface inter-
Sample B, E. coli
4e+2
Sample C, E. coli
stitial defects reduces the electrons or holes recombination and
consequently increase the antibacterial activities.
Residual servival

Sample A, S.aureus
Sample B, S. aureus
3e+2 Sample B, S.aureus
Acknowledgement

2e+2
The research grant provided by Islamic Azad University, Shah-
reza Branch is gratefully acknowledged.
1e+2
References
0
0 20 40 60 80 100 [1] N.L. Stock, J. Séller, K. Vinodgal, P.V. Kamat, Combinative sonolysis and
photocatalysis for textile dye degradation, Environ. Sci. Technol. 34 (2000)
Time (min) 1747–1750.
[2] J.Y. Kim, C. Lee, M. Cho, J. Yoon, Enhanced inactivation of E. coli and MS-2 phage
Fig. 8. Plot of the survival number of Escherichia coli and Staphylococcus aureus with by silver ions combined with UV-A and visible light irradiation, Water Res. 42
different catalysts at dark condition. (2008) 356–362.
 N. Talebian et al. / Journal of Photochemistry and Photobiology B: Biology 120 (2013) 66–73
[3] C.A. Murray, E.H. Goslan, S.A. Parsons, TiO2/UV: single stage drinking water
treatment for NOM removal?, J Environ. Eng. Sci. 6 (3) (2007) 311–317.
[4] Z. Huang, X. Zheng, D. Yan, G. Yin, X. Liao, Y. Kang, et al., Toxicological effect of
ZnO nanoparticles based on bacteria, Langmuir 24 (2008) 4140–4144.
[5] X. Bingshe, N. Meia, W. Liqiao, H. Wensheng, L. Xuguang, The structural
analysis of biomacromolecule wool fiber with Ag-loading SiO2 nano-
antibacterial agent by UV radiation, J. Photochem. Photobiol. A. Chem. 188
(2007) 98–105.
[6] R. Xu, X. Wang, D.S. Wang, K.B. Zhou, Y.D. Li, Surface structure effects in
nanocrystal MnO2 and Ag/MnO2 catalytic oxidation of CO, J. Catal. 237 (2006)
426–430.
[7] D.S. Wang, X. Wang, R. Xu, Y.D. Li, Shape-dependent catalytic activity of CuO/
MgO nanocatalysts, J. Nanosci. Nanotechnol. 7 (2007) 3602–3606.
[8] Ü. Özgür, Ya I. Alivov, C. Liu, A. Teke, M.A. Reshchikov, S. Doğan, V. Avrutin, S.-J.
Cho, H. Morkoç, A comprehensive review of ZnO materials and devices, J. Appl.
Phys. 98 (2005) 041301.
[9] J. Yu, X. Yu, Hydrothermal synthesis and photocatalytic activity of zinc oxide
hollow spheres, Environ. Sci. Technol. 42 (2008) 4902–4907.
[10] O. Seven, B. Dindar, S. Aydemir, D. Metin, M.A. Ozinel, S. Icli, Solar
photocatalytic disinfection of a group of bacteria and fungi aqueous
suspensions with TiO2, ZnO and Sahara desert dust, J. Photochem. Photobiol.
A Chem. 165 (2004) 103–107.
[11] O. Yamamoto, Influence of particle size on the antibacterial activity of zinc
oxide, Int. J. Inorg. Mater. 3 (2001) 643–646.
[12] R. Brayner, R. Ferrari-Illiou, N. Briviois, S. Djediat, M.F. Benedetti, F. Fievet,
Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO
nanoparticles colloidal medium, Nano Lett. 6 (2006) 866–870.
[13] N.M. Franklin, N.J. Rogers, S.C. Apte, G.E. Batley, G.E. Gadd, P.S. Casey,
Comparative toxicity of nanoparticulate ZnO, Bulk ZnO, and ZnCl2 to a
freshwater microalga (Pseudokirchneriella subcapitata): the importance of
particle solubility, Environ. Sci. Technol. 41 (2007) 8484–8490.
[14] N. Talebian, M.R. Nilforoushan, E. Badri Zargar, Enhanced antibacterial
performance of hybrid semiconductor nanomaterials: ZnO/SnO2
nanocomposite thin films, Appl. Surf. Sci. 258 (2011) 547–555.
[15] J. Sawai, S. Shoji, H. Igarashi, A. Hashimoto, T. Kokugan, M. Shimizu, H. Kojima,
Hydrogen peroxide as an antibacterial factor in zinc oxide powder slurry, J.
Ferment. Bioeng. 86 (1998) 521–522.
[16] S. Atmaca, K. Gul, R. Clcek, The effect of zinc on microbial growth, Turk. J. Med.
Sci. 28 (1998) 595–597.
[17] H.C. Poynton, J.M. Lazorchak, C.A. Impellitteri, M.E. Smith, K. Rogers, M. Patra,
K.A. Hammer, H.J. Allen, C.D. Vulpe, Differential gene expression in Daphnia
magna suggests distinct modes of action and bioavailability for ZnO
nanoparticles and Zn ions, Environ. Sci. Technol. 45 (2011) 762–768.
[18] K.H. Tam, A.B. Djurišić, C.M.N. Chan, Y.Y. Xi, C.W. Tse, Y.H. Leung, W.K. Chan,
F.C.C. Leung, D.W.T. Au, Antibacterial activity of ZnO nanorods prepared by a
hydrothermal method, Thin Solid Films 516 (2008) 6167–6174.
[19] C. Wu, L. Shen, Q. Huang, Y.C. Zhang, Synthesis of Na-doped ZnO nanowires
and their antibacterial properties, Powder Technol. 205 (2011) 137–142.
[20] J.S. Tawale, K.K. Dey, R. Pasricha, K.N. Sood, A.K. Srivastava, Synthesis and
characterization of ZnO tetrapods for optical and antibacterial applications,
Thin Solid Films 519 (2010) 1244–1247.
[21] K. Sue, K. Kimura, K. Murata, K. Arai, Effect of cations and anions on properties
of zinc oxide particles synthesized in supercritical water, J. Supercrit. Fluids 30
(2004) 325–331.
[22] G.R. Patzke, F. Krumeich, R. Nesper, Oxidic nanotubes and nanorods—
anisotropic modules for a future nanotechnology, Angew. Chem. Int. Ed. 41
(2002) 2446–2461.
[23] P. Tonto, O. Mekasuwandumrong, S. Phatanasri, V. Pavarajarn, P. Praserthdam,
Preparation of ZnO nanorod by solvothermal reaction of zinc acetate in various
alcohols, Ceram. Int. 34 (2008) 57–62.
[24] S. Biswas, S. Kar, S. Chaudhuri, Effect of the precursors and solvents on the size,
shape and crystal structure of manganese sulfide crystals in solvothermal
synthesis, Mater. Sci. Eng. B 142 (2007) 69–77.
[25] M. Rezapour, N. Talebian, Comparison of structural, optical properties and
photocatalytic activity of ZnO with different morphologies: effect of synthesis
methods and reaction media, Mater. Chem. Phys. 129 (2011) 249–255.
[26] Y. Wang, X. Li, N. Wang, X. Quan, Y. Chen, Controllable synthesis of ZnO
nanoflowers and their morphology-dependent photocatalytic activities, Sep.
Purif. Technol. 62 (2008) 727–732.
[27] D.R. Lidef (Ed.), Handbook of Chemistry and Physics, 81st ed., CRC Press, Boca
Raton, FL, 2000.
[28] S. Mridha, D. Basak, Effect of thickness on the structural, electrical and optical
properties of ZnO films, Mater. Res. Bull. 42 (2007) 875–882.
[29] L.J. Meng, M.P. Dos Santos, Investigations of titanium oxide films deposited by
d.c. reactive magnetron sputtering in different sputtering pressures, Thin Solid
Films 226 (1993) 22–29.
[30] D. Bao, H. Gu, A. Kuang, Sol–gel-derived c-axis oriented ZnO thin films, Thin
Solid Films 312 (1998) 37–39.
[31] T. Kawano, H. Imai, Fabrication of ZnO nanoparticles with various aspect ratios
through acidic and basic routes, Cryst. Growth Des. 6 (2006) 1054–1056.
[32] A. Pan, R. Yu, S. Xie, ZnO flowers made up of thin nanosheets and their optical
properties, J. Cryst. Growth 282 (2005) 165–172.
73
[33] J. Du, Z. Liu, Y. Huang, Y. Gao, B. Han, W. Li, G. Yang, Control of ZnO
morphologies via surfactants assisted route in the subcritical water, J. Cryst.
Growth 280 (2005) 126–134.
[34] H. Zhang, D. Yang, Y. Ji, X. Ma, J. Xu, D. Que, Low temperature synthesis of
flowerlike ZnO nanostructures by cetyltrimethylammonium bromide-assisted
hydrothermal process, J. Phys. Chem. B 108 (2004) 3955–3958.
[35] H. Zhang, D. Yang, X. Ma, Y. Ji, J. Xu, D. Que, Synthesis of flower-like ZnO
nanostructures by an organic-free hydrothermal process, Nanotechnology 15
(2004) 622–626.
[36] J. Tauc, R. Grigorovichi, A. Vancu, Optical properties and electronic structure of
amorphous germanium, Phys. Status Solidi 15 (1966) 627–637.
[37] P. Zhu, J. Zhang, Z. Wu, Z. Zhang, Microwave-assisted synthesis of various ZnO
hierarchical nanostructures: effects of heating parameters of microwave oven,
Cryst. Growth Des. 8 (2008) 3148–3153.
[38] K.V. Vanheusden, W.L. Warren, C.H. Seager, Mechanisms behind green
photoluminescence in ZnO phosphor powders, J. Appl. Phys. 79 (1996)
7983–7990.
[39] Q. Yang, K. Tang, J. Zuo, Y. Qian, Synthesis and luminescent property of single-
crystal ZnO nanobelts by a simple low temperature evaporation route, J. Appl.
Phys. 79 (2004) 1847–1851.
[40] K. Sharda, S. Jayanthi, Chawla, Synthesis of Mn doped ZnO nanoparticles with
biocompatible capping, Appl. Surf. Sci. 256 (2010) 2630–2635.
[41] Z. Chen, N. Wu, Z. Shan, M. Zhao, S. Li, C.B. Jiang, M.K. Chyu, S.X. Mao, Effect of
N2 flow rate on morphology and structure of ZnO nanocrystals synthesized via
vapor deposition, Scripta Mater. 52 (2005) 63–67.
[42] J. Liqiang, Q. Yichun, W. Baiqi, L. Shudan, J. Baojiang, Y. Libin, F. Wei, F.
Honggang, S. Jiazhong, Review of photoluminescence performance of nano-
sized semiconductor materials and its relationships with photocatalytic
activity, Sol. Energy Mater. Sol. Cells 90 (2006) 1773–1778.
[43] J. Zhang, K. Sasaki, E. Sutter, R.R. Adzic, Stabilization of platinum oxygen-
reduction electrocatalysts using gold clusters, Science 315 (5809) (2007) 220–
222.
[44] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, FEMS. Antimicrobial activities of
commercial nanoparticles against an environmental soil microbe,
Pseudomonas putida KT2440, Microbiol. Lett. 279 (2008) 71–76.
[45] N.M. Franklin, N.J. Rogers, S.C. Apte, G.E. Batley, G.E. Gadd, P.S. Casey,
Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a
freshwater microalga (Pseudokirchneriella subcapitata): The importance of
particle solubility, Environ. Sci. Technol. 41 (2007) 8484–8490.
[46] L. Zhang, L. Yin, C. Wang, N. Lun, Y. Qi, Solgel growth of hexagonal faceted
ZnO prism quantum dots with polar surfaces for enhanced photocatalytic
activity, Appl. Mater. Interfaces 2 (2010) 1769–1773.
[47] Y. Zheng, C. Chen, Y. Zhan, X. Lin, Q. Zheng, K. Wei, et al., Luminescence and
photocatalytic activity of ZnO nanocrystals: correlation between structure and
property, Inorg. Chem. 46 (2007) 6675–6682.
[48] K.B. Hewett, L.C. Anderson, M.P. Rosynek, J.H. Lunsford, Formation of hydroxyl
radicals from the reaction of water and oxygen over basic metal oxides, J. Am.
Chem. Soc. 118 (1996) 6992–6997.
[49] A.G. Rincón, C. Pulgarin, Bactericidal action of illuminated TiO2 on pure
Escherichia coli and natural bacterial consortia: post-irradiation events in the
dark and assessment of the effective disinfection time, Appl. Catal. B: Environ.
49 (2004) 99–112.
[50] A. Pal, S.O. Pehkonen, L.E. Yu, M.B. Ray, Photocatalytic inactivation of Gram-
positive and Gram-negative bacteria using fluorescent light, J. Photochem.
Photobiol. A: Chem. 186 (2007) 335–341.
[51] L.K. Limbach, L. Yuchun, R.N. Grass, T.J. Brunner, M.A. Hintermann, M. Muller,
D. Gunther, W.J. Stark, Oxide nanoparticle uptake inhumanlung fibroblasts:
effects of particle size, agglomeration, and diffusion at low concentrations,
Environ. Sci. Technol. 39 (2005) 9370–9376.
[52] T. Xia, M. Kovochich, M. Liong, L. Mädler, B. Gilbert, H. Shi, J.I. Yeh, J.I. Zink, A.E.
Nel, Comparison of the mechanism of toxicity of zinc oxide and cerium oxide
nanoparticles based on dissolution and oxidative stress properties, ACS Nano.
2 (2008) 2121–2134.
[53] Z. Yang, C. Xie, Osmotic regulation of 10 wheat (Triticum aestivum L.) genotypes
at soil water deficits, Colloids Surf., B 47 (2006) 132–139.
[54] T.J. Brunner, P. Wick, P. Manser, P. Spohn, R.N. Grass, L.K. Limbach, A. Bruinink,
W.J. Stark, In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos,
silica, and the effect of particle solubility, Environ. Sci. Technol. 40 (2006)
4374–4381.
[55] G. Applerot, A. Lipovsky, R. Dror, N. Perkas, Y. Nitzan, R. Lubart, A. Gedanken,
Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-
mediated cell injury, Adv. Funct. Mater. 19 (2009) 842–852.
[56] J. Sawai, H. Kojima, H. Igarashi, A. Hashimoto, S. Shoji, A. Takehara, T. Sawaki, T.
Kokugan, M. Shimizu, Escherichia coli damage by ceramic powder slurries, J.
Chem. Eng. Jpn. 30 (1997) 1034–1039.
[57] K. Hirota, M. Sugimoto, M. Kato, K. Tsukagoshi, T. Tanigawa, H. Sugimoto,
Preparation of zinc oxide ceramics with a sustainable antibacterial activity
under dark conditions, Ceram. Int. 36 (2010) 497–506.
[58] L. Huang, D.Q. Li, Y.-J. Lin, M. Wei, D.G. Evans, X. Duan, Controllable
preparation of nano-MgO and investigation of its bactericidal properties, J.
Inorg. Biochem. 99 (2005) 986–993.