Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185

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Journal of Photochemistry and Photobiology B: Biology

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

Photocatalytic antibacterial activity of nano-TiO2 (anatase)-based

thin films: Effects on Escherichia coli cells and fatty acids
Urmas Joost a,b,⇑,1, Katre Juganson c,d,1, Meeri Visnapuu a,c,1, Monika Mortimer c, Anne Kahru c,
Ergo Nõmmiste a, Urmeli Joost a, Vambola Kisand a,b, Angela Ivask c,⇑
a
Institute of Physics, University of Tartu, Ravila 14c, 50411 Tartu, Estonia
b
Estonian Nanotechnology Competence Center, Ravila 14c, 50411 Tartu, Estonia
c
Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
d
Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia

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

Article history: Titanium dioxide is a photocatalyst with well-known ability to oxidise a wide range of organic
Received 15 July 2014 contaminants as well as to destroy microbial cells. In the present work TiO2 nanoparticles with high
Received in revised form 26 November 2014 specific surface area (150 m2/g) were used to prepare nanostructured films. The TiO2 nanoparticle-based
Accepted 1 December 2014
film in combination with UV-A illumination with intensity (22 W/m2) comparable to that of the sunlight
Available online 17 December 2014
in the UV-A region was used to demonstrate light-induced antibacterial effects. Fast and effective
inactivation of Escherichia coli cells on the prepared thin films was observed. Visualization of bacterial
cells under scanning electron microscopy (SEM) showed enlargement of the cells, distortion of cellular
membrane and possible leakage of cytoplasm after 10 min of exposure to photoactivated TiO2. According
to the plate counts there were no viable cells as early as after 20 min of exposure to UV-A activated TiO2.
In parallel to effects on bacterial cell viability and morphology, changes in saturated and unsaturated
fatty acids – important components of bacterial cell membrane-were studied. Fast decomposition of
saturated fatty acids and changes in chemical structure of unsaturated fatty acids were detected. Thus,
we suggest that peroxidation and decomposition of membrane fatty acids could be one of the factors
contributing to the morphological changes of bacteria observed under SEM, and ultimately, cell death.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction can degrade different organic compounds [11–13], has antibacte-

With increasing appearance of antibiotic-resistant bacteria,
new and more potent antibacterial agents and materials are
needed. There are great hopes that nanotechnology could offer
new possibilities in this area [1,2]. Although nanoparticles (NPs)
of silver, zinc oxide and copper oxide [3] are already used in several
antimicrobial applications [4], those nanomaterials may also pose
hazard to other organisms when released to the environment
[3,5]. Kahru and Dubourguier [6] showed that among seven most
widespread nanomaterials (TiO2, ZnO, CuO, Ag, SWCNTs, MWCNTs
and C60-fullerenes) TiO2 NPs proved to be the most environmen-
tally benign. TiO2 has been studied extensively as a promising pho-
tocatalyst [7], solar cell material [8], anti-fogging and self-cleaning
coating material [9,10]. Due to its photocatalytic properties, titania
⇑ Corresponding authors at: Institute of Physics, University of Tartu, Ravila 14c,
50411 Tartu, Estonia (U. Joost). Laboratory of Environmental Toxicology, National
Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn,
Estonia (A. Ivask).
E-mail addresses: urmas.joost@ut.ee (U. Joost), angela.ivask@kbfi.ee (A. Ivask).
1 molecule during which superoxide anions (O2 ) are produced [24].
Equal participation.
rial [14,15] and photoinduced super-hydrophilic properties [9,10]
making it an ideal candidate for many applications where surfaces
are inaccessible for traditional cleaning and/or have to efficiently
inhibit potentially pathogenic bacteria [16]. One of the advantages
of using TiO2 in self-cleaning applications is that the material is
very stable, and that storage and repeated long term exposure to
UV has little or no effect on its antibacterial activity.
Antibacterial applications of TiO2 often use thin film coatings
that can be deposited by various methods including sputtering
[17], chemical solution deposition [18], pulsed laser deposition
[19] or sol–gel method [20–23].
The mechanism of TiO2 photocatalytic oxidation and conse-
quently, also antibacterial activity involves reactive oxygen species
(ROS); TiO2 in anatase phase has been shown as the most potent
form of TiO2 to produce ROS [24]. ROS production by TiO2 is induced
after absorption of a high-energy photon and subsequent excitation
of an electron from the TiO2 valence band to conduction band [24].
The next and rate determining step in formation of ROS is photoex-
cited electron transfer from conduction band of TiO2 to oxygen

http://dx.doi.org/10.1016/j.jphotobiol.2014.12.010
1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
 U. Joost et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185
Photoholes generated into the valence band during photoexcitation
oxidize surface absorbed H2O, AOH and surface titanol groups
(>TiOH) into highly reactive hydroxyl radicals (OH). Although other
ROS also contribute to TiO2 photocatalytic activity, the majority can
be attributed to OH radicals [25–27], that not only act on TiO2 sur-
face but can diffuse over short distances to the surrounding solution
near the photocatalyst surface and effectively degrade organic com-
pounds that are not directly in contact with the photocatalyst
[25,28,29]. Thus, hydroxyl radicals have been considered as the
main ROS species to cause both, photocatalytic activity as well as
antibacterial effects of TiO2 [30–32]. It is suggested that the main
reason for high antibacterial activity of TiO2 particles is photooxida-
tion of essential cellular components. Oxidation of one of the major
components of bacterial plasma membrane-phospholipids-by
photoactivated TiO2 has been reported in several studies [33,34].
However, it must be noted that studies presenting efficient and
rapid antibacterial activity of TiO2 have utilized short wavelength
UV-light (UV-C and UV-B) [27,35]. Studies where UV-A light that
has longer wavelength and, thus, is less harmful to living organisms
per se, was used to photoactivate TiO2 have reported relatively poor
antibacterial activity [16,36–39] or long exposure times needed for
achieving sufficient antibacterial effects [40,41].
In the current study, we investigated the time-dependent anti-
bacterial effect of TiO2 NP (anatase)-based thin films (hereafter des-
ignated as nano-TiO2 thin films) under UV-A light (315–400 nm). E.
coli, a potential pathogen and a sanitary indicator bacterium was
used as a model microorganism to evaluate the antimicrobial effi-
cacy of light-activated TiO2 films. We used recombinant lumines-
cent E. coli which allowed easy luminescence-based assessment of
cell viability. At different exposure times both, the number of viable
bacterial cells as well as changes in bacterial morphology were
determined by growth tests and scanning electron microscopy,
respectively. In parallel to the studies with bacterial cells the ability
of UV-A activated nano-TiO2 thin films to decompose saturated and
unsaturated fatty acids was evaluated by determining the changes
in fatty acid structure using X-ray photoelectron spectroscopy
(XPS). As these fatty acids are abundant in bacterial plasma mem-
brane the UV-light stimulated changes in fatty acid decomposition
may yield further information on the antimicrobial mechanism of
action of TiO2 thin films.
2. Materials and methods
2.1. Materials
Commercially available reagents: titanium(IV) butoxide
(Ti(OBu)4; Sigma–Aldrich, reagent grade), p-toluene sulfonic acid
(PTSA; Sigma–Aldrich, reagent plus), acetyl acetone (acac;
Sigma–Aldrich, reagent plus), butanol (Sigma–Aldrich), methanol
(Sigma–Aldrich P99.8%) and deionized water (MilliQ) were used
for the preparation of TiO2 NPs. Glutaraldehyde (Sigma–Aldrich,
70% in H2O) and anhydrous ethanol were used in sample prepara-
tion for electron microscopy study. Stearic acid (C18:0), oleic acid
(C18:1 cis-9; both from Cayman Chemical Company, purity >98%)
and linoleic acid (C18:2 cis-9,12; Sigma Aldrich, purity P99%)
(Fig. S1) were used in fatty acid degradation experiments. Butanol
was dried using CaH2 and distilled before utilization; all other
reagents were used as received. 0.9% NaCl solution that was pre-
pared using NaCl from Sigma Aldrich and MilliQ water was used
in bacterial viability assays.
2.2. Synthesis of titania nanoparticles and preparation of nano-TiO2
thin films
Titania NPs were synthesised using a method described by
Scolan and Sanchez [42] with slightly modified parameters. Briefly,
179
the molar ratio of PTSA and Ti(OBu)4 was set to 0.2, the molar ratio
of acac and Ti(OBu)4 was set to 3 and the molar ratio of water and
Ti(OBu)4 was set to 10. The reaction was carried out overnight at
reflux conditions. The particles were centrifuged for 0.5–1.5 h at
12,000g and subsequently washed 3 times with methanol. Finally,
the particles were dispersed in 95% ethanol and the attained dis-
persions were stable for months.
Thin films were prepared by spin-coating colloidal solution of
anatase TiO2 NPs on silicon (1 0 0) monocrystal substrates at ambi-
ent atmospheric conditions. The substrates were cleaned prior to
coating with 95% ethanol to remove small dust particles. The rota-
tion frequency during spin-coating was 3000 rpm and coating time
was 0.5 min. The obtained precursor films were aged at ambient
conditions for 4 days to allow the remaining solvent to evaporate
slowly in order to prevent cracking of the films. After ageing the
samples were annealed in a Nabertherm L5/11/S27 furnace at
400 °C and washed in deionized water for 10 min in ultrasonic bath
to remove organic residue. The obtained films were characterized
using Raman spectroscopy, atomic force microscopy (AFM), scan-
ning electron microscopy (SEM) and X-ray photoelectron spectros-
copy (XPS). The indirect optical band gap of the obtained anatase
thin films was evaluated using UV–Vis spectroscopy and Tauc plot
(Fig. S2). Characterization of the obtained nano-TiO2 thin films
with high specific surface area has been reported in detail in our
previous work [43], including the description of the cleaning meth-
ods of nano-TiO2 thin films.
Stearic, oleic and linoleic acid coatings for photoactivated deg-
radation studies were prepared by spin-coating 0.5% solution of
respective fatty acid in methanol on nano-TiO2 thin film substrate
(approximately 1 cm2) for 0.5 min at 3000 rpm, so that the whole
substrate surface was uniformly covered.
2.3. UV treatment
UV-irradiation of nano-titania films with fatty acids added was
conducted in climate chamber Memmert CTC 256 at 25 °C at a rel-
ative humidity of 70%. High relative humidity ensured the presence
of water molecules on the nano-titania surface. When bacteria on
Si-monocrystal substrates (negative control) or nano-TiO2 thin
films were exposed to UV in CTC 256 climate chamber the temper-
ature was set to 25 °C and the relative humidity to 90% to avoid
extensive drying of the droplet of bacterial suspension. The source
of UV-irradiation was a Philip Cleo 15 W fluorescent lamp with a
maximum intensity at 355 nm, i.e. in the UV-A region. Light inten-
sity at the sample surface was measured with a lux meter Delta
Ohm HD 2302.0 equipped with a sensor LP 471 UV-A or LP 471
UV-B accordingly. It was found that the light intensity in the
UV-A region (315–400 nm) was 22 W/m2 which is comparable to
the UV-light intensity of sunlight on the ground level according
to the ASTMG 173-03 table [44], and in the UV-B region
(280–315 nm) less than 0.1 W/m2. Due to the dominance of UV-A
light in the UV-spectra of the used UV-lamp, the applied light is
further designated as UV-A.
2.4. Evaluation of antibacterial activity of the nano-TiO2 thin films
Recombinant constitutively bioluminescent gram-negative bac-
teria E. coli MC1061 (pSLlux) [45] were used as test organisms to
evaluate the antibacterial activity of nano-TiO2 thin films. The cells
were cultivated in Luria-Bertani (LB) broth supplemented with
100 lg/mL of ampicillin at room temperature (23 ± 2 °C) overnight
without shaking. The cells were harvested in exponential growth
phase by centrifugation at 3500g for 10 min. The cells were washed
twice with 0.9% NaCl or with MilliQ water and were resuspended
either in equal volume of 0.9% NaCl or in MilliQ. According to direct
plate counting, the final number of viable bacterial cells either in
180 U. Joost et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185
0.9% NaCl or in MilliQ was about 108 colony forming units per
millilitre (CFU/mL). Antibacterial effects of nano-TiO2 thin films
to E. coli were determined quantitatively and visually.
In order to quantitatively evaluate the antibacterial activity of
nano-TiO2 thin films, 10 lL of E. coli MC1061 (pSLlux) suspension
in 0.9% NaCl was added onto nano-TiO2 thin film with approximate
surface area of 1 cm2. The actual contact area between the bacterial
suspension and the surface depended on the irradiation time and
therefore was difficult to determine accurately. The surfaces with
bacterial suspension were illuminated with UV-light as described
above (see Section 2.3) for 0, 5, 10, 15 and 20 min. For control, bac-
teria on Si-monocrystals with no TiO2 were illuminated identically.
For non-UV control, the cells were incubated on the same surfaces
for 20 min in the dark. After the exposure, the surfaces with bacte-
rial suspension were placed into a 15 mL round-bottom polypro-
pylene tube containing 1 mL of 0.9% NaCl and the bacteria were
separated from the surface by vortexing for 5 min. Then 15 lL of
the resultant bacterial suspension and its 10-, 100-, and 1000-fold
dilutions were spread onto LB agar plates containing 100 lg/mL of
ampicillin. The number of surviving luminescent bacterial colonies
(CFU) was counted in the dark after overnight incubation at 37 °C.
The experiment was conducted on two different days in three rep-
licates and the counts corresponding to a particular sample were
averaged.
Visual observation of antibacterial effects of nano-TiO2 thin
films was done using SEM (FEI Helios NanoLab 600). For SEM imag-
ing, 10 lL of E. coli MC1061 (pSLlux) suspension in MilliQ water
was added drop-wise onto the aforementioned surfaces. The expo-
sure conditions were the same as for the quantitative evaluation. In
addition, 40 and 60 min exposure times were applied. After the
exposure, the samples were moved to a chamber filled with water
vapour (see Fig. S3) and 20 lL of fixative (2.5% glutaraldehyde solu-
tion in ethanol) was pipetted onto each sample. The samples were
fixated for 2 h and then placed to a chamber filled with ethanol
vapours to initiate dehydration. After 15 min the samples were
allowed to dry at ambient conditions for 5 min under a cover.
The dehydration cycle was repeated twice. Next, the samples were
carefully rinsed with anhydrous ethanol and allowed to dry at
ambient conditions under a cover for three days before SEM imag-
ing. SEM images were acquired with an accelerating voltage of
1.0 kV. Prior to SEM imaging all samples were treated identically
(including glutaraldehyde treatment), therefore ‘‘bulged’’ bacteria
(see Fig. S4) which appeared after 10 min UV treatment were con-
sidered severely affected or dead. A number of images from differ-
ent places on the sample were taken and one image representing
the whole sample was presented.
2.5. Analysis of structural changes in fatty acids on nano-TiO2 thin
films using X-ray photoelectron spectroscopy (XPS)
XPS was used to investigate the chemical changes occurring in
stearic, oleic and linoleic acid coatings on nano-TiO2 thin films
upon exposure to UV-A illumination. XPS measurements were
conducted using a surface station equipped with an electron
energy analyser (SCIENTA SES 100) and a non-monochromatic twin
anode X-ray tube (Thermo XR3E2), with characteristic energies of
1253.6 eV (Mg Ka1,2 FWHM 0.68 eV) and 1486.6 eV (Al Ka1,2 FWHM
0.83 eV). All XPS measurements were conducted in ultra-high vac-
uum conditions. The binding energy scales for the XPS experiments
were referenced to the binding energy of Ti 2p3/2 (458.6 eV) photo-
emission line. To estimate overall atomic concentrations of differ-
ent compounds and elements Average Matrix Relative Sensitivity
Factors (AMRSF) procedure [46] and transmission function of the
instrument were used. Raw data were processed using Casa XPS
[47] software. Data processing involved removal of Ka and Kb
satellites, removal of background and fitting of components
[Gauss-Lorentz hybrid function (GL 70, Gauss 30%, Lorentz 70%)
was used for fitting and Tougaard background was used for back-
ground removal]. The residual carbon on nano-TiO2 surfaces was
taken into account by measuring the C 1s spectra of nano-TiO2 film
before covering it with fatty acids and subtracting it afterwards
from the spectra of samples covered with fatty acids. This was
done based on our previous work [43] where we noticed that after
washing the nano-TiO2 films in deionized water the composition
and the amount of residual carbon was always very similar. It
was not possible to measure the effects of UV-A radiation on
non-photocatalytic surface due to technical reasons (the fatty acids
evaporated from smooth Si(1 0 0) surface during pumping before
XPS spectra could be measured). However, it has been shown that
photochemical effects potentially occurring on Si-monocrystal sur-
face are at least 10 times smaller than photocatalytic effects that
take place on TiO2 surface and can be therefore neglected [48].
Due to surface region deviation from chemical homogeneity in
the working range of photoelectron spectroscopy (surface region
with thickness up to three electron mean free paths) the absolute
amounts of different chemical compounds were considered cau-
tiously and are presented in this study only to outline trends and
estimates of processes occurring during photo-oxidation.
3. Results and discussion
3.1. Characterization of nano-TiO2 thin films
Characterization of nano-TiO2 thin films has been reported in
our previous work [43]. Colloidal suspension of anatase TiO2 NPs
that was spin-coated onto the Si-monocrystal surface consisted
mainly of monodisperse particles with hydrodynamic diameter
less than 10 nm (Fig. S5). Taking into account the spherical nature
of the particles, the calculated surface area of the TiO2 particles was
approximately 150 m2/g. After deposition, the anatase nano-TiO2
particles (Fig. S6) were annealed at 400 °C and subsequently
washed with deionised water. The annealing step was necessary
for removal of organic material used during NP synthesis. This thin
film preparation methodology is suitable for most of the materials
that can be annealed at temperatures up to 400 °C. The thickness of
resulting TiO2 thin film was 115 nm (measured with AFM) [43]. It
is likely that the aggregation of 10 nm TiO2 particles during depo-
sition and annealing took place. Yet, the structural appearance of
thin film surface indicated high surface area of TiO2 also in the
form of a thin film after annealing (Fig. S7). High surface area of
the TiO2 nanomaterial ensured the presence of surface adsorbed
species that can act as electron and/or hole traps [24] increasing
thus the lifetime of electron/hole pairs and increasing the effi-
ciency of the photocatalyst, making this material an efficient pho-
tocatalyst. Optical band gap of prepared nano-TiO2 thin films was
3.5 eV (Fig. S2). This is wider than the 3.2 eV band gap of bulk
TiO2 and is characteristic to nano-sized TiO2 material [49].
3.2. Photoinduced antibacterial activity of nano-TiO2 thin films
Antibacterial effects of UV-treated nano-TiO2 thin films were
evaluated by determining the ability of exposed bacteria to form
bioluminescent colonies on agarized growth media (viability
assay) and also by visual inspection of bacterial morphology
(SEM images). Exposure of bacteria to UV-A light on nano-TiO2 thin
films had a distinct effect on their viability already after 5 min:
the number of colony forming bacteria decreased about 4 times,
from 7.9 ⁄ 107 to 2 ⁄ 107 CFU/mL (Fig. 1). Exposure for 10 min
caused a decrease of colony forming bacteria from 7.9 ⁄ 107 to
1.3 ⁄ 106 CFU/mL and no colony forming bacterial cells remained
after 20 min of exposure. To analyse the effect of UV-A light alone,
 U. Joost et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185
Fig. 1. Colony forming potential of Escherichia coli (pSLlux) in different exposure conditions. The effect of UV-irradiation length on colony forming potential of E. coli (pSLlux)
applied onto Si-monocrystal substrates (blue bars) or nano-TiO2 thin films (red bars). After 20 min of exposure on nano-TiO2 thin film to UV-A light no viable cells able to
grow on agar plate were observed (marker with ⁄). The colony forming potential of E. coli on surfaces kept in the dark (black and dark grey bars) was determined only in the
beginning (0 min) and at the end of the experiment (20 min). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
E. coli cells were illuminated for 20 min on Si-monocrystal surface
not coated with TiO2: 5, 10, 15 and 20 min illumination resulted in
a slight decrease of bacterial number (from 108 to 8.4 ⁄ 107,
5.9 ⁄ 107, 4.7 ⁄ 107 and 1.3 ⁄ 107 CFU/mL, respectively; Fig. 1).
Since silicon is considered one of the most biocompatible materials
[50] and silicon surface stored in atmosphere is completely passiv-
ated in the sense of photocatalytic action with amorphous SiO2
layer [51], this slight decrease was most likely not due to any anti-
bacterial effect of the silicon material but could be attributed to
drying of bacterial droplet.
We also studied the potential antibacterial effects of nano-TiO2
surface in dark conditions: incubation of bacteria for 20 min on
TiO2 surface decreased the bacterial number from 7.9 ⁄ 107 to
3.2 ⁄ 107 CFU/mL (Fig. 1). Similar decrease of viable bacteria (from
108 to 4.3 ⁄ 107 CFU/mL) was observed on Si(1 0 0)-monocrystal
surface in dark conditions, i.e. no TiO2 present (Fig. 1; Fig. S8).
Again, we proposed that this slight decrease could be attributed
to drying of 10 lL bacterial droplet on the surface of Si-monocrys-
tal and the TiO2 thin film. Very small proportion of non-viable cells
on nano-TiO2 thin films in dark conditions and on Si-monocrystal
surface under UV-A light suggest that high killing efficiency of
UV-irradiated TiO2 thin films was indeed due to photoactivation
of nano-TiO2 film. Therefore, we concluded that the TiO2 thin film
developed in this study was effective in killing bacterial cells after
20 min exposure to environmentally relevant UV-A light (22 W/m2
corresponds to UV-A in solar spectrum [44]). It is somewhat diffi-
cult to compare the results of this study with published literature
due to variable conditions used in each study. Only in two studies
TiO2 thin films were excited using UV-A light at intensity similar to
this study. Shiraishi et al excited anatase thin films using UV-A
(20 W/m2) with peak intensity at 352 nm and in these conditions,
90 min was required to completely inactivate bacterial cells [52].
Yeung et al used UV-A light (26.5 W/m2) with peak intensity at
365 nm and showed that 30 min was required to inhibit 99,7% of
B. subtilis and E. coli cells [37]. Thus, the thin films developed in
both of these previous studies were remarkably less effective in
bacterial inactivation than the thin film design in this study. A
number of published studies have utilized lower intensity UV-A
light (5–10 W/m2) and therefore, in these studies 60–90 min of
UV-irradiation has been applied for total inactivation of bacteria
even when rather diluted bacterial suspension was used [40,53].
In case of small volume of concentrated bacterial suspension and
very high UV-A light intensity (200 W/m2) 90% bacterial inhibition
on TiO2 thin film was reached very quickly, in 2 min [54]. On the
181
other hand, as this high UV-A intensity is not realistic in real con-
ditions, no conclusions could be made whether this material would
be also effective in practice.
Examination of photoactivated nano-TiO2 film exposed bacte-
rial cells by SEM (Fig. 2) showed that as bacterial number
decreased, morphology of bacterial cells changed. The shape of
the bacteria became more discursive and expanded, the diameter
and the length of the bacteria increased and the structure of the
bacterial cell membrane was distorted. In addition, a halo sur-
rounding the bacteria was observed that could be associated with
the leakage of organic material or ions for example K+ from the
bacterial cell what has also been observed in earlier studies
[14,40,55]. In general, imaging results correlated well with quan-
titative analysis of antibacterial activity, showing increasing num-
ber of bacteria with damaged cell wall when the length of UV-A
illumination was increased. 10-min exposure of bacteria to photo-
activated TiO2 surface, where first distorted cells were seen under
SEM, decreased the number of viable bacteria by two orders of
magnitude (Fig. 1). 20-min exposure distorted most of the cells
as visualized by SEM (Fig. 2) and resulted in 100% lethality of bac-
teria according to viable colony counts (Fig. 1). Although all the
bacteria were killed on photoactivated TiO2 thin films in 20 min,
i.e., no colonies were formed on agarized growth media, cellular
debris was still clearly visible under SEM and complete destruc-
tion of bacterial cells was not achieved even in 60 min (Fig. S9).
Therefore, our data showed that considerably longer time is
needed to completely decompose and degrade bacterial cells on
TiO2 surface than the time that is needed to affect bacterial
viability.
3.3. Chemical changes in fatty acids on photoactivated nano-TiO2 thin
film
Changes in chemical structure of stearic (C18:0), oleic (C18:1
cis-9) and linoleic acid (C18:2 cis-9,12) – the most abundant
fatty acids in bacterial plasma membrane [56,57] -induced by
UV-illuminated nano-TiO2 films were analysed by XPS. The assay
was performed by adding above-mentioned fatty acids on TiO2 thin
films and irradiating with UV-light. XPS spectra obtained from the
fatty acids before treatment corresponded well to their structure
and the number of carbon atoms in each different chemical state.
C 1s XPS band components from sp2 carbon (two single bonds
and one double bond) at 284.1 ± 0.1 eV [58], sp3 carbon (four single
bonds) at 284.8 ± 0.1 eV [59] and carboxylic group at 288.2 ± 0.1 eV
182 U. Joost et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185

Fig. 2. Images of surviving luminescent bacteria that were able to yield colonies after exposure to UV-light on Si-monocrystal substrates (upper block of panels) or on nano-
TiO2 thin films (lower block of panels). Different sectors of agar plates show the colonies of luminescent bacteria in 15 lL of 102, 103 and 104-fold dilutions of UV-exposed
bacterial suspension. The photos were taken in the dark. Inset on each image is an SEM image of the bacteria after respective exposure conditions. Scale bars on SEM images
correspond to 2 lm. SEM images show considerable morphological changes of bacterial cells already after 10-min UV-irradiation on nano-TiO2 thin film. See also Fig. S9 for
absence of analogous cellular damage in dark conditions.

Fig. 3. Decomposition and changes in chemical composition of stearic (C18:0), oleic (C18:1 cis-9) and linoleic (C18:2 cis-9,12)-acid after their exposure to UV-illumination on
nano-TiO2 films. a, b, c – X-ray photoelectron spectra; d, e, f, – percentages of different carbon species.

[58,60,61] could be identified. In oleic and linoleic acid C 1s spectra
contributions from both, sp3 and sp2 carbons as well as from car-
boxylic group could be detected (see division of C 1s experimental
spectra into the sub-bands, Fig. 3b and c); in stearic acid the con-
tributions from sp3 carbon and carboxylic group could be identified
(Fig. 3a, 0 min condition). In case of stearic acid C 1s spectra, posi-
tion of the contribution from the carboxylic group is at slightly
higher energy at 288.6 ± 0.1 eV (Fig. 3a). In the C 1s spectra of stea-
ric and oleic acid the intensity of the contribution from carboxylic
group is suppressed due to the tendency of fatty acids to form ori-
ented monolayers [62,63] where the hydrocarbon chain is located
perpendicular to substrate plane, carboxylic group is located on the
substrate and the length of molecule is comparable to the escape
depth of photoelectrons [64]. Chemical shifts in the O 1s spectra
are smaller than respective shifts in C 1s spectra, also O 1s signal
connected with fatty acids was mixed with O 1s signal coming
from TiO2. Therefore O 1s signal was not possible to use for similar
analysis.
 U. Joost et al. / Journal of Photochemistry and Photobiology B: Biology 142 (2015) 178–185
As XPS measurements of photo-oxidized fatty acids were con-
ducted in ultra-high vacuum (pressure in the order of 10 10 mbar),
photo-oxidation products with low molecular mass and molecules
that were not absorbed on the nano-titania surface were removed
leaving behind fatty acids directly linked to the surface of nano-
TiO2 films. The latter enabled the monitoring of chemical changes
that occurred to fatty acids without the interference from other
photo-oxidation products.
XPS spectra of the fatty acids after their exposure to
UV-illumination on nano-TiO2 films (Fig. 3) suggested that
photo-oxidation of unsaturated and saturated fatty acids was dif-
ferent. Photo-oxidation of stearic acid did not induce any changes
in the structure of the fatty acid. Only decrease in sp3 carbons and
carbon in carboxylic groups (marked as OAC@O) was observed
(Fig. 3a). Thus, in case of this saturated fatty acid no other chemical
changes than shortening of the alkyl chain resulting finally in total
mineralization of the molecule was detected during photo-
oxidation.
However, photo-oxidation of oleic (Fig. 3b) and linoleic acid
(Fig. 3c) was different due to the radical reactions associated with
carbon double bond. In the C 1s spectra of oleic and linoleic acid a
shoulder related to CAO bond [58,60] appeared at 286.2 ± 0.1 eV
already after 1 min of UV-illumination. The appearance of CAO
bonds during photo-oxidation of unsaturated fatty acids can be
linked to the formation of peroxides, as is proposed by several
authors [32,34,65]. Formation of peroxides in oleic and linoleic
acids was most likely driven by OH radicals attacking a hydrogen
atom in RAH and by that, creating a carbon radical R. In the next
step, molecular oxygen is added to R creating a peroxyl radical
ROO, peroxyl radical abstracts a hydrogen from the RAH bond cre-
ating a lipid hydroperoxide ROOH. Each lipid hydroperoxide con-
tains one CAO bond between carbon and oxygen [34]. Formation
of CAO bond was relatively fast: during the first three minutes of
photo-oxidation the relative number of CAO bonds in oleic and lin-
oleic acid layers increased (Fig. 3(e) and (f)), and then started to
decrease as the total carbon composition decreased due to
photo-oxidation. After 3-min exposure the amount of CAO carbon
became similar to the remaining amount of sp2 carbon for both
oleic and linoleic acid layers indicating that the change in the
chemical composition of unsaturated fatty acids was quite exten-
sive. One OH radical can initiate a process resulting in peroxida-
tion of several fatty acids (radical chain reaction); thus, even very
low concentrations of OH radicals can cause significant oxidative
damage to the components of bacterial cellular membrane. The
time required for total photo-mineralization was similar for all
three fatty acids. After 10 min of exposure to UV-illumination on
nano-TiO2 thin film the peaks of all carbon compounds had disap-
peared in the XPS spectra suggesting total mineralization of fatty
acids.
As stearic, oleic and linoleic acids are the main components of
bacterial membranes [57,66] it can be suggested that the chemical
changes that were observed in those fatty acids upon exposure to
photo-activated nano-TiO2 thin film were also taking place in E. coli
cells. Thus, it is reasonable to suggest that already after short-time
exposure of E. coli to photo-activated nano-TiO2, unsaturated fatty
acids first change their chemical composition followed by mineral-
ization whereas saturated fatty acids just mineralize. Different
behaviour of saturated and unsaturated fatty acids during photo-
oxidation process has also been shown earlier; Leung et al. [14]
and Kiwi and Nadtochenko [34] showed that cell membrane’s sus-
ceptibility to TiO2 photo-oxidation has been linked to the degree of
unsaturation of fatty acid chains of phospholipids.
A recent report by Kubacka et al. [27] showed that the cell wall
and cell membrane composition and integrity were one of the main
biological processes that were affected by photo-activated nano-
TiO2 thin film in pathogenic bacterium Pseudomonas aeruginosa.
183
This statement is in line with our observation on damaged surfaces
of bacterial cells treated with photo-activated TiO2 thin films
(Fig. 2). We also propose that a halo surrounding the damaged bac-
terial cells that was visible on SEM images (Fig. 2) may reveal the
leakage of intracellular components. Thus, our study suggested that
the final result of antibacterial effect of photo-activated nano-TiO2
thin film was loss of the integrity of the main cellular outer barriers
and bacterial cell envelope.
4. Conclusions
This study investigated time-dependent antibacterial properties
of photo-activated nano-TiO2 thin films using two different biolog-
ical systems: viable bacterial cells (in vivo approach) and crucial
components of bacterial plasma membrane-fatty acids.
Total inactivation of bacteria on nano-TiO2 thin films after
20 min of UV-A irradiation confirmed the remarkable photoactive
nature of the nano-TiO2 and developed thin films. SEM investiga-
tions confirmed the results of the viability tests, showing that dam-
age to bacterial cell envelope occurred in the same time frame as
inactivation of bacteria. XPS analysis indicated that both, saturated
as well as unsaturated fatty acids commonly present in bacterial
plasma membrane, decomposed within 10 min of exposure to
photoactivated nano-TiO2 thin films. In addition, before complete
mineralization peroxidation of unsaturated fatty acids, as evi-
denced by the formation of CAO bonds, took place. Our results
indicated that fast and effective peroxidation and decomposition
of membrane fatty acids could be one of the factors contributing
to the morphological changes and leakage of the bacterial cell com-
ponents as observed under SEM, and ultimately, bacterial death.
Though in membranes fatty acids are bound to form hydrophobic
tails of phospholipids, their decomposition should be similar to
the decomposition of free fatty acids. As fatty acids are present
in most biological membranes it is likely that the UV-light-acti-
vated nano-TiO2 thin films described in this study are applicable
for removal of most of undesired organisms. Considering the fast
and efficient oxidative damage mediated inactivation of bacteria
on the nano-TiO2 thin films demonstrated in this study, our data
supports further development and application of nanosized TiO2
based novel surface coating materials.
Acknowledgements
Financial support by the Estonian Ministry of Education and
Research (target-financed themes IUT2-25 and IUT23-5), Estonian
Science Foundation (Grants ETF8561 and 8737), Estonian
Nanotechnology Competence Center (EU29996), ERDF projects
(‘‘IRGLASS’’ 3.2.1101.12-0027, ‘‘TRIBOFILM’’ 3.2.1101.12-0028,
‘‘Nano-Com’’ 3.2.1101.12-0010, ‘‘Mesosystems: Theory and
Applications’’ TK114, ‘‘High-technology Materials for Sustainable
Development’’ TK117), Graduate School ‘‘Functional Materials
and Technologies’’ (European Social Fund project 1.2.0401.09-
0079), Development Fund of University of Tartu, is gratefully
acknowledged. Markus Laars is gratefully acknowledged for
technical assistance.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014.
12.010.
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