Real Time, in situ Observation of the Photocatalytic Destruction of Saccharomyces cerevisiae Cells by Palladium-modified Nitrogen-doped Titanium Oxide Thin Film

Available online at ScienceDirect

ScienceDirectJ. Mater. Sci. Technol., 2014, -(-), 1e7

Real Time, in situ Observation of the Photocatalytic

Destruction of Saccharomyces cerevisiae Cells by

Palladium-modified Nitrogen-doped Titanium Oxide

Thin Film

Jingtao Zhang1), Qi Li1)*, Ronghui Li1), Jian Ku Shang1,2)

1) Environment Functional Materials Division, Shenyang National Laboratory for Materials Science,Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China

2) Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign,Urbana, IL 61801, USA[Manuscript received April 2, 2014, in revised form May 29, 2014, Available online xxx]

* Corresp23971211005-03JournalLimited.http://dx

Please

Palladium-modified nitrogen-doped titanium oxide (TiON/PdO) thin film was synthesized by the ion-beam-assisted deposition technique, which enabled a heavy nitrogen doping and the subsequent light absorptionextension to w700 nm for a better usage of the solar spectrum. Based on TiON/PdO thin film and a phasecontrast microscope, a micro-reaction chamber was developed, which allowed the simultaneous opticalexcitation of the photocatalytic thin film and the phase contrast image observation of cells in it. The real time,in situ observation of the photocatalytic destruction of Saccharomyces cerevisiae (S. cerevisiae), an essentialeukaryotic unicellular model of living cells, was conducted with this new observation technique, whichdemonstrated clearly that the photocatalytic destruction effect was much stronger than the photodamageeffect caused by the visible light irradiation alone in the disinfection process.

KEY WORDS: Ion-beam assisted deposition (IBAD); TiON/PdO thin film; Micro-reaction chamber; Visible-light; Photocatalytic destruction

1. Introduction

In 1985, Matsunaga and his coworker[1] reported the firstsuccessful photocatalytic microorganism disinfection with tita-nium dioxide (TiO2). Since then, extensive researches had beenconducted on TiO2-based photocatalysts for various microor-ganism disinfections[2e5]. It is generally believed that the pho-tocatalytic microorganism disinfection relies on the interactionbetween microorganisms and reactive oxygen species (ROS),such as hydroxyl radicals ð$OHÞ, superoxide ion ðO$�

2 Þ andhydrogen peroxide H2O2, generated by photocatalysts upon theiractivation from proper light illumination. So the photocatalyticmicroorganism disinfection is a general approach to disinfectvarious microorganisms, while many widely used antibioticsmay not be effective when they are used to disinfect

onding author. Ph.D.; Tel.: þ86 24 83978028; Fax: þ86 245; E-mail address: [email protected] (Q. Li).02/$e see front matter Copyright� 2014, The editorial office ofof Materials Science & Technology. Published by ElsevierAll rights reserved..doi.org/10.1016/j.jmst.2014.08.003

cite this article in press as: J. Zhang, et al., Journal of Materials Scien

microorganisms with antibiotic resistance[6]. For example, it hadbeen demonstrated that the antibiotic resistant bacteria such asmethicillin-resistant Staphylococcus aureus could be disinfectedeffectively by TiO2

[7]. This technology could also degrade toxinsproduced by bacteria and cyanobacteria[8,9], and produce nodisinfection by-product (DBP). Thus, this technology may offera DBP-free alternative for the control of pathogens for envi-ronmental applications with further development.Due to the high physical and chemical stability, low cost and

good photocatalytic efficiency, TiO2 is considered as one of thebest photocatalyst candidates suitable for energy conversions andenvironmental remediation[10e13]. However, the photocatalyticcapability of TiO2 is limited to ultraviolet (UV) illumination dueto its relatively wide band gap at w3.2 eV, so only a smallportion (3%e4%) of the whole solar spectrum could be utilizedby pure TiO2. Thus, it is of great interest to develop photo-catalysts that could yield high reactivity under visible light thataccounts for around 45% of solar spectrum. With extensiveresearch efforts in the last several decades, TiO2 and its modifiedforms had been successfully applied to microorganism disin-fection in water and air purification[14,15], hospital surfacedisinfection[16], and food processing[16,17]. Although excellent

ce & Technology (2014), http://dx.doi.org/10.1016/j.jmst.2014.08.003

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2 J. Zhang et al.: J. Mater. Sci. Technol., 2014, -(-), 1e7

disinfection effects on various microorganisms had beendemonstrated, no report was available for the real time, in situobservations of the photocatalytic disinfection process due to thelack of proper observation techniques.In this study, we synthesized palladium-modified nitrogen-

doped TiO2 (TiON/PdO) thin film by the ion-beam-assisteddeposition (IBAD) technique. By this technique, nitrogencould be heavily doped into TiO2 thin film for a much strongerabsorption in the visible-light range. The morphology and crystalstructure of these thin films were characterized by scanningelectron microscopy (SEM) and atomic force microscopy (AFM)and X-ray diffraction (XRD) techniques, respectively. Thechemical states of nitrogen and palladium in these TiON/PdOthin films were investigated by X-ray photoelectron spectroscopy(XPS). A micro-reaction chamber was constructed, and anobservation technique was developed to reveal the real time, insitu photocatalytic disinfection process of Saccharomyces cer-evisiae cells by TiON/PdO thin film under visible light illumi-nation. The real time, in situ observation demonstrated clearlythat the photocatalytic destruction effect was much stronger thanthe photodamage effect caused by the visible light irradiationalone in the disinfection process.

2. Experimental

Fig. 1 Schematic illustration of the micro-reaction chamber constructedto allow the photocatalytic treatment and the optical observationto occur simultaneously.

2.1. Thin film fabrication

The IBAD system used in this study consisted of a 3-kWelectron-beam evaporator, 3-cm-diameter Kaufman-type ionsource, and a substrate holder with a heater made of tantalum. Aquartz-crystal monitor was used to control the film thickness aswell as the deposition rate. Microscope glass slides were used assubstrates. They were ultrasonically cleaned in acetone andmethanol baths successively, and blow dried under nitrogenpurge. Prior to deposition, the substrate was cleaned by 1000-eVArþ ions for 5 min to remove surface contamination. Nitrogen-doped titanium oxide with palladium oxide modification wasdeposited by electron-beam evaporation of TiO2/PdO pellet andsimultaneously bombarding the growing film with nitrogen ions.TiO2/PdO pellet was prepared by thoroughly mixing titanium(IV) oxide (anatase, 99.9%, Alfa Aesar, USA) and palladium (II)oxide monohydrate (99.9%, Alfa Aesar, USA) at the Pd/Tiatomic ratio of 0.5%, followed by dry pressing, isostatic press-ing, and calcination at 750 �C for 4 h successively to removesolvent and obtain a dense structure. During the deposition, thenitrogen partial pressure was controlled at a stable value in therange of 1.33 � 10�2e3.99 � 10�2 Pa (1.0 � 10�4e3.0 � 10�4 Torr). The working pressure and the deposition ratewere adjusted to regulate N incorporation. The deposition wascarried out at a substrate temperature of 450 �C to crystallize thinfilm samples.

2.2. Thin film sample characterization

The crystal structure of the thin film sample was analyzed bythe glancing angle X-ray diffraction (GAXRD) using a D/MAX-2004 X-ray diffractometer (Rigaku Corporation, Tokyo, Japan)with Ni-filtered Cu (l ¼ 0.15418 nm) radiation at 56 kV and182 mA. The morphology of these films was examined by SEMand AFM. SEM observation was made with an SEM LEO SU-PRA 35 (Carl ZEISS Inc., Oberkochen, Germany) at an accel-eration voltage of 20 kV. Before imaging, the samples were

Please cite this article in press as: J. Zhang, et al., Journal of Materials Scien

sputtered with gold for 120 s to enhance their surface conduc-tivity (Cressington Sputter Coater 208HR, Cressington ScientificInstrument Ltd., Watford, U.K.). AFM observation was madewith an AFM PicoScan 2500 (Agilent, Palo Alto, America). TheUV-Vis spectra of these films were measured on a UV-2550spectrophotometer (Shimadzu Corporation, Kyoto, Japan).Compositions of these samples were analyzed by an ESCA-LAB250 XPS (Thermo Fisher Scientific Inc., Waltham, MA)with an Al K anode (1486.6 eV photon energy, 300 W). To getrid of contaminants on the thin film surface, Arþ ion sputteringwas conducted for 60 s before the XPS survey.

2.3. Cell culture and real time, in situ observation of yeast cellsunder photocatalytic treatment

Wild type S. cerevisiae cells were kindly provided by Prof.Xiaoping Zhang, College of Resources and Environment,Sichuan Agricultural University. Yeast cells (2% v/v final) wereinoculated into yeast extract/peptone/dextrose (YPD or YEPD)media. The cells were incubated on a rotary shaker (KYC-100C,Shanghai CIMO Medical Instrument Manufacturing Co., Ltd.)under 120 r/min and 26 �C for 12 h. After overnight culture, cellswere harvested by centrifugation at 4000 r/min for 5 min at roomtemperature, washed twice with a phosphate buffer solution(PBS, pH 6.0), and diluted to a cell suspension (ca. 107 CFU/ml)in PBS before its use in photocatalytic inactivation experiments.All solid/liquid materials had been autoclaved for 30 min at121 �C before use.Nikon 80i upright research-grade fluorescence microscope

with phase contrast condenser was used for the real time, in situobservation of yeast cells under photocatalytic treatment. The U-LH100HG 100 W mercury lamp was used as the light source,and the BV-2A filter block was used to provide visible lightillumination with 400e440 nm wavelength. The light intensitystriking the cells was at ca. 25 mW/cm2, as measured by the FZ-A optical radiometer. Overnight-grown yeast cells were washedand resuspended in PBS solution to ca. 107 CFU/ml prior to thephotocatalytic treatment. TiON/PdO thin film was used as thephotocatalyst in the real time, in situ observation. 20 ml cellsuspensions were dropped onto the TiON/PdO thin film, then aquartz cover slip was put on the suspension drop and theredundant suspension was swabbed. The yeast cell suspensionwas sealed by applying microscopic oil along the cover slip andthe TiON/PdO thin film to avoid the solution loss and evapo-ration during the experiment. Thus, a micro-reaction chamberwas constructed to allow the photocatalytic treatment and theoptical observation to occur simultaneously (see Fig. 1). Atregular time intervals, images were recorded through the ColorCooled Digital Camera Head (DS-Fi1c) with a 100 � oil im-mersion lens. Control runs were also carried out when no pho-tocatalyst was on the glass slide. The obtained images were


J. Zhang et al.: J. Mater. Sci. Technol., 2014, -(-), 1e7 3

analyzed by the imaging software NIS-Elements (version 3.13,Nikon Company).

3. Results and Discussion

3.1. Characterization of TiON/PdO thin film

The XRD pattern of TiON/PdO film corresponds to an anatasephase as shown in Fig. 2(a), and there is no rutile phase observedin this sample. No obvious XRD diffraction peak of PdO wasobserved in the XRD pattern, which may be due to the lowatomic ratio of Pd/Ti (0.5%) and the signal noise from the glassslide beneath the thin film sample. The semi-quantitativechemical composition of the sample was analyzed by XPS.Arþ ion sputtering was adopted to remove the surface contam-inations on the sample to explore the chemical state of elementsin the thin sample. Fig. 2(b) shows the representative XPS sur-vey spectrum of TiON/PdO thin film, which confirms the pres-ence of Ti, Pd, O, and N in the sample. The relative elementcomposition ratio was determined by multiplex high-resolutionscans over Ti 2p, Pd 3d, O 1s, and N 1s spectral regions, andthe average atomic ratios of N/Ti and Pd/Ti were w74.7%and w0.3%, respectively. The high nitrogen concentrationdemonstrated that nitrogen could be heavily doped into the thinfilm samples by the IBAD technique[18]. Fig. 2(c) and (d) showshigh-resolution XPS scans over N 1s and Pd 3d spectral regions,respectively. A strong peak at w396 eV was observed in the N1s XPS spectrum, which was originated from the substitution ofnitrogen for lattice oxygen by forming TieN bonds[19]. Thebinding energy of Pd 3d 5/2 was w336.8 eV, which suggestedthat Pd additive existed as PdO in the TiON/PdO thin filmsample. Fig. 2(e) and (f) shows the SEM and AFM images of theTiON/PdO thin film surface, respectively, which demonstrates

Fig. 2 (a) XRD pattern of TiON/PdO thin film (A: anatase phase), (b) represresolution XPS scan spectrum over N 1s and Pd 3d spectral region oTiON/PdO thin film, respectively.


that the TiON/PdO thin film has a dense surface structure and thegrain size is w10e50 nm.

3.2. Optical properties of TiON/PdO thin film

Fig. 3(a) shows the optical properties of TiON/PdO thin film,compared with that of TiO2 thin film, which were investigated bymeasuring the optical absorbance spectrum with a UV-Vis spec-trophotometer in the transmission mode. The TiO2 thin film showsits characteristic spectrum with the major absorbance in the UVrange, whereas the TiON/PdO thin film shows a clear shift ofabsorbance into the visible-light range (l > 400 nm). The heavydoping of substituted nitrogen could largely increase the visiblelight absorption of TiO2

[20,21]. Fig. 3(b) shows the Tauc plot(ðahvÞ0:5 versus hv)[22] constructed fromFig. 3(a), which shows thelinear Tauc region just above the optical absorption edge to deter-mine the semiconductor band gap shown by the dotted lines. Theband gap of the TiON/PdO thinfilm isw2.27 eV,which agreeswellwith their good visible light absorbance, while that of the TiO2 thinfilm is w3.26 eV. The band gap change of TiON/PdO thin filmcould be attributed to both the nitrogen-doping[21] effect and thetransition metal modification enhancement from SPR effect[23].

3.3. Real time, in situ observation of photocatalytic destructionof yeast cells under visible light irradiation

The excellent photocatalytic disinfection effect of TiON/PdOphotocatalyst on various microorganisms had been demonstratedin our previous work[24e28]. However, no report was available onthe real time, in situ observation of its photocatalytic destructionprocess of microorganisms. In this work, we designed a micro-reaction chamber (see Fig. 1), which allowed the simultaneousphotocatalytic reaction and phase contrast microscopic

entative XPS survey spectrum of TiON/PdO thin film, (c) and (d) high-f TiON/PdO thin film, respectively, (e) and (f) SEM and AFM image of


Fig. 3 (a) Optical absorbance (in terms of KubelkaeMunk equivalent absorbance units) of TiON/PdO thin film, compared with that of TiO2 thin film,(b) Tauc plots constructed from (a).


observation to happen. Thus, a real time, in situ observation ofthe morphology change of S. cerevisiae cells during the photo-catalytic destruction progress could be achieved.Fig. 4 shows the representative phase contrast images of

S. cerevisiae cells under visible light irradiation with no photo-catalytic treatment at different time intervals till 22 h. At theinitial moment of the irradiation, the vacuoles in S. cerevisiaecells are relatively small and not very evident. After being irra-diated for 2 h, vacuoles in all of these S. cerevisiae cells enlargeand become distinct. This observation is consistent with a pre-vious report by Logg et al.[29], in which enlarged/more distinctvacuoles were found for S. cerevisiae cells under visible lightillumination (l ¼ 470 � 20 nm). After being irradiated for alonger time, however, both vacuoles and cells begin to shrink,which could be attributed to the photodamage[30,31]. Forexample, both vacuoles and cells shrink obviously after 11 hvisible light illumination. However, the existence of vacuoles in

Fig. 4 Real time, in situ phase contrast observations of S. cerevisiae cells undintervals (t from 0 to 22 h). The cell a was selected for the quantitativethe process.


these cells indicated that they did not break. At this stage, thevisible light illumination was shut down, and the cells were keptin dark overnight. No significant change was observed on bothvacuoles and cells after being kept in dark overnight, whichfurther demonstrated that the photodamage observed was causedby the visible light illumination. When the visible light illumi-nation was resumed, both vacuoles and cells continued to shrinkwith the further increase of the illumination time. After 22 h,some cells finally lose their vacuoles, indicating the loss of theirviability duo to the photodamage.Fig. 5 demonstrates the representative phase contrast images

of S. cerevisiae cells under visible light irradiation with photo-catalytic treatment by TiON/PdO thin film at different time in-tervals till 22 h. Compared with that without photocatalytictreatment, different morphological change pattern is found forthese S. cerevisiae cells under photocatalytic treatment. Themorphological change of these S. cerevisiae cells is largely

er visible light illumination (400e440 nm, 25 mW/cm2) at different timeanalysis of the cell’s apparent vacuolar and cellular areas changes during


Fig. 5 Real time, in situ phase contrast observations of S. cerevisiae cells under photocatalytic treatment by TiON/PdO thin film with visible lightillumination (400e440 nm, 25 mW/cm2) at different time intervals (t from 0 to 22 h). The cells b, c, and d were selected for the quantitativeanalysis of their vacuolar and cellular areas changes during the process.


accelerated when the photocatalytic treatment is present. Afteronly being photocatalytically treated for half an hour, vacuoles inthese S. cerevisiae cells enlarge and become distinct. Then, mostof these S. cerevisiae cells lose their vacuoles within only 2 hphotocatalytic treatment, while some S. cerevisiae cells can keeptheir vacuoles even after 22 h visible light illumination when nophotocatalyst is present. This observation indicated that thesecells were broken due to serious damages caused by the photo-catalytic treatment, while photodamages caused by only visiblelight illumination were much milder. With the further increase ofthe photocatalytic treatment time, all cells lose their vacuoles andthey continue to shrink. The visible light illumination was shutdown after 11 h treatment, and the cells were also kept in darkovernight. No significant change was observed on these cellsafter being kept in dark overnight, which suggested that theleakage of biomolecules inside these cells was not the majorcontributor to their large shrinkage. When the visible light illu-mination was resumed, these cells continued to shrink with thefurther increase of the photocatalytic treatment time. After 22 h,all these cells become much smaller compared with their originalsize, which should come from the photocatalytic biomoleculedegradation of these cells. These S. cerevisiae cells can remainpartial existence even after 22 h photocatalytic treatment, which


indicates that they have much stronger oxidation resistance thansome other microorganisms we studied before[24e27]. Thisobservation is consistent with a previous report by Kogan et al.,in which they found that the (1/ 3)-b-D-glucan (the componentof the yeast cell wall) had the antioxidant properties[32]. In ourprevious work, we demonstrated that TiON/PdO had a highlyeffective photocatalytic disinfection of Fusarium graminearummacroconidia under visible light illumination and the disinfec-tion mechanism was due to the cell wall/membrane damagecaused by the attack from ROSs[28]. The photocatalytic disin-fection of S. cerevisiae cells followed the same mechanism.These images were further analyzed by the imaging software

NIS-Elements to quantitatively describe the morphologicalchanges of S. cerevisiae cells under different treatments. Four cellswere chosen for the analysis of their apparent vacuole and cellularareas, respectively. Cell a was chosen from S. cerevisiae cellsunder only visible light illumination (see Fig. 4), while cells b, c,and d were chosen from S. cerevisiae cells under photocatalytictreatment (see Fig. 5). Fig. 6(a) demonstrates the relative apparentvacuole area changes of these four cells with increasing treatmenttime. They all increase firstly and then decrease. For cell a, itsapparent vacuole area increases up to w169% after 4 h visiblelight irradiation, then gradually decreases to w80.2% after 22 h


Fig. 6 Relative changes of apparent vacuolar (a) and cellular (b) areas for S. cerevisiae cell a, b, c, and d as labeled in Figs. 4 and 5.


visible light irradiation. For cell b, c, and d, their apparent vacuoleareas increase up to w204.9%, w155.4%, and w151.4%,respectively, after 40 min photocatalytic treatment, then largelydecrease to zero after only 70 min photocatalytic treatment.Fig. 6(b) demonstrates the relative apparent cellular area changesof these four cells with increasing treatment time. They also showan initial increase followed with a gradual decrease. For cell a, itsapparent cellular area increases up to w107.4% after 2 h visiblelight irradiation, then gradually decreases to w77.4% after 22 hvisible light irradiation. For cell b, c, and d, their apparent cellularareas increase up to w121.1%, w106.5%, and w109.4%,respectively, after only 1 h photocatalytic treatment, then decreaseto w25.7%, w18.7%, and w29.5%, respectively, after 22 hphotocatalytic treatment. The much higher relative changes oftheir apparent vacuole and cellular areas with photocatalytictreatment demonstrated that the photocatalytic destruction effectwas much stronger than the photodamage effect, which was themajor contributor to the disinfection of these S. cerevisiae cells.

4. Conclusion

TiON/PdO thin film was synthesized by the IBAD technique,which demonstrated a largely enhanced light absorption tow700 nm, covering most of the visible light region. A micro-reaction chamber was built based on TiON/PdO thin film anda phase contrast microscope for the real-time, in-situ observationof photocatalytic disinfection process of S. cerevisiae cells. After22 h photocatalytic treatment, the apparent cellular area of thesecells was about one quarter of their original size, and theirvacuoles disappeared. The much higher relative changes of theirapparent vacuole and cellular areas with photocatalytic treatmentdemonstrated that the photocatalytic destruction effect was muchstronger than the photodamage effect. This real-time, in situobservation technique could be further applied to examine thechanges of microorganisms during the photocatalytic treatment,beneficial to the understanding of their photocatalytic disinfec-tion process and mechanism.

AcknowledgmentsThe experimental assistance on S. cerevisiae cell culture by

Ms. Mian Song and Ms. Shuang Jiao was greatly appreciated.This study was supported by the National Natural ScienceFoundation of China (Grant No. 51102246), the KnowledgeInnovation Program of Institute of Metal Research, ChineseAcademy of Sciences (Grant No. Y0N5A111A1), the Youth


Innovation Promotion Association, Chinese Academy of Sci-ences (Grant No. Y2N5711171), and the Scientific ResearchFoundation for the Returned Overseas Chinese Scholars, StateEducation Ministry, China.

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Documents

Real Time, in situ Observation of the Photocatalytic Destruction of Saccharomyces cerevisiae Cells by Palladium-modified Nitrogen-doped Titanium Oxide Thin Film