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TESTING METHODS FOR ANTIMICROBIAL ACTIVITY OF TiO2PHOTOCATALYST

Siniia L. Markov, Ana M. Vidakovit*

UniversityofNovi Sad, Faculty ofTechnology Novi Sad,Bulevar cwaLazaral,21000 Novi Sad, Serbia

In recent years, a lot of commercial TiO2 photocatalyst products haye been develo-ped and extensively studied for prospective and safe antimicrobial application in dailylife, medicine, laboratories, food and pharmaceutical industry, waste water treatmentsand in development of new self-cleaning and antimicrobial materials, sudaces andpaints. This paper reviews the studies published world'vrtide on killing microorganisms,methods for testing the antimicrobial activity, light sources and intensities, as well as

calculation methods usually used when evaluating the antimicrobial properties of theTiOrbased products. Additionally, some strengths and weaknesses of the availablemethods for testing the antimicrobial activity of TiO2 photocatalyst products have beenpointed out.

KEY WORDS: antimicrobial activity, titanium dioxide, light sources, calculations,testing methods

INTRODUCTION

Titanium(IV) oxide, TiO2, commonly known as titania or titanium dioxide, is natu-rally occurring oxide of titanium, which exists in three forms: rutile, anatase and brookite.All these forms are usually linked to minerals such as qurarlz, tourmaline, barite, hematite,silicates, feldspar, chalcopyrite, and sphene (1). Scientific studies have approved that ana-tase appears to be the most photoactive and stable for widespread applications, whereasrutile is photocatalytically less active, although it expresses photoactive selectivity in so-me cases (2). TiO2 powders have been used as a white pigment from ancient times. Also,it is widely used to provide whiteness in lacquers, plastic and paper. In addition, nanopar-ticles oftitania are also used as an opacifier in textiles, leather, glass and porcelain ena-mels. Moreover, TiO2 is a permitted dye in the food indusfiy as 8171, and it is commonlyfound in pharmaceuticals, cosmetics, and skin care products (3).

Since the magnificent discovery of photocatalytic cleavage of water on TiOz electro-des in 1972 by Fujishima and Honda (4), this research field has received greater aware-ness in the recent years. The breakthrough work of Matsunaga et al. in 1985, reporting

* Coresponding author: Ana M. Vidakovid, University of Novi Sad, Faculty of Teohnology Novi Sad, BulevarcuaLazara 1, 21000 Novi Sad, Serbi4 e-mail: anav@nns.ac.rs

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the application of TiO2 photocatalysis for the deskuction of Lactobacillus acidophilus,Saccharomyces cerevisiae and Escherichia coli using Pt-loaded TiOz, established for thefirst time antimicrobial properties of TiO2 (5). Since then, research efforts have been ma-de in order to improve the efficiency of the TiOz photocatalysis by doping it with variousmetals (6,7) andnonmetals (8).

There are many conf,trmed advantages of using photocatalytic methods. Firstly, allreactions are carried out under ambient temperature and pressure. Additionally, minerali-zation of organic compounds is completed in an environmentally friendly way withoutcreating any secondary pollutants, as final reaction product. Also, TiO2 catalyst is charac-terized by non-toxicity, high stability and low cost (9). Because of the numerous benefits,TiO2 as a photocatalyst has been extensively studied, and its applications in medicine,water treatment, food and pharmacy have been reported (10).

Although there are plenty of TiO2 coated products on the market with antimicrobialactivity, there has been no standard method, except Japanese standard JIS 22801:2000(11), with good experimental basis suggested for testing antimicrobial activity. For thisreason, researchers use different experimental sefups, light sources, and calculations ofthe antimicrobial activity.

The current review will focus on four important segments of studying and testing theantimicrobial activity of TiOz photocatalyst: (i) photokilling mechanisms, (ii) experimen-tal setup, (iii) light sources and intensity, and (iv) calculation methods.

Photokilling mechanisms

The irradiation of TiO2 with ultraviolet light, whose wavelength is less than 385 nm,leads to generation ofan election-hole pair on the TiO2 surface. The electrons and holesreact on the surface and convert water and oxygen into reactive oxygen species (RoS)guch as hydroxyl radicals (olD, superoxide io-n (o;) and hydrog; peroxide (i{ror)(12). The primary oxidizing agents are short-living hydroxyl radicali that can bind to thesurfacg. Those hydroxyl radicals have significantly short life span (10-e), which preventsthem from diffusing to a long distance. Consequently, only microbial cells that adhere tothe surface of the TiO2 catalyst may react with the hydroxyl radical that usually causesthe loss of membrane integrrty (13). In tum, superoxide ions are long-lived, although dueto the negative charge, they cannot penetrate into the cell membrane. The penetraiion ispossible by hydrogen peroxide (14).

In 1988, three years after publishing the first results of killing microbial cells bycontact with the TiO2-Pt catalyst upon illumination with near UV light, the su-" group ofscientists successfully constructed a practical photochemical device in which TiO, pow-der was immobilized on an acetylcellulose membrane (15). An E. coli cell suspensionfollowing through the device was completely killed; therefore, Matsunaga and coworkers(5,15) proposed the first killing mechanism. They believed that direit photochemicaloxidation of intracellular coenryme A to its dimeric form was the reason oithe decreasesin the respiratory activities that led to cell death. ln the next years, Saito et al. (16) pro-posed that TiO2 photochemical reaction leads to the disruption of the cell membrane-andcell wall of Streptococcus sobrinus, caused by leakage of intracellular K* ions. On theother hand, Sunada et al. (17) recommend that the primary cause of the cell death during

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the TiO2 photochemical reaction may be the destruction of the E. coli endotoxin, an

integral component of the outer membrane. If the TiO2particles are sufficiently small,they can penetrate into the microbial cell, where the photocatalytic process takes place.

Also, free TiO2 particles can attack intracellular components directly if it is enhancedwith UV irradiation. This may lead to physical and chemical damages of DNA and RNA,causing the conversion of the pyrimidine and purine bases to carbon dioxide, ammonia

and nitrate ions (18,19). Although there are at least three hypotheses of the antimicrobialeffect of TiO2 photocatalytig reaction, that mechanism is still to be proved.

EXPERIMENTAL

Heterogeneous photocatalysis, an Advanced Oxidation Technology that uses lightsources, commonly lIV, and TiO2, has emerged in the last decade as an innovativemethod of disinfection. The antimicrobial activity of TiO2 has been assayed in severalbacteria and yeasts including Escherichia coli (5,20-24), Lactobacillus acidophilus (5),Bacillus subtilis, Pseudomonas putida, Staphylococcus aureus, Listeria innocua (refe-rences in (23), Enterobacter cloacae Q5), Candida albicans (24), and Saccharomycescerevisiae (5). On the other hand, only a few studies have explored the ability of TiOzphotocatalysis to inactivate resistant microbial forms such as fungal spores, protozoan

cysts and oocysts or bacterial spores. The main reasons for this are the thickness andstructure complexity of their wall. However, Maneerat and Hayata (26)have reported theantifungal effect of TiO2 against Penicillium expqnsum in fruit root and proposed thepotential treatment for post-harvest disease control. Other researchers reported about theresistance of fungi spores including Fusarium sp. and Aspergillus niger Q7,28,29). Zhaoand coworkers (30) reported photocatalytic inactivation of Bacillus cereus spores. Also, ithas been confirmed that TiOz photocatalyst has antiviral activity, which is very importantfor the application of the photocatalysts in hospitals (20). Although the antimicrobialproperties of the TiO2 photocatalyst have been established, it is diffrcult to compareresults among authors due to different experimental setups. Different experimental setupsare a consequence of the absence of a universal standard method with good practicalsettings.

Generally, in the available literature there are at least three methods for testing theantimicrobial efficiency of TiOz photocatalyst (17,20,24,31). The most used methods arethin-fikn method and adhesion-test method with hlm, while the inhibition-zone methodhas been recently used.

During studying the antimicrobial effect of the TiOz photocatalyst, Sunada et al.(17,32) have mainly used the thin film method. TiO2 thin films were prepared byconventional dip-coating technique on silica-coated soda-lime glass plate. The coatedglass was placed on a Petri dish and 150 pl of the E. coli cell suspension was applicatedonto it. The system was put in a chamber and inadiated upon Pyrex window. In order tomaintain the humidity, l0 ml of water was poured below the Petri dish (Figure 1a). Thisgroup of scientists has obtained the complete sterilization within one hour of the inadia-tion. In the absence of TiOz, the UV irradiation caused only 50 o/o sterilization in 4 hours.The same experimental setting has been extensively used in the next years (20,23).

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The same group of workers modified the previous system in order to improve it (17,

32). Namely, they separated 2 ml of the E. coli cell suspension from the TiOz surface by aporous 50 pm thick PTFE membrane (Figure 1b). Also, the complete system was

irradiated from below. According to the obtained results, a similar antimicrobial efficien-cy was observed as in the system without the membrane. It is necessary to emphasize thatthe'OH reactive species are probably deactivated before traversing the 50 pm thickness,

due to their short life (13). Therefore, other active oxygen species (Oz'- and H2O2) are

responsible for the sterilization in the membrane system (12).

'totry-|.

ffi"&.

'tgq4dd-

&-J&fudl

Figure 1. Schematic illustration of the inadiation system for antimicrobial testing of thephotocatalyst: thin fikn method (a) Petri dish system and

(b) PTFE membrane-separated system

The adhesion test with film is a newly developed method by Kim and coworkers (31).They placed the photocatalytic product (TiO, coated glass, paper and plastic on a Petridish. Afterwards, 0.5 ml of the E coli cell suspension was placed onto them. The adhe-sion film was placed on the suspension in order to facilitate the attachment of the micro-organism to the TiOz surface. Everything was covered wi*r the Pefi dish lid in order tomaintain the humidity at more than 90 %. In the work, three types of the adhesion filmwere used: polyethylene (PE), polypropylene (PP) and acrylic. The Black Light Blue(BLB) lamps were used for light irradiation and positioned above the Petri dish (Figure2). The antimicrobial efficacy of TiO2 coated glass, paper and plastic was determined byadhesion film method after 3 hours of irradiation, and it was 99.5 oh,99.8 %o and 60 Yo,

respectively. Also, the PP frlm was determined to be optimal adhesion film for thismethod. The adhesion film method is relatively new, although its application has beenreported (31,33).

Two groups of researchers Q9,34) employed the inhibition zone method in order totest the antimicrobial activity of the TiOz-coated products upon UV irradiation. Theinhibition zone method, also called Kirby-Bauer Test, is usually used clinically tomeasure antibiotic resistance, as well as industrially in order to test the ability of solidsand textiles to inhibit microbial growth. In this method, sterilized culture media is pouredin Petri dishes and, after solidification of the media the suspension of test microorganismis, spread on the plate using an L-rod. The TiO2 coated products are placed into the Petri

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dishes with the coated zone on the culture media and incubated. After the incubationperiod, diameter of the inhibition zone (the zone without microbial growth) is measured(Figure 3) (29,34).

:r--"-- ,![Eliryd

Figure 2. Schematic illustration of the photocata\ttic TiO2 adhesion test method

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wy.. ii$q$.irqi4qqrsryB!ry

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Figure 3. Inhibition zone method for antimicrobial testing ofphotocatalytic activity of TiO2

Light sources and intensity

\n 1877, Downes and Blunt (35) discovered by chance that sunlight could kill bac-teria. They noticed that sugar water placed on the window remained cloudy in the night,while in the sun the water became clear. By microscopy, they determined that the numberof bacteria was significantly higher during the night than during the day. In 1g92, Mar-

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shall Ward demonstrated that the bactericidal actions were from the ultraviolet part of thelight spectrum. Since then, the application of UV light is widespread in many areas suchas: medicine, laboratory sterilization, food and pharmacy industry, etc. (10,36).

Photocatalysis, especially with TiO2, is a modern method for inactivating microbes onsurfaces. In order to manifest its photocatalytic and antimicrobial effect, TiOz requireslight as an excitation source. Ifthe photocatalysis is used indoor, it is necessary to take allthe protective steps. According to The American Conference of Govermental IndustrialHygiensts (ACGIH) for safety indoor application, UVA (ultraviolet radiation at 320-400nm) exposure must be less than l0 Wm2 for periods lasting more than 1000 s (30).

Because of the ACGIH requirements, the light irradiance effect on photocatalysis isvery important. Rincon and Pulgarin reported about an effect of uvA and uvA/Tio2inactivation (37). Namely, the study showed linear trends for the UVA inactivation aloneand nonlinear trends for combined inactivation with UVA and photocatalyst. Based onthe obtained results, they first suggested the presence of an optimum between the lightintensity and duration of irradiation (37). Another explanation was proposed by Zhao etal. (30), who considered the generation of ROS by the photocatalyst upon UV irradiation.when wA irradiation is low, the generation rate of RoS by UVA is low as well. Theincrease in the UVA irradiance increases the energy input and the rate of ROS genera-tion. On the other hand, the radical generation by photocatalysis requires very low energyinput, and is therefore predominant at low UVA inadiation. When the UVA inadiation ishigh, the effectiveness ofthe photocatalysis is usually reduced (30). Therefore, Kim et al.(31) determined the optimal light intensity and irradiation time for testing the antimicro-bial activity of Tioz photocatalysis to be 1.0 mWcm2 and 3 hours, respeitively. Rajsgo-pal et al. (37) also reported that the light intensity affects not only the photoinactivationrate, but also the behavior of bacteria after stopping irradiation. Namely, after the inadia-tion of TiO2 coated samples, the number of bacteria keeps decreasing in the dark. Never-theless, the recovery of bacteria is observed on the samples without TiO2. Also, inactiva-tion of the bacterial cells on the samples without catalyst addition was more affected bythe increase in the intensity as compared to the photocatalytic system (37).

However, one of the biggest problems in this area is the presence of non-comparableresults of antimicrobial effect among authors. Different authors use different microorga-nisms, photocatalysts, light sources, irradiation intensities and time, as well as distancesbetween the samples and light sources during testing. A choice of experimental condi-tions of some researchers is presented in Table 1.

Testing the antimicrobial activity of TiO2 photocatalysts is performed using at leastfive different light sources (Table l). Additionally, the irradiation intensity is in the rangefrom 0.1 to 30 Wm2, while the irradiation time varies from 0 to 1440 minutes. Also, blgoversight of the experiments is the distance between the tested samples and the lightsource, which is reported only in a few papers (Table l). Moreover, the number of lightsources is different from paper to paper, and it varies from one to sixteen (23-25,30-3 l).In the future, all these parameters have to be optimized and even standardized in order toallow the work to be repeated by others.

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Table 1. Chosen experimental light settings for testing the antimicrobial efficiency of' TiOz-basedphotocatalysts

Test microorganism Photocatalyst LightIrradiationintensitv(tlr'/-2i

lrradiationtime(minl

Distance(cm) Ref.

Bacterial biofilm Tio. Polvchromatic 250- 2-t0 15 (37\E. coli (ATCC 8739\ Desussa P25 WA 0.1 0-360 (31)

E. coli (NCC25922),5.aureus. P. outida. L. innotn Degussa P25 UVA g 30-300 l0 (23)

B. cereus (NICC 2 Deeussa P25 Solar 30 150-330 (30)E. coli K-12 Deeussa P25 WA 8 30 (22\P. exoansum TiOr UVA 0.1 1) (26\

E. coli Ae,O/TiON Metal-halogen 0.16 30 (38)E. cloacae Degussa P25 TIVA 0.55 40 (2s\

P. aerusinosa Tio? UVA 0.08 330-1440 33 (39)E. coli, P. aeruginosa, S.

aureus, S. cerevisiae, C.albicans, A. niser

Tio2 Sodium 0.04' 240 l0 (28)

S. aureus, S. tlexneri, A.bautnannii cnio2 lncandescent 9 40 5 (40)

E. coli, P, aeruginosa, S.

aureus, E. faecium, C,albicans

Degussa P25 UVA 2 x 0.015' 60 (24)

Total flux (kW)

Calculation methods

Generally, both qualitative and quantitative assessments of the antimicrobial efficacyof the Tio2-based photocatalysts have been performed. A qualitative estimation of theantimicrobial efficacy of the TiO2-based photocatalyts is obtained by comparison of thePetri dishes inoculated with the irradiated and non-irradiated suspension ofthe test micro-organism taken from the coated surface ofthe photocatalyst (37). Also, Chung et al. (41)srggested comparison of the inoculated Petri dishes after testing samples with andwithout TiO2.

The same group of researchers employed IIS 22801:2000 as a standard to test theantimicrobial efficacy (40,41). According to the Japanese standard, the results of a quan-titative evaluation of the antimicrobial efficacy should be calculated as follows (l l):

Na=Cax[oxYo Il]R: [og(Ns/Na) - log(Nq/Na)] : [log(Ns/Ng)] 12)

where: Na - the number of viable bacteria after inoculation, Cr - the number of bacteriacolonies, Da - the fold of dilutioh, ve - the volume (ml) of the dilute bufler, R - antimic-robial activity, N6 - the number of viable bacteria of the uncoated sample after irradia-tion, Ns - the number of viable bacteria of the coated sample after irradiation.

This kind of qualitative evaluation of the antimicrobial activity is performed by manyresearchers with or without small modifications (24,39,41). An obvious oversighi of thlproposed calculation is not taking into account the number of viable bacteria after inocu-

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lation, Na (gq. (Z)). This parameter is very important to get an objective picture of thebacterial number reduction in all steps of the experiment.

Pal et al. (42) proposed the bacterial inactivation efficiency followed first order ki-netics with respect to bacterial colony count (N) which is shown by Eq. (3):

ln(N/No)= -kt t3l

where: N - the number of CFUs (Colony-forming units) after inadiation for t min, No -the number of CFUs at 0 min, k - the inactivation rate constant, Nt/No - the survival ratio.The survival ratio was calculated by normalizing the resultant CFUs on any plate to thaton the plate without exposure to light.

On the other hand, Kim et al. (31) suggested that the inactivation percentage is asufficient indicator of the antimicrobial activity of TiO2-based photocatalyst and theinactivation ratio should be calculated as:

\(%)= ((M"-Mu)/IvI") x10O t4I

where: I, - inactivation ratio, Mn - the initial concentration of microorganism, M6 - theconcentration of the microorganism on the TiOz coated sample.

The results of the antimicrobial activity of TiO2-based products in the ffiibition zonemethod are accessible as millimeters of the zone around the test sample without the mic-robial growth (the inhibition zone) (34). An improvement in measuring the inhibitionzone is recently published by Vudetii et al. (29). The group of researchers imported theimages of the Petri dishes after the incubation period into the software Matlab R20l2a,where the images were converted to binary images. The percentage of the surface covera-ge is calculated by a specifically designed program code, which determines the ratio ofthe black and white pixels. The black pixels correspond to the surface covered with mic-robial growth (29). This kind of calculation of the antimicrobial efficacy of the TiO2-ba-sed products is significantly simplier and easier. Moreover, subjective error is remarkablyreduced by using the designed program code.

CONCLUSIONS

This study reviews the killing mechanisms, experimental setups for testing the anti-microbial efficiency of TiOz coated, light sources and intensity, as well as the pertainingcalculation methods. On analyzing the investigations that have been done worldwide, itclearly comes out that great effort should be put in the optimization and improvement ofall segments of the available antimicrobial testing methods of TiOz photocatalysts. In thefuture, a new testing method with good settings basis that can be used in many researchfields should be developed. In that way, the problem of the comparison of the obtainedresults among researches could be solved.

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Acknowledgement

The financial support of the Ministry of Education, Science and Technological Deve-lopment of the republic of Serbia (Contract No. III45008) and of the Provincial Secreta-riat for Science and Technological Development of Vojvodina Region (Contract No 114-4 5 I -3 426 I 20 I 3 -02) are gratefu lly acknowledged.

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l5l

APTEFF, 45, t-283 (20t4)DOI: 10.2298IAPT l445l4lM

UDC : 6 I 5.28 I :5 46.A+542.92' 73BIBLID: 1450-71 Rg (2O1 4\ 47 141 -l \2

Original scientifi c paper

METOAE 3A LICIIUTUBABE AHTIIMI4KPOBHE AKTUBHOCTIT TiO2OOTOKATAJIII3ATOPA

Cuumaa JI. Mapxoe, Aua M. Budarcoeuh

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MaHe TpeIIyTHO rOpuUrheurur MeroAa 3a r{crrrrTlrB€uLe aHTuMr,rKpo6ne axrr,rsHocTu IIpo-ryKara na 6asn TiO2.

K.rry.rne peqlr: aHT[MuKpo6Ha aKruBuocr, TnrarrlljyM,uroKcHA, [r3Bop[r 3parrerb4 npo-parryHll, MeToAe IIcuIrtI{BaILa

Received: 17 September 2014.Accepted: 21 October 2014.

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