Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16

Comparative study of homogeneous and heterogeneous photo-oxidative treatment on bacterial cell via multianalytical techniques

R. Shwetharani, R. Geetha Balakrishna *Centre for Nano and Material Sciences, Jain University, Jakkasandra Post, Kanakapura Taluk, Bangalore Rural District 562112, India

A R T I C L E I N F O

Article history:Received 12 June 2014Received in revised form 13 August 2014Accepted 26 August 2014Available online 4 September 2014

Keywords:Salmonella typhimuriumTiO2

PhotocatalystSemiconductorBactericidalPhoto-Fenton

A B S T R A C T

The present article demonstrates the comparative efficiency of homogeneous and heterogeneousphotocatalyst on cell membrane destruction using electron microscopy and spectroscopy techniques tounderstand the mechanism and evolve an effective water disinfection technique. The antibacterialactivity of a heterogeneous photocatalyst nano-TiO2 was compared with a homogeneous neutralPhoto-Fenton reagent (Fe2+/3+, H2O2) against Salmonella typhimurium, a gram negative water bornebacterium. To confirm the extent of both types of photooxidation treatment on bacterial cell, productionof MDA, release of RNA, DNA and protein were carefully studied and analyzed. Based on the aboveexperimental results, heterogeneous photocatalyst shows better performance than homogeneousneutral Photo-Fenton system and the difference is attributed to the mode of disinfection in the twosystems. The damage of cell membrane and release of inner cell components is higher in heterogeneoussystem in contrast to the neutral Photo-Fenton system which causes disinfection through both cellmembrane and internal cell damage. The entrapped RNA/DNA within the cell up to 80% in Fe2+ treatedbacteria substantiates destruction via internal cell damage.

ã 2014 Published by Elsevier B.V.

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology A:Chemistry

journal homepa ge: www.elsev ier .com/ locate / jphotochem

1. Introduction

Harmful microorganisms are a serious problem in drinkingwater. Hence, there is an immediate need for an appropriatedisinfection technique. Salmonella typhimurium is a typhoid fevercausing Gram negative, rod-shaped bacterium and the salmonellainfections in humans are divided into two categories – Salmonellatyphi (S. typhi) and Salmonella paratyphi (S. paratyphi). The diseaseis transmitted through the ingestion of infected foods andcontaminated water. Recently, the Photo-Fenton (PF)(Fe2+/3+/H2O2/hg) system under sunlight, at physiological pH wasreported [1,2] to be an effective option for bactericidal activity. Theantibacterial activity of Photo-Fenton, Fe3+/hg was associated withadsorption of Fe3+ ions on the bacterial cell membrane, togetherwith formation of iron-bacteria exciplexes, thus, leading to oxidationof cell membrane, in contrast to Fe2+/hg, which causes diffusion ofFe2+and by intracellular dark Fenton reactions, leads to generation of�OH radicals resulting in direct damage of DNA. Photo-Fenton wasdemonstrated to be highly efficient not only for the acceleration of

* Corresponding author. Tel.: +91 8027577212; fax: + 91 8027577211.E-mail addresses: geethabalakrishna@yahoo.co.in,

br.geetha@jainuniversity.ac.in, http://cnms.jainuniversity.ac.in(R. Geetha Balakrishna).

http://dx.doi.org/10.1016/j.jphotochem.2014.08.0131010-6030/ã 2014 Published by Elsevier B.V.

solar disinfection of water, but also for elimination of natural organicmatter [3–9]. But since, decades, titanium dioxide has beenextensively used for surface coating as a self-cleaning andself-disinfecting material, as these materials have many attractivefeatures, such as optical properties, low cost, high photocatalyticactivity, chemical stabilityand non-toxicity [10,11]. Titanium dioxidein the anatase crystalline form behaves as a classical semiconductor.Illumination of TiO2 in water with light of �400 nm generateselectrons in the conduction band (e�cb) and positive “holes” in thevalence band (hþ

vb) [12]. This potent redox power characteristicallyresults in the lysis of bacteria and other organic substances[13–16]. Spuhler et al. [1] in their extensive study in 2010 claimedthat solar Photo-Fenton system at low concentration of reagentand at near neutral pH proved efficient for both bacterialinactivation and mineralization of model organic matter. Although,for the last couple of years there are few papers reporting the effectof Photo-Fenton and TiO2 (Degussa P25) individually on organicdegradation/antibacterial activity, a comparative study of theirefficiency on a single pathogen to evaluate the best disinfectionsystem and its mechanism is reported for the first time to the bestof our knowledge. Also a highly efficient, single phase of anataseand nano-titania with a high surface area, which is uniformlysized and regularly shaped, has been experimented to validate thetrue comparative capability of nano-TiO2 photocatalyst overPhoto-Fenton reagents in causing disinfection of water.

Fig. 1. Extent of photoinactivation of S. typhimurium.

12 R. Shwetharani, R. Geetha Balakrishna / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16

2. Experimental materials and methods

2.1. Material

TiCl4 (99.5% Loba Chemie) was used as a titanium source for thepreparation of TiO2. The pure culture of S. typhimurium(ATCC14028) was obtained from Defense Food Research Laborato-ry, Mysore, India. Other chemicals, namely, sulphuric acid (98%, 36N), sodium hydroxide (97%), hydrogen peroxide (30%), ferricchloride (96%), ferrous sulphate heptahydrate (99%) and ammoni-um hydroxide (30%), were of Merck brand. The photocatalyst TiO2

was prepared as per our earlier reported protocol [17].

2.2. Preparation of bacterial culture

The photocatalytic performance of the synthesized TiO2 wasevaluated on S. typhi ATCC14028, prepared by picking a loop full ofexponential growth phase of pure culture colonies into 500 mL ofbrain–heart infusion (BHI) broth and incubated at 37� C for 15 h.The density of the bacterial suspension was adjusted approxi-mately equivalent to 107 CFU/mL. The bacterial cells were collectedin an exponential phase by centrifugation and the bacterial pelletwas washed three times with double distilled water. Finally thebacterial pellet was resuspended in double distilled water. Thebacterial suspension (�107 CFU/mL) was placed in the dark at 25 �Cunder stirring for 30 mins to allow the bacteria to acclimatize to thenew matrix, also to allow die-off of the most stress sensitivespecies and in all to stabilize the bacterial population.

2.3. Photoreactions

Experiments were conducted in six of the reactor vessels havingan exposure area of 176.62 cm2 into which the sunlight was directlyfocused. The concentration of photocatalysts used was as follows:0.4 g/L TiO2 as heterogeneous catalyst, 0.06 g/L of ferrous sulphateheptahydrate and ferric chloride as Fe2+ and Fe3+ PF sourcesrespectively, with 0.01 g/L of H2O2. The photocatalytic reactionswere conducted on intense sunny days in the month of Marchbetween 11 AM and 2 PM in Bangalore city (13�00.570N and77�34.150E) which is about 1800 km from New Delhi, the capital ofIndia. The average solar intensity was around 900 W/m2 and nosteps were taken to maintain the intensity of sunlight during thereaction. The reaction mixture was stirred continuously with amagnetic stirrer in atmospheric oxygen during photoreaction. Thesamples were collected every 15 mins for various analysis such asstandard plate count method (SPC), dye exclusion, lipid peroxida-tion, protein, RNA/DNA estimation and TEM/SEM analysis. Theprotocols used for the above estimations are as mentioned in ourearlier papers [18,19]. The above tests were conducted in triplicategiving fairly good reproducibility. The quantitative detection ofhydroxyl radicals produced by various photocatalysts is estimatedby using coumarin as a probe molecule [20], which was convertedto a highly fluorescent 7-hydroxy coumarin (7HC) when reactingwith OH radicals.

3. Results and discussion

3.1. Comparative bacterial inactivation mechanism under near-neutral Photo-Fenton (homogeneous) and TiO2 (heterogeneous)photocatalysis

The synthesized photocatalyst used for the present study hasbeen characterized by XRD, EDS, SEM, TEM, BET and spectral studyin depth and is as reported in our earlier paper [17]. Fig. 1 depictsthe extent of bacterial inactivation by cultivability measurements,where cultivability is N/No (N is the number of colonies obtained

after an irradiation time period t and No is the number of coloniesobtained before any irradiation). The heterogeneous catalysiscauses complete inactivation within 120 mins of reaction. It isobserved that all the reagents cause rapid decrease in the initial25 mins of irradiation due to quick action of the catalyst and 80%of inactivation occurs within that period. The remaining 20%takes another 95 mins for completion. The difference in theefficiency of the photocatalysts (either heterogeneous orhomogeneous) with regard to loss of cultivability appears marginalin Fig. 1. This may be because, both the internal damage due todiffusion (by homogenous catalysis) and cell damage due tooxidation (by heterogeneous catalysis) leads to ultimate loss ofcultivability and hence the marginal difference.

Fig. 2 represents the accumulation of malondialdehyde (MDA),one of the most abundant aldehyde forms originating fromperoxidation of bacterial lipid membranes. The heterogeneouscatalyst is almost on par with other catalysts up to 60 mins beyondwhich there is a tremendous increase in MDA content. The highefficiency of the TiO2 photocatalyst to generate ROS and causemembrane oxidation is in agreement with the high rate of lipidperoxidation occurring by hydroxyl radicals. The final MDAconcentration reaches 99.323 nmol/mL on treatment with theTiO2 photocatalyst, being much very higher than 17.478 nmol/mLobtained without the photocatalyst. The order of MDA concentra-tion obtained with different compositions of Photo-Fenton reagentis 55.251 nmol/mL > 49.064 nmol/mL > 40.187 nmol/mL > 32.154nmol/mL for Fe3+/H2O2, Fe3+, Fe2+/H2O2 and Fe2+ respectively.

The same has been substantiated by hydroxyl radical scaveng-ing investigation as shown in Fig. 2a. The spectrum denotes thehydroxyl radicals produced during a photocatalytic reactionhighlighting the ability to generate both �OH and O

��2 radicals

proportionately with time. The plot indicates the generation ofhydroxyl radicals to be highly linear with time for the heteroge-neous catalyst unlike the PF reagents, and this is clearly indicativeof the enhanced inactivation (membrane oxidation and membranepermeability) occurring with TiO2, which in fact evidences to behighly efficient in producing hydroxyl radicals. The phenomenonpoints to a classical mechanism of the light induced promotion ofvalence band electron to conduction band which yields valenceband holes and produce hydroxyl radicals at the particle surface.The �OH radicals (along with other ROS such as O

��2 and H2O2

generated on the illuminated TiO2 surface) can attack polyunsatu-rated components in S. typhi, leading to cell membrane breakage.

Fig. 2. (a) Concentration of malondialdehyde released from lipid peroxidation during photoinactivation. (b) Concentration of OH radicals production in the presence ofphotocatalysts.

R. Shwetharani, R. Geetha Balakrishna / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16 13

Also holes of the catalyst can disturb cell membrane leading tooxidative stress and eventually cell death. The same disinfection ofS. typhi by Degussa P25 has been reported by Moncayo-Lasso et al.[3] in 2012 to cause only partial disinfection in 120 mins, althoughin higher intensity of simulated solar light irradiation, unlike thepresent nano-TiO2 which causes complete disinfection. However,MDA is a relatively unstable product; under photocatalyticreactions it undergoes further metabolic transformations. MDAhas two aldehyde groups and the degradation products areexpected to be ring cleavage products, such as semialdehyde,

Fig. 3. TEM micrographs of S. typhi (a) untreated (control), t

malonate, monoaldehyde (formaldehyde, acetaldehyde), mono-ketons (acetone) and carboxylic acids (formic and/or acetic acid) orcomplete mineralization products such as CO2 and H2O [18].Therefore, the MDA values in each peroxidation lipidic experimentare the net result of its production and photocatalytic degradationin oxidative condition. Thus, eventually, the plots depict theexceeded rate of MDA production over the rate of MDA destruction.TiO2 photocatalytic treatment effectively degrades MDA at betterrates than PF reagents. However its accumulation is exceeded bydegradation beyond a particular time of treatment.

reated with (b) nano- TiO2, (c) Fe3+/H2O2, (d) Fe2+/H2O2.

Fig. 4. Extent of DNA release during photocatalytic inactivation.

Fig. 5. Extent of RNA release during photocatalytic inactivation.

14 R. Shwetharani, R. Geetha Balakrishna / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16

Photo-Fenton can act on bacteria in three ways depending on itscomposition,

i. Fe2+ ions with a positive charge and smaller size can stronglyinteract with cell membrane, diffuse into the cells (or) reactwith oxygen leading to the generation of �OH via intracellularFenton reactions, with metabolic H2O2. The diffusion of Fe2+ ismore probable due to the lower charge density of Fe2+

compared to Fe3+. In the presence of sunlight, the synergisticinteraction of Fe2+ and sunlight leads to the easy diffusion ofFe2+ into the cells. The diffused Fe2+ reacts with the intracellularH2O2 (which is always produced in respiration) followed byformation of toxic �OH and in the presence of light causes directDNA damage and photoexcitation of endogenous (cytochrome,flavin, tryptophan) photosensitizers. The excited photosensi-tizer reacts with cellular biomolecules, protein and cellmembrane components like membrane lipids leading to lipidperoxidation [21]. The insolubility of Fe3+ at higher pHprecludes its mobility as a metal ion into microorganisms. Tomanage the transport of Fe3+ into the cell, S. typhi (gramnegative bacteria) has several specific outer membrane bindingproteins. Once in the cytoplasm, siderophores, specificallychelate to Fe3+ (e.g. hydroxamates, catecholates, carboxylates orheterocyclic compounds). Fe3+ cannot move freely into the cells,once inside the cell. The reaction with H2O2 is considerably lessrapid. Also Fe3+ can be used for facultative respiration by S. typhiand hence, intuitively understandable that the bactericidalactivity due to loss of internal cell integrity is more pronouncedfor Fe2+ and can be substantiated from cultivability studies.Unlike the other studies as further reported, a marginal increasein loss of cultivability is observed for Fe2+. The Fig. 1 inSupporting data indicates the existence of increased VBNC(viable but non-culturable) bacteria and can be expected fromFe2+ treatment which causes the bacteria to undergo internaldamage with a provision of repair and become active ifirradiation was stopped. However estimation of cell compo-nents (which may have leaked due to membrane damage)remains higher in Fe3+ system. This can also be substantiated byTEM in Fig. 3 where most of the cells indicated higher rupture incell membrane of Fe3+ treated systems unlike the loss ofintegrity between the cell wall and cell membrane occurring inFe2+ treated cells. The TiO2 treated cells appeared decomposed,completely shrunk and the interior of the cell was tending tobecome transparent, indicating the damage of the outermembrane and leading to a leakage of intracellular compo-nents, validating the highest catalytic efficiency of nano-titania.

ii. Fe3+ may adsorb on bacteria instead of diffusing inside the cells,leading to the formation of Fe3+- bacteria exciplexes (photo-active complex) which are formed and this leads to photo-generation of �OH radicals that attack cell membranes, causinglipid-peroxidation. The significant cellular component accu-mulation with Fe3+ under sunlight irradiation is probably due tothe Fe3+ binding to bacterial membrane proteins and theircarboxylic end groups forming Fe3+ - bacteria photo-activecomplex. The photoactive complexes contribute to the bacterialinactivation either by direct oxidation of the membraneconstituents or by the generation of Fe2+, H2O2 and �OH withinspitting distance of the target bacteria. In photoactive com-plexes, the excitation of Fe3+ chelate into a LMCT (ligand tometal charge transfer) state by light irradiation, followed byphotoinduced electron transfer leading to reduction of Fe3+ toFe2+ via oxidation of the chelating ligand. For the classicaldegradation of chemical pollutants by Fenton systems(Fe2+/3+/hg), it is from a mechanistic point of view, meaninglessto distinguish ferrous from ferric type iron reactions, due to thefast oxidation of Fe2+ to Fe3+ in the presence of H2O2. The

distinction is significant for Photo-Fenton like reactionsbecause of dissimilarity in the affinity for the formation ofphotoactive complexes. The differentiation is also relevant forbiological systems (S. typhi bacteria) because of differentiatedways of action for each species: Fe3+ has a higher probability toadsorb on cells and to form exciplexes on the bacterialmembrane because it cannot easily diffuse into the cells andcan bind to specific proteins; Fe2+ can diffuse into cells,especially in the presence of osmotic forces [22,23].

iii. The presence of H2O2 increases the extent of bacterialmembrane permeability for all the three photo reagents. Itallows better Fe2+ diffusion and enhanced roughness for Fe3+

attachment, thereby enhancing the efficiency of photoinacti-vation. H2O2 increases the production of �OH radicals, inducingoxidation reactions. H2O2 is a long living reactive oxygenspecies, thus, relatively stable (unlike �OH) and uncharged(unlike O

��2 ) and when added to the bulk solution can penetrate

into the bacterial membrane and diffuse into cells, therefore,H2O2 stress arises inside cells, whenever H2O2 is present in theextracellular habitat. When excess H2O2 reacts with free orloosely bound intracellular Fe2+ via Haber–Weiss reaction [1,2],generation of �OH takes place, thus, enhancing the oxidationprocess of cell membrane.

Figs. 4 and 5 indicates the extent of leakage of internalcomponents DNA and RNA respectively. The plot indicates 55% of

Fig. 6. Extent of protein release during photocatalytic inactivation.

R. Shwetharani, R. Geetha Balakrishna / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16 15

DNA that could be traceable and hence 45% of DNA was stilltrapped within the cell when treated with TiO2. Although all thesystems achieved 90% loss of cultivability within 120 mins, systemswith Fe2+/H2O2, Fe2+ could not give out more than 15% of DNA at120 mins implying the non-leakage and non-permeability of thecell membrane. Therefore TiO2 photocatalysis was very efficient incausing membrane damage and permeability.

Fig. 6 shows the extent of protein leakage during photocatalyticreaction. The highest amount of protein released was for sample

Fig. 7. SEM micrographs of S. typhi (a) untreated (control), t

treated with the TiO2 photocatalyst after 200 mins of irradiationamounting to 1389.221 mg/L as against 815.364 mg/L,711.345 mg/L, 689.968 mg/L and 436.124 mg/L for Fe3+/H2O2,Fe3+, Fe2+/H2O2 and Fe2+respectively.

This indicates that the major destruction occurred at 160 minsof treatment. The maximum protein release occurs between30 mins and 60 mins, the catalyst sites initially, are completelyavailable for catalysis and hence, most of the protein releasehappens in the 30–60 mins of degradation in both Photo-Fentonand TiO2. Therefore it is reasonable to assume that the membraneis mostly damaged by reactive oxygen species such as superoxide(O

��2 ) or hydroxyl radicals (�OH) which are known to be produced

by TiO2 photocatalysis. The release of high amount of protein innano-titania treated sample indicates that it is a very effectivephotocatalyst for cell membrane destruction . SEM micrographs ofphotocatalysts treated bacteria is as shown in Fig. 7, great changeshad taken place to the morphology of S. typhi. Fig. 7 depicts asmooth surface, with very little damage in the cell structure of thecontrol (only sunlight) experiment. The titania treated bacterialcells shows significant changes like rough surface or a hollowshape, loss of cell wall, cell membrane and leakage of cell contentsindicating severe damage by nano-titania and Fe3+/H2O2 treatedbacteria also shows cell membrane breakage but it is not asprominent as nano-titania.

There is no remarkable damage in Fe2+/H2O2 treated bacteriafrom the control. The SEM/TEM microscopy shows that TiO2

causes damage to the cell structure followed by leakage of innercell components leading to bacterial destruction. However, theexact difference in cell damage due to heterogeneous andhomogeneous catalysis was very difficult to observe from SEMstudies.

reated with (b) nano- TiO2, (c) Fe3+/H2O2, (d) Fe2+/H2O2.

16 R. Shwetharani, R. Geetha Balakrishna / Journal of Photochemistry and Photobiology A: Chemistry 295 (2014) 11–16

4. Conclusions

The synthesized nano-titania with a better size, shape, surfacearea and surface charge over the previously reported DegussaP25 has been proved to be highly efficient in causing both thedamage of cell membrane and internal components leading to acomplete loss of viability and cultivability unlike the Photo-Fentonreagents. The results of cell membrane damage by lipid peroxida-tion/protein leakage, loss of cell permeability by estimation ofDNA/RNA and loss of cultivability by SPC were carefully analyzedand the mechanism of action has been elucidated to validate theactivity of photocatalyst. Fe3+/H2O2 is proved to be a betterdisinfectant than Fe2+/H2O2 as it binds to the cellular membraneand generates localized ROS that can attack the membrane.Conversely Fe2+ can diffuse into the cell and participate inintracellular Fenton's reactions leading to generation of �OHradicals. The study gains significance in proving and elucidatingthe mechanism for highly efficient nano-titania causing disinfec-tion of water over Photo-Fenton reagents.

Acknowledgements

The authors greatly acknowledge DST, New Delhi and JainUniversity for the financial support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jphotochem.2014.08.013.

References

[1] D. Spuhler, J.A. Rengifo- Herrera, C. Pulgarin, The effect of Fe2+, Fe3+, H2O2 andthe photo-Fenton reagent at near neutral pH on the solar disinfection (SODIS)at low temperatures of water containing Escherichia coli K12, Appl. Catal. B 96(2010) 126–141.

[2] A. Moncayo- Lasso, J. Sanabria, C. Pulgarin, N. Benitez, Simultaneous E. coliinactivation and NOM degradation in river water via photo-Fenton process atnatural pH in solar CPC reactor. A new way for enhancing solar disinfection ofnatural water, Chemosphere 77 (2009) 296–300.

[3] A. Moncayo- Lasso, L.E. Mora- Arismendi, J.A. Rengifo- Herrera, J. Sanabria,N. Benitez, C. Pulgarin, The detrimental influence of bacteria (E. coli,Shigellaand Salmonella) on the degradation of organic compounds (and vice versa) inTiO2 photocatalysis and near-neutral photo-Fenton processes under simulatedsolar light, Photochem. Photobiol. Sci. (2012) , doi:http://dx.doi.org/10.1039/c2pp05290c (in press).

[4] S. Barreca, J.J.V. Colmenaresc, A. Pacea, S. Orecchioa, C. Pulgarinc, Neutral solarphoto-Fenton degradation of 4- nitrophenol oniron-enriched hybrid

montmorillonite-alginate beads (Fe- MABs), J. Photochem. Photobiol. A 282(2014) 33–40.

[5] M. Punzi, B. Mattiasson, M. Jonstrup, Treatment of synthetic textile wastewaterby homogeneous and heterogeneous photo-Fenton oxidation, J. Photochem.Photobiol. A 248 (2012) 30–35.

[6] A. Moncayo- Lasso, A.G. Rincon, C. Pulgarin, N. Benitez, Significant decreaseof THMs generated during chlorination of river water by previousphoto-Fenton treatment at near neutral pH, J. Photochem. Photobiol. A229 (2012) 46–52.

[7] M. Jonstrup, M. Punzi, B. Mattiasson, Comparison of anaerobic pre-treatmentand aerobic post-treatment coupled to photo-Fenton oxidation for degrada-tion of azo dyes, J. Photochem. Photobiol. A 224 (2011) 55–61.

[8] Y.Y. Zhang, C. He, J.H. Deng, Y.T. Tu, J.K. Liu, Y. Xiong, Photo- Fenton-likecatalytic activity of nano- lamellar Fe2V4O13 in the degradation of organicpollutants, Res. Chem. Intermediat. 35 (2009) 727–737.

[9] P. Bouras, P. Lianos, Synergy effect in the combined photodegradation of an azodye by titanium dioxide photocatalysis and photo-Fenton oxidation, Catal.Lett. 123 (2008) 220–225.

[10] A. Fujishima, T.N. Rao, D.A. Tryk, Titanium dioxide photocatalysis, J.Photochem. Photobiol. C 1 (2000) 1–21.

[11] J.C. Ireland, P. Klostermann, E.W. Rice, R.M. Clark, Inactivation of Escherichia coliby titanium dioxide photocatalytic oxidation, Appl. Environ. Microbiol. 59(1993) 1668–1670.

[12] M. Cho, H. Chung, W. Choi, Different inactivation behaviors of MS-2 phage andEscherichia coli in TiO2 photocatalytic disinfection, Appl. Environ. Microbiol. 71(2005) 270–275.

[13] K. Shiraishi, H. Koseki, T. Tsurumoto, Antibacterial metal implant with aTiO2- conferred photocatalytic bactericidal effect against Staphylococcusaureus, Surf. Interface Anal. 41 (2008) 17–22.

[14] S. Ede, L. Hafner, P. Dunlop, J. Byrne, G. Will, Photocatalytic disinfection ofbacterial pollutants using suspended and immobilized TiO2 powders,Photochem. Photobiol. 88 (2012) 728–735.

[15] S. Swetha, S.M. Santhosh, R. Geetha Balakrishna, Synthesis and comparativestudy of nano- TiO2 over Degussa P-25 in disinfection of water, Photochem.Photobiol. 86 (2010) 628–632.

[16] S. Swetha, S.M. Santhosh, R. Geetha Balakrishna, Enhanced bactericidal activityof modified titania in sunlight against Pseudomonas aeruginosa, a water-bornepathogen, Photochem. Photobiol. 86 (2010) 1127–1134.

[17] P. Laveena, S. Shree, R. Geetha Balakrishna, Bifunctional titania float for metalion reduction and organics degradation, via sunlight, Ind. Eng. Chem. Res.(2013) , doi:http://dx.doi.org/10.1021/ie402592k (in press).

[18] R. Shwetharani, M.S. Jyothi, P.D. Laveena, R. Geetha Balakrishna, Photoactivetitania float for disinfection of water; evaluation of cell damage bybioanalytical techniques, Photochem. Photobiol. (2014) , doi:http://dx.doi.org/10.1111/php.12277 (in press).

[19] S. Swetha, M.K. Singh, K.U. Minchitha, R. Geetha Balakrishna, Elucidation of cellkilling mechanism by comparative analysis of photoreactions on differenttypes of bacteria, Photochem. Photobiol. 88 (2012) 414–422.

[20] Q. Xiang, J. Yu, P.K. Wong, Quantitative characterization of hydroxylradicals produced by various photocatalysts, J. Colloid Interface Sci. 357(2011) 163–167.

[21] C. Venkobachar, L. Iyengar, A.V.S.P. Rao, Mechanism of disinfection, Water Res.9 (1975) 119–124.

[22] B.C. Faust, R.G. Zepp, Photochemistry of aqueous iron(III)- polycarboxylatecomplexes: roles in the chemistry of atmospheric and surface waters, Environ.Sci. Technol. 27 (1993) 2517–2522.

[23] W. Feng, D. Nansheng, Photochemistry of hydrolytic iron(III) speciesand photoinduced degradation of organic compounds. A minireview,Chemosphere 41 (2000) 1137–1147.