1

gcr�todi

N

s

J

Downloa

Julián A. Rengifo-HerreraLaboratory for Environmental Biotechnology,

Ecole Polytechnique Fédèrale de Lausanne(EPFL),

CH-1015 Lausanne,Switzerland

Janeth Sanabria

Fiderman Machuca

Carlos F. Dierolf

Grupo de Investigación en Procesos Avanzadosde Oxidación para Tratamientos Químicos y

Biológicos,Facultad de Ingeniería,Universidad del Valle,

A.A. 25360, Cali,Colombia

Cesar PulgarinLaboratory for Environmental Biotechnology,

Ecole Polytechnique Fédèrale de Lausanne(EPFL),

CH-1015 Lausanne,Switzerland

e-mail: cesar.pulgarin@epfl.ch

Guillermo OrellanaLaboratory of Applied Photochemistry,

Department of Organic Chemistry,Faculty of Chemistry,

Complutense University of Madrid,28040 Madrid,

Spaine-mail: orellana@quim.ucm.es

A Comparison of SolarPhotocatalytic Inactivation ofWaterborne E. coli Using Tris„2,2�-bipyridine…ruthenium„II…,Rose Bengal, and TiO2Background. The development of alternative processes to eliminate pathogenic agents inwater is a matter of growing interest. Current drinking water disinfection procedures,such as chlorination and ozonation, can generate disinfection by-products with carcino-genic and mutagenic potential and are not readily applicable in isolated rural commu-nities of less-favored countries. Solar disinfection processes are of particular interest towater treatment in sunny regions of the Earth. Solar light may be used to activate aphotocatalyst or photosensitizer that generates, in the presence of molecular oxygendissolved in water, reactive oxygen species (ROS), such as the HO• radical, singletoxygen �1O2�, or superoxide �O2

• �, which are toxic to waterborne microorganisms.Method of Approach. Wild and collection-type Escherichia coli have been selected asmodel bacteria. Inactivation of such bacteria by either TiO2 nanoparticles, water-solubletris(2,2�-bipyridine)ruthenium(II) dichloride or Rose bengal (RB) subject to simulatedsunlight have been compared. Although TiO2 is the prototypical material for heteroge-neous photocatalysis, the other two dyes are known to generate significant amounts of1O2 by photosensitization but have different chemical structures. The concentration ofdye, illumination time, photostability, presence of scavengers, and post-treatment re-growth of bacteria have been investigated. Results. After 1 hr of solar illumination theRu(II) complex produced a strong loss of E. coli culturability monitored with solid selec-tive agars. Both the collection- and wild-type bacteria are sensitive to the treatment with2–10 mg L−1 of dye. This photosensitizer showed a better inactivation effect than TiO2and the anionic organic dye RB due to a combination of visible light absorption, photo-stability, and production of 1O2 and other ROS when bound to the bacterial membrane.A complete loss of culturability was observed when the initial concentration was103 CFU mL−1, with no bacteria regrowth detected after 24 hr of the water treatment. Athigher initial microorganism levels, culturability still remains and regrowth is observed.Scavengers show that the HO• radical is not involved in bacteria inactivation by photo-sensitization. Conclusions. A higher quantum yield of ROS generation by the sensitizingdyes compared to the semiconductor photocatalyst determines the faster sunlight-activated water disinfection of photodynamic processes. The homogeneous nature of thelatter determines a more efficient interaction of the toxic intermediates with the targetmicroorganisms. Solid supporting of the Ru(II) dye is expected to eliminate the potentialsproblems associated to the water-soluble dye. �DOI: 10.1115/1.2391319�

Keywords: solar water disinfection, photodynamic action, photocatalysis,photosensitization, E. coli, bacteria inactivation

Introduction

The development of alternative processes to eliminate patho-enic agents in water is a matter of growing interest. Indeed,onventional drinking water disinfection procedures, such as chlo-ination and ozonization, can generate disinfection by-productsDBPs� with carcinogenic and mutagenic potential �1�. Severalreatments such as UV light irradiation, chlorine derivatives, andther oxidizing agents are being employed as alternatives. Solarisinfection processes are of particular interest to water treatmentn sunny regions of the Earth. Solar light may be used to activate

Contributed by the Solar Energy Division of ASME for publication in the JOUR-

AL OF SOLAR ENERGY ENGINEERING. Manuscript received July 27, 2005; final manu-

cript received December 12, 2005. Review conducted by Sixto Malato.

ournal of Solar Energy Engineering Copyright © 20

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

a photocatalyst or photosensitizer agent that generates, in the pres-ence of molecular oxygen dissolved in water �ca. 8 mg L−1 atambient pressure and temperature�, the so-called reactive oxygenspecies �ROS� �2�. The hydroxyl radical �HO•� and singlet mo-lecular oxygen �1O2� are some of the ROS produced under illu-mination of organic and inorganic absorbers of light. Hydroxylradicals produced by semiconductor photocatalysis in the UV-Ahave recently shown biocidal activity against waterborne bacteria�3,4�. Pure singlet oxygen is a known toxic to virtually any kind ofcell examined thus far from prokaryotic to mammalian �5,6�, as itcan attack nucleic acids �7,8� and membrane lipids �9�.

Common photocatalytic processes for water treatment are thosebased on corrosion-resistant semiconductor nanoparticles such asTiO2, ZnO, or WO3. Illumination of the photocatalyst with light

�typically below 400 nm� produces an electron-hole pair that ini-

FEBRUARY 2007, Vol. 129 / 13507 by ASME

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm

th�asr

Ttptcattftb

shcamdoadslrleAasuittca

2

�sbBua

eRtc

1

Downloa

iates a series of electrochemical reactions in which ROS, such asydroxyl �HO•� and superoxide �O2

• � radicals are generated �Eqs.1�–�3� �10,11�. The high oxidizing power of the HO• radical isble to mineralize most organic compounds and oxidize inorganicpecies. Both radicals are toxic for bacteria�12,13�, and previouseports have shown good results in water disinfection �3,4,14,15�.

TiO2 + h��UV� → TiO2�e− + h+� �1�

TiO2�h+� + H2O�ads� → HO�ads�· + H+ �2�

TiO2�e−� + O2 → O2·− �3�

he photodynamic processes use an organic or inorganic sensi-izer �S�. The sensitizer molecule absorbs UV and/or visible lightroducing an electronic excited state �S*, Eq. �4��. This species ishen quenched by molecular oxygen via electron transfer �photo-hemical “type I” reaction, Eq. �5�� to yield superoxide radicalnion �O2

• � and the oxidized sensitizer, or via energy transfer �pho-ochemical “type II” reaction, Eq. �6�� to generate the lowest elec-ronic excited state of O2 �singlet molecular oxygen, 1�g or 1O2or short� �16,17�. Type II processes are indeed photocatalytic ashe sensitizer species returns to its �initial� ground electronic statey the O2 quenching reaction.

S + h��UV� → S* �4�

S* + O2 → S+ + O2·− �type I� �5�

S* + O2 → S + 1O2 �type II� �6�The most commonly used photosensitizers are organic dyes,

uch as methylene blue, Rose bengal, or porphyrins. These dyesave exhibited ability to inactivate bacteria such as Escherichiaoli via a type II mechanism, using both solar simulators �18–20�nd direct solar light �21,22�. Metal complexes with polyazaaro-atic chelating ligands, such as tris�2,2�-bipyridine�ruthenium�II�

ication �Ru�bpy�32+�, have shown high quantum yields of singlet

xygen production �up to 1.0 in organic solvents and up to 0.5 inqueous solution �23,24��. Moreover, these blue light-absorbingyes show a unique combination of photochemical, redox andpectroscopic characteristics, and are significantly stable under il-umination �25�. However, Ru�II� complexes have only been usedecently for efficient bacteria inactivation under visible and sun-ight illumination �26,27�. The aim of the present basic study is toxplore bacterial inactivation of two E. coli strains �namely,TCC 25922 and wild� in water, using the three different photo-ctive materials �Ru�bpy�3

2+�, Rose bengal �RB�, and TiO2 underimulated sunlight. Although the first two are water soluble andndergo type-II photosensitization in the presence of O2 and vis-ble radiation, but have very different chemical structures, the lat-er is the most widespread semiconductor for photocatalytic waterreatment in heterogeneous phase. The results will show the bestombination of photocatalyst and operational conditions tochieve water disinfection with sunlight.

Experimental

2.1 Materials. The photosensitizing dyes Rose bengal4,5,6,7-tetrachloro-2� ,4� ,5� ,7�-tetraiodofluorescein disodiumalt, Sigma-Aldrich, USA, �95%� and tris�2,2�-ipyridine�ruthenium�II� dichloride hexahydrate �Acros Organics,elgium, �99%� were used as received. The TiO2 photocatalystsed �Degussa P-25, Germany� is mainly anatase, with a surfacerea of 50 m2 g−1.

2.2 Disinfection Experiments. For the different disinfectionxperiments, the RB concentration was 2 mg L−1,u�bpy�3Cl2 ·6H2O was added in the 2–10 mg L−1 range, and for

he TiO2 slurry a concentration of 1 g L−1 was selected. Two E.

oli strains in stationary phase of their culture were used, namely,

36 / Vol. 129, FEBRUARY 2007

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

ATCC 25922 �abbreviated ATCC E. coli� and a wild strain �ab-breviated wild E. coli�. The wild E. coli was isolated from a do-mestic wastewater treatment plant, located in Ginebra, Valle delCauca, Colombia. Both strains were grown in casein peptonebroth until they reached stationary phase and a concentration ofca. 107 CFU mL−1. Then a suitable amount of the cell suspensionwas inoculated into Pyrex® glass reactors containing 50 mL ofsterile distilled water to yield an initial concentration of microor-ganism of 103 or 105 CFU mL−1.

Illumination �950 W m−2� of the 50 mL water samples was pro-vided by a Hanau Suntest �AM-1� solar simulator. The lamp has aspectral distribution with �0.5% of emitted photons at wave-lengths shorter than 300 nm �UV-C range�, and �4% between300 nm and 400 nm �UV-B and UV-A ranges�. The distribution ofthe photons emitted between 400 nm and 800 nm follows the so-lar spectrum. The photoreactor Pyrex walls are opaque in theUV-B and -C regions.

The �static� samples were illuminated for 1 hr or 6 hr undercontinuous air injection while the solution temperature increasedduring the experiments from 28 to 38°C. Samples were taken atdifferent time intervals, and serial dilutions were performed inpeptoned water. The samples were spotted onto Chromocult® E.coli plates �Merck� and spread using standard microbiologicaltechniques. The plates were incubated at 37°C for 24 hr prior tocolony counting. All the experiments were carried out three times,and the pH of the initial solution was 6.8–7.0. The standard de-viation of the results is equal or lower than 8%.

2.3 Regrowth Tests. After the illumination period, the Pyrexglass reactors were protected from light and maintained in thedark for 24 hr. Then, samples were taken, serial dilutions weremade in peptoned water and aliquots were spotted in plates usingthe same media �Chromocult�. The plates were incubated at 37°Cfor 24 hr prior to colony counting.

3 Results and Discussion

3.1 Bacteria Photoinactivation by „Ru„bpy…32+…. Figure 1

shows that illumination of �Ru�bpy�32+� with the solar simulator

leads to strong phototoxicity on the ATCC E. coli strain. TheUV-A and B components of the lamp have, in the absence of the�Ru�bpy�3

2+�, a weak effect on the cell culturability and there is noeffect of the photosensitizer dye in the dark at the concentrationtested �2.0 mg L−1� of Ru�bpy�3Cl2 ·6H2O�. The positivelycharged metal complex in water binds to the bacterial wall �28�

Fig. 1 Phototoxic effect of 2.0 mg L−1 Ru„bpy…3Cl2 ·6H2O onATCC E. coli „initial concentration: 105 CFU mL−1

…: -�-Ru„bpy…3

2++light, -�- only light, -�- Ru„bpy…32+ without light

and singlet molecular oxygen is produced on illumination �the

Transactions of the ASME

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm

qeobt�apa

nihg

par

Fd†

=

J

Downloa

uantum yield of 1O2 generation by �Ru�bpy�32+� in air-

quilibrated water is 0.22, but increases circa threefold in polarrganic solvents �23� so that it might approach this value whenound to the cell wall�. As photoinduced electron transfer to O2 ishermodynamically allowed for this dye ��G=−54 KJ mol−1

23��, superoxide radical anions �O2• � might also be generated in

queous medium. Both ROS attack the bacterial membrane, com-romising the cell viability and producing a loss of their cultur-bility after sufficient illumination time.

3.2 Comparison Between the Photocatalytic and Photody-amic Disinfection Processes. A comparison between the photo-

nduced inactivation processes tested was made using TiO2 aseterogeneous photocatalyst, and �Ru�bpy�3

2+� and RB as homo-eneous photosensitizers. The results are depicted in Fig. 2.

Perturbation on the culturability of ATCC E. coli was moreronounced during the photodynamic treatment with the dyes thans a consequence of the TiO2 photocatalysis. Several possibleeasons may explain the dramatic difference observed:

�a� The solar photocatalytic process with TiO2 has a hetero-geneous nature. Interaction between the suspended cata-lyst nanoparticles and bacteria is a requisite for cell inac-tivation �15�, as the extremely short-lived hydroxylradicals are produced from water or OH− molecules ad-sorbed on the catalyst surface according to Eq. �2�. Thus,the attack of the strongly oxidizing HO• radical �E0=2.80 V versus NHE� is possible only over a few sites ofthe bacterial membrane �where there is a cell-catalyst sur-face interaction�, not always producing a lethal damage.On the other hand, the photodynamic treatment eitherwith solubilized Rose bengal or the Ru�II� complex is notsubject to such limitations, and the generated ROS cansimultaneously attack the bacterial membrane in manypoints, producing a more widespread damage and risingthe probability of a lethal collision.

�b� There are differences in the quantum yield of photody-namic and photocatalytic processes. Photocatalysis withTiO2 has a small quantum yield of HO• generation �0.04��29,30� due, in part, to efficient e− /h+ recombinationwithin the semiconductor material. The TiO2 particlesalso produce scattering of the incident UV light, loweringthe photon absorption by the catalyst. In contrast, the

ig. 2 Comparison between photocatalytic and photodynamicisinfection processes, using 105 CFU mL−1 ATCC E. coli,Ru„bpy…3Cl2 ·6H2O‡=2 mg/L−1, †RB‡=2 mg/L−1, †TiO2‡

1 g/L−1, -�- Ru„bpy…32+, -�- Rose Bengal „RB…, -�- TiO2

photodynamic processes have a higher quantum yields

ournal of Solar Energy Engineering

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

for the production of the reactive singlet molecular oxy-gen �0.22–0.73 �23,24� and 0.75 �31� for Ru�bpy�3

2+ andRB, respectively, depending on the exact nature of themicroenvironment around the dye: solvating species, ag-gregation,…�. Moreover, both species produce other ROSin addition to 1O2 that increase the probability of celldamage and generate secondary reactants by attack to thebacterial membrane �e.g., endoperoxides, hydroperox-ides, dioxetanes, etc.� �3�.

�c� The fraction of UV-A and UV-B light of our solar simu-lator responsible for the TiO2 electronic excitation is onlyca. 4% of the total incident photonic flux �Fig. 3�. Incontrast, the fraction of UV-VIS energy from the solarsimulator being absorbed by RB or Ru�bpy�3

2+ is signifi-cantly higher.

�d� The observed differences in the E. coli inactivation ratebetween Rose bengal and Ru�bpy�3

2+ photosensitizersmight be due to the capacity of the later cationic dye tobind to the �anionic� cell membrane of bacteria, as it hasbeen shown for other Ru�II� complexes incubated witheukaryotic cells �28,33�. This is a key issue in the photo-dynamic inactivation of microorganisms, since the prox-imity allows an efficient attack of the photogeneratedshort-lived ROS on various targets �membrane lipids andproteins� raising the chances of causing lethal damage tothe cell �34� �the lifetime of 1O2 in water is only 3–4 �s,allowing it to cover just a 100 nm free mean path length�.On the contrary, Rose bengal is an anion at pH 7.0 �35�so that the interaction between negatively-charged dyesand the cellular envelope of the gram-negative bacteria isnot possible due to its highly negative-charged surface,thus reducing the inactivating effect �17�.

�e� Figure 3 shows that the absorption of Rose bengal in thevisible region ��max=90,400 L mol−1 cm−1 at 559 nm�36�� is stronger than that of Ru�bpy�3

2+ ��max=14,000 L mol−1 cm−1 at 452 nm �25��, yet the photody-namic effect of the latter seems to be higher for similardye concentrations. As demonstrated in Fig. 4, this isprobably due to the fact that, unlike the Ru dye, RB un-dergoes a fast modification during the illumination, itsphotochemical products displaying much lower absorp-tion of light and photosensitization properties.

3.3 Effect of the Ru Complex Concentration and 1O2Scavengers on Photodynamic Water Treatment. As expected, a

2+

Fig. 3 ---- Spectral distribution of the xenon lamp used in theexperiments and UV-Vis absorption spectra of --- Rose bengaland—Ru„bpy…3

2+ in water

rise of the initial Ru�bpy�3 concentration produced a faster loss

FEBRUARY 2007, Vol. 129 / 137

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm

osocop

�eTalrssae

Fas-

Fai2R

1

Downloa

f culturability of the tested ATCC E. coli �Fig. 5�. At higherensitizer concentration, the amount of photogenerated singletxygen increases, raising the probability of critical damage to theell �the likelihood of a lethal hit of pure 1O2 with a cell is in therder of 10−13 �32� but should be much higher if other ROS areroduced simultaneously�.

When 0.26 mM thiourea, an effective 1O2 scavenger �kq=1108 M−1 s−1 �37��, is added to the solution, a weak �negative�

ffect on the inactivation rate of ATCC E. coli is observed �Fig. 5�.aking into account that under such conditions the scavenger isble to quench only 10% of the photogenerated 1O2 �the 4 �sifetime of this species in water yields a 2.5�105 s−1 deactivationate constant�, we suggest the possible participation of other ROSuch as O2

•− in the bacteria inactivation process, as the excitedtate quenching of Ru�bpy�3

2+ by molecular oxygen may occurlso by electron transfer in addition to energy transfer by electronxchange to generate 1O2 �see above and Eqs. �5� and �6�.

ig. 4 Evolution of the absorbance of Rose bengal „2 mg/L−1…

nd Ru„bpy…3Cl2 ·6H2O „2 mg/L−1… in air-equilibrated aqueous

olution on illumination at 550 nm and 450 nm, respectively:�- Rose Bengal „RB…, -�-Ru„bpy…3

2+.

ig. 5 Effect of the Ru„bpy…32+ photosensitizer concentration

nd the addition of thiourea on E. coli ATCC „105 CFU mL−1…

nactivation by simulated sunlight: -�-Ru„bpy…3Cl2 ·6H2Omg/L−1; -�-Ru„bpy…3Cl2 ·6H2O 10 mg/L−1; -�-

−1 −1

u„bpy…3Cl2 ·6H2O 10 mg/L +thiourea 20 mg/L

38 / Vol. 129, FEBRUARY 2007

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

3.4 Postirradiation Events after Photodynamic TreatmentWith Ru„bpy…3

2+, Effect of the Initial Bacteria Levels and Ef-ficient Disinfection Time (EDT). During a dark period of timeafter the photodynamic treatment, both wild and ATCC E. coliundergo full recovery of their culturability when the initial con-centration of microorganisms is 105 CFU mL−1 �Fig. 6�.

The Ru�bpy�32+ dye is not able to penetrate the intact outermost

membrane of bacteria �28,38�. The experimental data can be ac-counted for if the complex accumulates in the cell membranebefore starting illumination. When the light is turned on and 1O2is produced, the damage on the wall begins allowing the ruthe-nium complex to penetrate the compromised cell membrane anddecreasing bacterial viability. Such mechanism has been sug-gested also by Schafer et al. in the photodynamic inactivation ofE. coli using RB �19�.

During 60 min of simulated solar illumination �Fig. 6�, the lightalone only leads to inactivation when the initial bacteria level is103 CFU mL−1. During the subsequent dark period, a slower re-growth is observed for both wild and ATCC E. coli strains. Thisregrowth can be explained if full inactivation of the whole bacte-ria population has not been achieved �i.e., the remaining cell countis under the detection limit of our measuring technique�.

When the initial concentration was 105 CFU mL−1, the photo-dynamic effect of Ru�bpy�3

2+ did not produce complete loss ofculturability for either strain as regrowth was observed after stop-ping the illumination �Fig. 6�. This regrowth could be caused by a

Fig. 6 Evolution of the bacterial population at different initiallevels after photoinactivation with 10 mg/L−1

Ru„bpy…3Cl2 ·6H2O or sunlight of „a… wild E. coli: -�-Ru„bpy…3

2++light and E. coli 4.0Ã105 CFU mL−1, � E. coli 4.8Ã105 CFU mL−1+light; -�- Ru„bpy…3

2++light and E. coli 4.0Ã103 CFU mL−1; -�- E. coli 4.2Ã103 CFU mL−1+light. „b… ATCCE. coli: -�- Ru„phen…3

2++light and E. coli 2.7Ã105 CFU mL−1; �E. coli 4.1Ã105 CFU mL−1+light; -�- Ru„bpy…3

2++light and E.coli 2.0Ã103 CFU mL−1; -�- E. coli 1.7Ã103 CFU mL−1+light

partial damage of the outer cell membrane causing the microor-

Transactions of the ASME

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm

gpittp6sl�itu

dpbittctrsn1

cpbipt

4

tcwadmbs

FocR

J

Downloa

anism to enter into a so-called viable but noncultivable �VBNC�rotective state �39,40�. This bacteria state, where the microorgan-sms do not grow in solids agars but its viability remains unal-ered, is due to the oxidative stress �nonlethal hits� produced byhe 1O2 attack. When the stress is turned off �i.e., during the darkeriod�, bacteria can recover their culturability. In comparison,0 min illumination with the solar simulator of a TiO2 aqueoususpension containing 108 CFU mL−1 E. coli, leads to bacteriaevels close to 1 CFU mL−1 with no regrowth in the dark for 60 hr3�. Production of other �stable� oxidizing species, such as H2O2n addition to HO• by the photosemiconductor, may account forhe different behavior observed when the Ru�II� photosensitizer issed instead.

The inactivation efficiency may be expressed by the effectiveisinfection time �EDT�, defined in a previous paper using TiO2hotocatalysis �3� as the time elapsed to observe reactivation ofacteria subject to the sunlight/photocatalyst treatment. The EDTs dependent of the initial bacterial population �Fig. 6�. Reactiva-ion was measured after 24 hr, when the initial E. coli concentra-ion was 105 CFU mL−1. However, an ETD24 was achieved in ourase when a 103 CFU mL−1 E. coli suspension was illuminated inhe presence of Ru�bpy�3

2+, as at such an initial level no bacteriaeactivation in the dark has been observed after 24 hr. It must betressed that the ETD24 was not reached by increasing the illumi-ation time up 6 hr for either wild or ATCC E. coli at05 CFU mL−1 �Fig. 7�.At high initial bacteria concentrations, dead cells and their ex-

reted intracellular components may compete for oxidation by thehotogenerated 1O2, leading to protection of the remaining activeacteria against the photodynamic treatment. Moreover, when thenitial bacterial concentration is low, there are, on average, morehotosensitizer molecules bound per microorganism, increasinghe accumulated damage responsible for the phototoxic effect.

Conclusions

The Ru�bpy�32+ photosensitizer has a better inactivating effect

han TiO2 and Rose bengal on both laboratory- and wild-type E.oli strains. Efficient photocatalytic disinfection would require aider interaction between bacteria and the TiO2 nanoparticles to

llow the highly oxidative action of the HO• radical. The photo-ynamic processes do not display this limitation due to their ho-ogeneous nature. The higher quantum yield of ROS generation

y the prototypical sensitizing dyes compared to the classical

ig. 7 Postirradiation events after 6 h of sunlight illuminationf waterborne „-�-… ATCC 4.3Ã105 CFU mL−1 or „-�-… wild E.oli 4.1Ã105 CFU mL−1 in the presence of 10 mg/L−1

u„bpy…3Cl2 ·6H2O

emiconductor photocatalysts determines the faster sunlight-

ournal of Solar Energy Engineering

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

activated water disinfection of photodynamic processes. In spiteof their lower quantum yields of singlet molecular oxygen produc-tion, ruthenium polypyridyl complexes display a better perfor-mance that the prototypical Rose bengal photosensitizer. As theformer is significantly more photostable than the polyhalogenatedtriarylmethane dye, a sustained generation of ROS toxic to bacte-ria is achieved. Moreover, the cationic nature of the rutheniumcomplex allows interaction with the bacterial wall enhancing thephotodynamic inactivation process. Under appropriate photosen-sitizer concentration and low bacteria levels, 60 min of solar illu-mination suffice for waterborne E. coli inactivation with absenceof regrowth for 24 hr.

The problems associated with the practical use of soluble Ru�II�dyes for domestic water disinfection with sunlight-generated sin-glet oxygen can be eliminated by immobilizing the photosensitiz-ers on a suitable polymer support, provided the latter promotesinteraction with the target bacteria. Closeness of the 1O2 photoge-nerator and the target microorganism is essential to achieve effi-cient disinfection due to the microseconds lifetime of this reactiveoxygen species in water. Such an approach has only recently beenrealized with promising results �26,27�.

AcknowledgmentThe authors thank the Swiss Agency for Development and Co-

operation for its support to the BIOSOLARDETOX project,OFES No. 02.0030 and No. 02.0316 within the “SOLWATER”�Contract No. ICA4-CT-2002-10001� and “AQUACAT” �ContractNo. ICA3-CT-2002-10016� European Union research projects, re-spectively, and the Environment cooperation program EPFL-Colombia for financial support. G. Orellana also acknowledgessupport by the Spanish Ministry of Education and Science �GrantNo. PPQ-2000-0778-C02-01�. We thank I. Monroy, D. Acevedoand J. Tintinago for their collaboration in some laboratory experi-ments and helpful comments. J. Rengifo-Herrera is very gratefulto B. Herrera, F. Herrera, M. L. Molina, and F. Rengifo for theircontinuous encouragement.

References�1� Richardson, S. D., and Ternes, T. A., 2005, “Water Analysis: Emerging Con-

taminants and Current Issues,” Anal. Chem., 77, pp. 3807–3838.�2� Faust, B. C., 1999, “Aquatic Photochemical Reactions in Atmospheric, Sur-

face, and Marine Waters: Influences on Oxidant Formation and Pollutant Deg-radation,” The Handbook of Environmental Chemistry, Boule, P. ed., Springer-Verlag, Berlin, Vol. 2, Part L: Environmental Photochemistry, pp. 101–122.

�3� Rincon, A. G., and Pulgarin, C., 2004, “Bactericidal Action of IlluminatedTiO2 on Pure Escherichia coli and Natural Bacterial Consortia: Post Irradia-tion Events in the Dark and Assessment of the Efficient Disinfection Time,”Appl. Catal., B, 49, pp. 99–112.

�4� Rincon, A. G., and Pulgarin, C., 2004, “Field Solar E. coli Inactivation in theAbsence and Presence of TiO2: Is UV Solar Dose an Appropriate Parameterfor Standardization of Water Solar Disinfection?,” Sol. Energy, 77, pp. 635–648.

�5� Dahl, T. A., Midden, W. R., and Hartman, P. E., 1987, “Pure Singlet OxygenCytotoxicity for Bacteria,” Photochem. Photobiol., 46, pp. 345–352.

�6� Dahl, T. A., 1993, “Direct Exposure of Mammalian Cells to Pure ExogenousSinglet Oxygen �1�gO2�,” Photochem. Photobiol., 57, pp. 248–254.

�7� Sies, H., and Menck, C., 1992, “Singlet Oxygen Induced DNA Damage,”Mutat Res., 275, pp. 367–375.

�8� Hergueta-Bravo, A., Jiménez-Hernández, M. E., Oliveros, E., Montero, F., andOrellana, G., 2002, “Singlet Oxygen-Mediated DNA Photocleavage WithRu�II� Polypyridyl Complexes,” J. Phys. Chem. B, 106, pp. 4010–4017, andreferences therein.

�9� Geoffroy, M., Lambelet, P., and Richert, P., 2000, “Role Of Hydroxyl Radicalsand Singlet Oxygen in the Formation Primary Radicals in Unsaturated Lipids:A Solid-State Electron Paramagnetic Resonance Study,” J. Agric. Food Chem.,48, pp. 974–978.

�10� Mills, A., Davies, R., and Worsley, D., 1993, “Water Purification by Semicon-ductor Photocatalysis,” Chem. Soc. Rev., 22, pp. 417–426.

�11� Fujishima, A., Rao, T. N., and Tryk, D. A., 2000, “Titanium Dioxide Photo-catalysis,” J. Photochem. Photobiol. C, 1, pp. 1–21.

�12� Cabiscol, E., Tamarit, J., and Ros, J., 2000, “Oxidative Stress in Bacteria andProtein Damage by Reactive Oxygen Species,” Int. Microbiol., 3, pp. 3–8.

�13� Blake, D. M., Maness, P.-Ch., Huang, Z., Wolfrum, E. J., Huang, J., andJacoby, W. A., 1999, “Application of The Photocatalytic Chemistry of Tita-nium Dioxide to Disinfection and Killing of Cancer Cells,” Sep. Purif. Meth-

ods, 28, pp. 1–50.

FEBRUARY 2007, Vol. 129 / 139

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm

1

Downloa

�14� Srinivasan, C., and Somasundaram, N., 2003, “Bactericidal and Detoxificationof Irradiated Semiconductor Catalyst TiO2,” Curr. Sci., 85, pp. 1431–1439.

�15� Huang, Z., Maness, P.-Ch., Blake, D. M., Wolfrum, E. J., Smolinski, S., andJacoby, W. A., 2000, “Bactericidal Mode of Titanium Dioxide Photocatalysis,”J. Photochem. Photobiol., A, 130, pp. 163–170.

�16� Schweitzer, C., and Schmidt, R., 2003, “Physical Mechanisms of Generationand Deactivation of Singlet Oxygen,” JAMA, J. Am. Med. Assoc., 103, pp.1685–1758.

�17� Jori, G., and Brown, S. B., 2004, “Photosensitized Inactivation of Microorgan-isms,” Photochem. Photobiol. Sci., 3, pp. 403–405.

�18� Acher, A. J., and Juven, B. J., 1977, “Destruction of Coliforms in Water andSewage Water by Dye-Sensitized Photooxidation,” Appl. Environ. Microbiol.,33, pp. 1019–1022.

�19� Schafer, M., Schmitz, C., Facius, R., Horneck, G., Milow, B., Funken, K.-H.,and Ortner, J., 2000, “Systematic Study of Parameters Influencing the Actionof Rose Bengal With Visible Light on Bacterial Cells: Comparison Betweenthe Biological Effect and Singlet-Oxygen Production,” Photochem. Photobiol.,71, pp. 514–523.

�20� Bonnet, R., Buckley, D. G., Burrow, T., Galia, A. A. B., Saville, B., andSongca, B., 1993, “Photobactericidal Materials Based on Phorphyrins and Ph-talocyanines,” J. Mater. Chem., 3, pp. 323–324.

�21� Cooper, A., Goswami, D. Y., 2002, “Evaluation of Methylene Blue and RoseBengal for Dye Sensitized Solar Water Treatment,” ASME J. Sol. Energy Eng.,124, pp. 305–311.

�22� Acher, A., Fischer, E., Turnheim, R., and Manor, Y., 1997, “EcologicallyFriendly Wastewater Disinfection Techniques,” Water Res., 31, pp. 1398–1404.

�23� García-Fresnadillo, D., Georgiadou, Y., Orellana, G., Braun, A. M., and Oliv-eros, E., 1996, “Singlet Oxygen Production by Ruthenium�II� Complexes Con-taining Polyazaheterocyclic Ligands in Methanol and in Water,” Helv. Chim.Acta, 79, pp. 1222–1238.

�24� Gutiérrez, M. I., Martínez, C. G., García-Fresnadillo, D., Castro, A. M., Orel-lana, G., Braun, A. M., and Oliveros, E., 2003, “Singlet Oxygen �1�g� Pro-duction by Ruthenium�II� Complexes in Microheterogeneous Systems,” J.Phys. Chem. A, 107, pp. 3397–3403.

�25� Juris, A., Balzani, V., Barigelletti, F., Campagna, S., Belser, P., and vonZelewski, A., 1988, “Ru�II� Polypyridine Complexes. Photophysics, Photo-chemistry, Electrochemistry and Chemiluminescence,” Coord. Chem. Rev.,84, pp. 85–277.

�26� Orellana, G., Jiménez-Hernández, M. E., and García-Fresnadillo, D., 2003,“Photocatalytic Material and Method for Water Disinfection,” Spanish PatentNo. 2,226,576.

40 / Vol. 129, FEBRUARY 2007

ded 02 Aug 2007 to 128.178.84.129. Redistribution subject to ASM

�27� Jiménez-Hernández, M. E., Manjón, F., García-Fresnadillo, D., and Orellana,G., 2005, “Solar Water Disinfection by Singlet Oxygen Photogenerated WithPolymer-Supported Ru�II� Sensitizers,” Sol. Energy, 80, pp. 1382–1387.

�28� Jiménez-Hernández, M. E., Portolés, T., Montero, G., and Orellana, G., 2000,“Cell Viability Measurements Using Luminescent Ruthenium�II� Probes,”Photochem. Photobiol., 72, pp. 28–34.

�29� Grela, M. A., and Colussi, A. J., 1996, “Kinetics and Stochastic Charge Trans-fer and Recombination Events in Semiconductor Colloids: Relevance to Pho-tocatalysis Efficiency,” J. Phys. Chem., 100, pp. 18214–18221.

�30� Schwarz, P. F., Turro, N. J., Bossmann, S. H., Braun, A. M., Abdel Wahab,A.-M. A., and Durr, H., 1997, “A New Method to Determine the Generation ofHydroxyl Radicals in Illuminated TiO2 Suspensions,” J. Phys. Chem. B, 101,pp. 7127–7134.

�31� Nowakowska, M., Kepczynski, M., and Szczubialka, K., 2001, “New Poly-meric Photosensitizers,” Pure Appl. Chem., 73, pp. 491–495.

�32� Dahl, T. A., 1994, “Examining the Role of Singlet Oxygen in PhotosensitizedCytotoxicity,” Aquatic and Surface Photochemistry, Helz, R. G., and Crosby,D. G., eds., CRC Press, Boca Raton, pp. 241–258.

�33� Jiménez-Hernández, M. E., 2001, “Luminescent Complexes of Ru�II� WithDibenzodipyridophenazine: DNA Interaction, Cell Viability Determinations,and Singlet Oxygen �1�g� Photosensitization,” Ph.D. thesis, Universidad Com-plutense, Madrid.

�34� Salmon-Divon, M., Nitzan, Y., and Malik, Z., 2004, “Mechanistic Aspects ofEscherichia coli Photodynamic Inactivation by Cationic Tetra-Meso�N-methylpyridyl�porphine,” Photochem. Photobiol. Sci., 3, pp. 423–429.

�35� Lambert, C. R., and Kochevar, I., 1997, “Electron Transfer Quenching of theRose Bengal Triplet State,” Photochem. Photobiol., 66, pp. 15–25.

�36� Seybold, P. G., Gouterman, M., and Callis, J., 1969, “Calorimetric, Photomet-ric and Lifetime Determinations of Fluorescence Yields of Fluorescein Dyes,”Photochem. Photobiol., 9, pp. 229–242.

�37� Dorfman, L. M., and Adams, G. E., 1973, “Reactivity of Hydroxyl Radical inAqueous Solutions,” National Standards Reference Data Service, National Bu-reau of Standards, No. C13.48:46, Gaithersburg, MD.

�38� Dobrucki, J. W., 2001, “Interaction of Oxygen Sensitive Luminescent ProbesRu�phen�3

2+ and Ru�bpy�32+ With Animal and Plant Cells In Vitro: Mechanism

of Phototoxicity and Conditions for Non-Invasive Oxygen Measurements,” J.Photochem. Photobiol., B, 65, pp. 136–144.

�39� Piette, J., 1991, “Biological Consequences Associated with DNA OxidationMediated by Singlet Oxygen,” J. Photochem. Photobiol., B, 11, pp. 241–260.

�40� Boaretti, M., Lleo, M. M., Bonato, B., Signoretto, C., and Canepari, P., 2003,“Involvement of RpoS in the Survival of Escherichia coli in the Viable butNon-Cultivable State,” Environ. Microbiol., 5, pp. 986–996.

Transactions of the ASME

E license or copyright, see http://www.asme.org/terms/Terms_Use.cfm