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Photocatalytic bacterial inactivation by polyoxometalates

Eunyoung Bae a, Jae Won Lee b, Byeong Hee Hwang b, Jiman Yeo a, Jeyong Yoon c, Hyung Joon Cha a,b,*, Wonyong Choi a,*
a

School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea c School of Chemical Engineering, Seoul National University, Seoul 151-742, Republic of Korea Received 17 November 2007; received in revised form 30 January 2008; accepted 30 January 2008 Available online 17 March 2008

Abstract The photocatalytic inactivation (PCI) of Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) was performed using polyoxometalate (POM) as a homogeneous photocatalyst and compared with that of heterogeneous TiO2 photocatalyst. Aqueous suspensions of the microorganisms (107108 cfu ml1) and POM (or TiO2) were irradiated with black light lamps. The POM-PCI was faster than (or comparable to) TiO2-PCI under the experimental conditions employed in this study. The relative eciency of POM-PCI was species-dependent. Among three POMs (H3PW12O40, H3PMo12O40, and H4SiW12O40) tested in this study, the inactivation of E. coli was fastest with H4SiW12O40 while that of B. subtilis was the most ecient with H3PW12O40. Although the biocidal action of TiO2 photocatalyst has been commonly ascribed to the role of photogenerated reactive oxygen species such as hydroxyl radicals and superoxides, the cell death mechanism with POM seems to be dierent from TiO2-PCI. While TiO2 caused the cell membrane disruption, POM did not induce the cell lysis. When methanol was added to the POM solution, not only the PCI of E. coli was enhanced (contrary to the case of TiO2-PCI) but also the dark inactivation was observed. This was ascribed to the in situ production of formaldehyde from the oxidation of methanol. The interesting biocidal property of POM photocatalyst might be utilized as a potential disinfectant technology. 2008 Elsevier Ltd. All rights reserved.
Keywords: Photocatalytic disinfection; Heteropoly acid; Deactivating microorganisms; Escherichia coli; Reactive oxygen species

1. Introduction Homogeneous or heterogeneous photocatalysis plays a central role in many photochemical conversion processes. As for heterogeneous photocatalysis, semiconductor oxides including TiO2 have been widely investigated for the complete oxidation of toxic contaminants in water and air (Ollis and Al-Ekabi, 1993; Homan et al., 1995; Choi, 2006). Since Matsunaga et al. (1985) reported the rst application of TiO2 photocatalysis to the inactivation of Escherichia coli, a number of studies on photocatalytic inactivation (PCI) of microorganisms have been conducted (Wei et al., 1994; Kikuchi et al., 1997; Cho et al., 2004,
Corresponding authors. Tel.: +82 54 279 2283; fax: +82 54 279 8299. E-mail addresses: hjcha@postech.ac.kr (H.J. Cha), wchoi@postech.ac.kr (W. Choi). 0045-6535/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.01.071
*

2005). The PCI of biological cells can be similarly compared with the photocatalytic degradation of chemical compounds. The photo-induced radical chemistry involving reactive oxygen species drives not only the degradation of chemical compounds but also the inactivation or the death of microbial cells. It is generally believed that the hydroxyl radical, which is the major oxidant of TiO2 photocatalysis, should attack and disrupt the cell wall or membrane to initiate the inactivation process (Ireland et al., 1993; Bekbo let, 1997; Lee et al., 1997; Cho et al., 2004). Polyoxometalates (POMs) have been studied as a homogeneous photocatalyst (Maldotti et al., 1994; Weinstock, 1998; Androulaki et al., 2000; Hiskia et al., 2001a) and often similarly compared with its heterogeneous counterpart, TiO2 (Kim et al., 2004; Park and Choi, 2005; Lv and Xu, 2006). POM is a well-organized metaloxygen cluster anion, which initiates a variety of redox reactions

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under UV-illuminated condition (Yamase, 1998; Hiskia et al., 2001b; Song and Barteau, 2004). Common POMs that have been investigated as photocatalysts include tungstosilicic acid (H4SiW12O40), phosphotungstic acid (H3PW12O40), and phosphomolybdic acid (H3PMo12O40) (Hiskia et al., 2001a,b; Song and Barteau, 2004). POM and TiO2 share the similar photochemical mechanisms in their operation. Photoexcited POMs carry a strong oxidant power to directly abstract an electron from substrates or to generate OH radicals through water oxidation as the bandgap excited TiO2 does. Similarities between the homogeneous and heterogeneous photocatalysts (POMs vs TiO2) have been recognized, and a few comparative studies on their photocatalytic behaviors were carried out (Ozer and Ferry, 2002; Kim et al., 2004; Park and Choi, 2005; Lv and Xu, 2006). Although the photocatalytic biocidal eects of TiO2 have been widely recognized and investigated (Wei et al., 1994; Cho et al., 2004, 2005), the similar eects of POMs have not been reported yet. In the present work, a comparative study of POMs and TiO2 as an inactivation photocatalyst was done using E. coli and Bacillus subtilis as a representative of Gram-negative and positive bacteria, respectively. 2. Experimental section 2.1. Chemicals and materials H3PW12O40 (Aldrich), H3PMo12O40 (Fluka), and H4SiW12O40 (Aldrich) were used as homogeneous photocatalysts without any further treatment. Each POM is abbreviated as PW12, PMo12, and SiW12, respectively, throughout the text. TiO2 (Degussa P25), a mixture of 80% anatase and 20% rutile with an average surface area of 50 15 m2 g1, was used as a heterogeneous photocatalyst. Methanol (MeOH; Samchun, Korea) was used as received. Deionized water was ultrapure (18 MX cm) and prepared by a Barnstead purication system. All glassware used in these experiments were washed with distilled water, and then autoclaved at 121 C for 15 min. E. coli (ATCC 8739), a well-known indicator for Gram-negative bacterium, and B. subtilis (NRRL B-23049), a well-known indicator for Gram-positive bacterium, were chosen as the test microorganisms for PCI. 2.2. Photocatalytic inactivation experiments POM (or TiO2) was dissolved (or dispersed) in distilled water by simultaneous sonication and shaking for 30 s in an ultrasonic cleaning bath. E. coli and B. subtilis were grown in Luria Bertain-medium (Merck) containing 10 g l1 tryptone, 5 g l1 yeast extract, and 10 g l1 NaCl at 37 C with shaking at 200 rpm overnight. We used only harvested cells that were separated from the medium to avoid the interference of the LB-medium components in PCI reaction. B. subtilis that was used in this PCI test was cultured under the condition where the spore forma-

tion was not favored. The pH of TiO2/E. coli suspension was adjusted to 7.1 using a phosphate buer (KH2PO4/ NaOH). In the absence of the phosphate buer, microorganisms sampled from the TiO2 suspension were not cultured well. It seems that the strong anity between TiO2 and microorganisms hinders the culturing process. Therefore, PCI experiments employing suspended TiO2 particles were usually carried out in the phosphate buer solutions (Matsunaga et al., 1985; Cho et al., 2004, 2005). On the other hand, when the pH of POM/E. coli suspension was adjusted to 7.1 using the same phosphate buer used in the TiO2-PCI experiment, no PCI of microorganisms was observed. The PCI activity of POM seems to work only at acidic condition since POMs are stable only at acidic conditions. Therefore, all POM-PCI experiments were conducted at acidic pH without using the phosphate buer. POM-PCI and TiO2-PCI experiments were carried out and compared at dierent pH because their optimal operating conditions were dierent. Incidentally, the pH eect on the TiO2-PCI eciency seems to be insignicant. A previous PCI study employing the same TiO2 (P25 TiO2 g l1) as a photocatalyst showed that the E. coli inactivation kinetics was not aected by pH (5.6, 7.1, and 8.2) at all (Cho et al., 2004). Unlike TiO2, POMs have the intrinsic biocidal eect even in the absence of light as Table 1 shows. The viability of E. coli under the dark environment was not inhibited by POM up to [PW12] = 0.7 mM, [SiW12] = 0.1 mM, and [PMo12] = 0.05 mM. However, when [POM] increased above this critical value, E. coli was signicantly inactivated even in the dark condition. Therefore, most PCI experiments in this work were conducted with [POM] at which the dark biocidal eect was not observed. The employed concentration of each POM was dierent: [PW12] = 0.35, [SiW12] = 0.1, and [PMo12] = 0.05 mM. When we tried to compare the PCI activity of three POMs at the same POM concentration, some PCI activity was so high that the time proles of the PCI could not be obtained. Therefore, the employed POM concentrations were the result of the adjustment so that the time proles of Log (N/N0) in POM-PCI can be comparable in the same time scale (040 min) among dierent POMs and TiO2.
Table 1 The dark inactivation of E. coli (in 20 min) in the presence of POMs (PW12, SiW12, PMo12) SiW12 [POM] (mM) 0 0.05 0.1 0.25 0.3 0.5 E. coli (Log (N/N0)) 0 0 0 3.14 3.4 4.7 PMo12 [POM] (mM) 0 0.05 0.1 0.5 1 E. coli (Log (N/N0)) 0 0 2.7 4.3 n.v.c.a PW12 [POM] (mM) 0 0.35 0.5 0.7 1 2 E. coli (Log (N/N0)) 0 0 0 0 3.11 3.21

N, concentration of microorganisms (in cfu per milliliter); N0, initial N. a No viable cells.

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Each POM was dissolved in water to yield an acidic pH (2 3) and the initial pH slightly depended on [POM]. The initial pH of the microbial suspensions with PW12, SiW12, and PMo12 (at 0.35, 0.1, and 0.05 mM, respectively) was 3.5, 2.7 and 2.3, respectively. Although such an acidic condition is generally not favorable for microorganisms, the dark control tests (POMs/E. coli) under the same [POM] and pH condition where the POM-PCI experiments were carried out showed no marked sign of E. coli inactivation up to 6 h. Initial concentrations of E. coli and B. subtilis were adjusted to 107 (with TiO2) or 108 cfu ml1 (with POM) for PCI. The initial cell concentration employed in POMPCI was 10 times as high as that in TiO2-PCI because some POM-PCI kinetics for E. coli was so fast that the colony counting could not be easily done with the same cell concentration employed in TiO2-PCI. It should be noted that since we are comparing the PCI activity of three POMs and TiO2 at dierent conditions (e.g. catalyst concentration, cell concentration, pH) for the reasons described above, the direct comparison of the PCI activity among different photocatalysts is not very meaningful. The main focus of this work is not to provide a quantitative comparison among dierent POMs and TiO2, but to demonstrate that the POM-PCI works eciently like the well-established TiO2-PCI. The PCI experiments were carried out in a 30-ml Pyrex reactor (20 ml solution) with magnetic stirring. The light source for the bacterial inactivation was black light blue lamps (BLB 10 W 4; Philips) which emit mainly in the 300420 nm range. The UV absorption spectrum of each POM and the BLB lamp emission spectrum are compared in Fig. 1. The UV absorption spectra of POMs were recorded with a UVvis spectrophotometer (UV-2401PC, Shimadzu) and the relative emission spectrum of the BLB lamp was measured using a radiometer (Ocean Optics, USB2000). The incident light intensity was measured by the standard ferrioxalate actinometry (Hatchard and

Parker, 1956) and determined to be 3.3 106 E l1 s1. All three POMs have absorption spectra that overlap with the BLB emission and PW12 shows the highest UV absorption. When the eect of dissolved oxygen was investigated, the reactor was purged with nitrogen gas for 30 min prior to the UV irradiation and sealed from the ambient air during the irradiation. When needed, MeOH (30 mM) was added as a hydroxyl radical scavenger. The in situ generated formaldehyde concentration was determined colorimetrically by using chromotropic acid as a reagent (Feigl, 1956). Two microlitre of sample solution was transferred into a test tube and then 100 ll of 1% chromotropic acid and 2.5 ml concentrated H2SO4 were added. The test tubes containing the sample or the standard were heated for 10 min in a steam bath then cooled to room temperature. The resulting coloration (violetred) was quantied by the absorbance at 572 nm and calibrated against the standard. The microbial cell concentration was measured without separating the microorganisms from POM (or TiO2 particles) as previously described by Wei et al. (1994). To determine the concentrations of E. coli and B. subtilis, an aliquot of 1 ml sample solution was withdrawn intermittently during irradiation and diluted. One-tenth of a milliliter of the undiluted and diluted solutions was used to count the number of microbial cells. The cell numbers were determined by spreading the sample on an agar plate and counting colony numbers which appeared after 24 h incubation at 37 C. All the PCI experiments were repeated at least three times under the identical condition with using the dierent culture batch of bacteria and the average concentration with the statistical deviation were used for the data analysis. 2.3. Amplication of 16S rDNA by PCR To investigate whether the cell disruption happens in PCI, we carried out polymerase chain reaction (PCR) tests. The primer sets (sense 16S 27F: 50 AGAGTTTGATCMTGGCT-30 , antisense 16S 1100R: 50 -GGGTTGCGCTCGTT-30 ) were used in PCR to amplify 1073 bp 16S rDNA fragment. Total 20 ll reaction mixture consisted of 1 ll of the PCI-sample supernatant as template, 1 U Taq DNA polymerase (Bioneer, Korea), 250 mM of dNTPs, 1.5 mM of MgCl2, 1 ll of each primer (10 pmol), and 10X polymerase buer. For each reaction, the reaction mixture was pre-denatured at 95 C for 5 min to ensure the complete dissociation of the template DNA. The amplication was performed based on 30 cycles of amplication that consists of denaturation at 95 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 1 min. For the last cycle, the elongation was prolonged to 7 min to ensure proper extension of the bases. Five microlitre from the total 20 ll of the PCR mixture was electrophoresed on 1% (w/v) agarose gel containing 0.2 mg ml1 of ethidium bromide (Sigma). The gel was then run in a Trisborate EDTA buer at 100 V with 5 ll of a standard 100 bp DNA ladder marker.

3.0
PMo12

1.0
SiW12 PW12

2.5

0.8

Black light

Absorbance

2.0 0.6 1.5 0.4 1.0 0.5 0.0 250 0.2

300

350

400

0.0 450

Wavelength (nm)
Fig. 1. The relative emission spectrum of the BLB lamp and the UV absorption spectra of aqueous POMs at the concentration employed in the PCI experiment. [PW12] = 0.35 mM, [SiW12] = 0.1 mM, [PMo12] = 0.05 mM.

Intensity (a.u.)

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3. Results and discussion POM is very similar to TiO2 photocatalyst in its light absorption and the band-edge positions (Hiskia et al., 2001b; Kim et al., 2004; Park and Choi, 2005). POM is excited by absorbing UV photons and subsequently induces the electron transfer reactions (Maldotti et al., 1994; Weinstock, 1998; Androulaki et al., 2000). The UV excitation of POM induces a ligand (oxygen)-to-metal charge transfer with promoting an electron from the highest occupied molecular orbital to the lowest unoccupied molecular orbital. The resulting charge-transfer excited state POM* has a highly oxidizing power that is strong enough to oxidize a variety of organic compounds (D) or to generate OH radicals reactions ((1)(3)). POM hm ! POM POM D ! POM D POM H2 O ! POM H OH

1 2 3

Three types of POMs (PW12, SiW12, PMo12) were tested for the PCI of E. coli under the identical irradiation condition. We compared the kinetics of the PCI of E. coli among three POMs and TiO2 in air-equilibrated water (Fig. 2a). The employed concentration of TiO2 was 1 g l1 which cor-

0 -1 -2 -3 -4 -5 -6 0 10 20 30
UV Control TiO2 PW12 SiW12 PMo12 Dark control

40

50

b
0 -1

Log (N/N0)

-2 -3 -4 -5

UV Control TiO2 PW12 SiW12 PMo12

Dark control

10

20

30

40

50

Irradiation time (min)

Fig. 2. Photocatalytic inactivation of (a) E. coli and (b) B. subtilis in POM and TiO2 suspensions. N, concentration of microorganisms (in cfu per milliliter); N0, initial N. [PW12] = 0.35 mM, [SiW12] = 0.1 mM, [PMo12] = 0.05 mM, [TiO2] = 1 g l1.

responds to 12.5 mM of monomeric TiO2 units. Although the concentrations of POMs (homogeneous catalyst) and TiO2 (heterogeneous catalyst) cannot be directly compared, [PW12] = 1 mM and [TiO2] = 0.5 g l1 were similarly compared in the photocatalytic degradation of 4-chlorophenol (Kim et al., 2004). Even though the POM concentrations used in this study are much lower than 1 mM, POM was at least as eective as TiO2 (1 g l1) in PCI. The control test in which either POM or light was absent showed no inactivation of E. coli. Fig. 2a shows that E. coli cells were inactivated faster with POMs than with TiO2 under UV light. In particular, PMo12 and SiW12 are extremely eective with causing $3 log decrease within a minute. PW12 is markedly less active in PCI although it absorbed more UV photons than the others. This clearly indicates that the POM-PCI activity is not correlated with the light absorption. Photocatalytic activity in general does not show a direct correlation with the absorbed light intensity since the dynamics of the charge recombination/transfer following the photoexcitation and the catalyst interfacial properties are very dierent from catalyst to catalyst. Fig. 3 shows the dependence of the PCI activity for E. coli on [POM]. The higher POM concentration is apparently more eective in PCI and each POM exhibits dierent concentration-dependence. The POM-PCI eciency of E. coli decreased in the order of PMo12 > SiW12 > PW12 at the same POM concentration (0.1 mM). PMo12 was particularly active and induced more than 4 log decrease in just 10-min irradiation with [PMo12] = 0.1 mM. Note that a similar level of PCI with TiO2 (0.5 g l1) could not be obtained even after 40-min irradiation (Fig. 2a). As for the TiO2-PCI of E. coli, the inactivation eciency increased with [TiO2 (P25)] up to 1 g l1 above which the PCI eect was saturated (Wei et al., 1994; Cho et al., 2004). We also compared the PCI of Gram-positive B. subtilis (under spore-free condition) using three POMs and TiO2 in air-equilibrated water (Fig. 2b), which can be directly compared with that of Gram-negative E. coli (Fig. 2a). Gram-negative and Gram-positive bacterial cells are dierent, particularly with respect to their cell wall structure. Gram-positive bacteria have thick single ($90% peptidoglycan) layer cell wall while Gram-negative bacteria have thin multi-layer cell wall composed of peptidoglycan (520%), lipopolysaccharide, and proteins. The signicant dierence in the cell wall structure might aect the bactericidal eciency and mechanism depending on the type of microbial species and main reactive oxygen species. Note the marked dierence between the two PCI cases. PW12 showed the highest PCI activity for B. subtilis whereas it was the least active for the PCI of E. coli. The PCI activity of SiW12 and PMo12 for B. subtilis was much reduced from that for E. coli. On the contrary, TiO2-PCI activity for B. subtilis was higher than for E. coli. Therefore, the PCI eciency of each photocatalyst is apparently species-dependent. Considering that the photocatalytic mechanisms of organic compounds degradation can be very dierent depending on the type of substrate and catalyst (Kim and Choi,

Log (N/N0)

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a
0 -1

-2

-3
Dark control PW12

-4

-5 0.0 0.2 0.4 0.6 0.8

b
0 -1

-2 -3
Dark control

-4 -5 0.00

SiW12

0.03

0.06

0.09

0.12

0.15

c
0 -1

Log (N/N0)

nents and the probability of the disinfectants penetration into the cell. The cell wall-disinfectant interaction seems to be responsible for the species specicity in PCI. The PCI process may occur through several dierent mechanisms. As for TiO2-PCI, it is widely accepted that the destruction of the cell membrane is involved as the critical step. To investigate whether cell death is due to the cell membrane disruption in this PCI study, we employed the PCR method. If bacterial cells are disrupted during the PCI reaction, chromosomal DNA molecules will be released into solution and a specic DNA sequence based on the released chromosomal DNA (template) can be amplied using PCR. We selected 16S rDNA as the target DNA for amplication because 16S rDNA is the typical nger print of microorganisms. While 16S rDNA band was not signicantly amplied in the TiO2/dark/E. coli system (Fig. 4a, lane 7), the band amplication was outstanding in the TiO2/UV/E. coli (lane 8). The control cell lysis was induced by the surfactant treatment (sodium dodecylsulfate: SDS) (lane 9) in the dark, which showed a PCR result similar to the case of TiO2/UV treatment (lane 8). This conrms that TiO2 disrupts microbial cells under UV light, which is consistent with the previous reports (Sunada et al., 2003; Cho et al., 2005; Nadtochenko et al., 2005). However, all three POMs showed very dim bands (lanes 46) whose intensity is similar to the case of dark (lane 1) and UV alone (lane 2) systems. The same results were obtained with B. subtilis (data not shown). The PCR results indicate that the TiO2-PCI involves the cell lysis but the POM-PCI does not. Alternatively, the absence of 16S rDNA band may be ascribed to the photocatalytic degradation of the DNA molecules by POM, not

Log (N/N0)

Log (N/N0)

-2 -3

M
Dark control

-4 -5 0.00

PMo12

1,073 bp
0.02 0.04 0.06 0.08 0.10

[POM] (mM)
Fig. 3. Eect of POM [(a) PW12, (b) SiW12, and (c) PMo12] concentration on the inactivation of E. coli. reaction time: 10 min. N, concentration of microorganisms (in cfu per milliliter); N0, initial N. The UV control refers to the direct photo-inactivation in the absence of POM.

2002; Lee and Choi, 2004), it is reasonable to believe that the PCI mechanisms can be also species-dependent. Accordingly, Cho et al. (2005) reported that the TiO2-PCI mechanism is dierent depending on the type of microorganisms. In general, the overall PCI process should be inuenced by both the cell wall structure (depending on the type of bacterial species) and the type of main reactive oxygen species responsible for the PCI action. Since the cell wall is the primary barrier for the disinfectant, the PCI activity should be related with the reactivity of the disinfectant (mainly reactive oxygen species) with the cell wall compo-

1,073 bp

Fig. 4. Amplication of 16S rDNA using PCR from the supernatant of the photocatalyst-E. coli suspensions (a) without and (b) with MeOH. (a) M: 100 bp size marker, 1: dark, 2: UV alone, 3: PW12/dark, 4: PW12/UV, 5: SiW12/UV, 6: PMo12/UV, 7: TiO2/dark, 8: TiO2/UV, 9: 0.02% SDS treatment in the dark. (b) M: 100 bp size marker, 1: TiO2/UV, 2: TiO2/ MeOH/UV, 3: PW12/MeOH/UV, 4: PW12/UV. [PW12] = 0.35 mM, [SiW12] = 0.1 mM, [PMo12] = 0.05 mM, [TiO2] = 1 g l1, reaction time 20 min.

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to the absence of the cell lysis. To test this possibility, we performed control photocatalysis experiments using 16S rDNA as the starting substrate and found that 16S rDNA molecules were not signicantly degraded by either TiO2 or POM (PW12) under the PCI condition we employed. Therefore, the absence of 16S rDNA band indicates the lack of cell lysis. POM and TiO2 photocatalysts are similar in their action mechanisms but they are dierent in many ways. For example, Kim et al. (2004) compared the photocatalytic degradation of 4-CP in the deaerated solution between the POM and TiO2 systems and showed that the degradation of 4-CP in the POM/N2 system was little dierent from that in the POM/air system whereas no degradation of 4-CP was observed in the TiO2/N2 system. The excited POM can abstract an electron from substrates and hold the electron. This property enables the anoxic activity of POM photocatalyst in the absence of dissolved O2 (electron acceptor) whereas the TiO2 photocatalytic activity is mostly inhibited in the deaerated suspensions. We compared the PCI kinetics of E. coli with POMs and TiO2 under the nitrogen-saturated condition (Fig. 5). The anaerobic PCI activity was higher with POMs than TiO2 as expected. More than 90% inactivation could be achieved within 20 min even in the deaerated POM solution. However, the anaerobic POM-PCI activity was markedly reduced compared with that of the aerated condition (Figs. 2a and 5). In particular, the activity of SiW12 and PMo12 was highly reduced in the absence of O2. Like TiO2 photocatalyst, POMs demonstrate to be a good inactivation photocatalyst for both Gram-negative and Gram-positive bacteria in this study. TiO2 photocatalysis has been reported to induce a number of functional alterations in cells including the cellular membrane permeability (Satio et al., 1992; Wamer et al., 1997), release of macromolecules (Satio et al., 1992), and cytotoxicity (Wamer et al., 1997). Hydroxyl radicals are considered as the primary oxidant in TiO2 photocatalysis for not only chemical substrates but also microorganisms. For example, Cho et al. (2004) showed that the TiO2-PCI eciency for
0.0 -0.5

Log (N/N0)

-1.0 -1.5 -2.0 -2.5 -3.0

UV control TiO2
PW12 SiW12 PMo12

10

15

20

Irradiation time (min)

Fig. 5. Photocatalytic inactivation of E. coli in N2-saturated suspensions of POM and TiO2. N, concentration of microorganisms (in cfu per milliliter); N0, initial N. [PW12] = 0.35 mM, [SiW12] = 0.1 mM, [PMo12] = 0.05 mM, [TiO2] = 1 g l1.

E. coli is linearly correlated with the steady-state concentration of OH radicals generated in UV-illuminated TiO2 suspension, which conrms that the OH radical is the primary species responsible for E. coli inactivation. The OH radicals are also considered as the main oxidant in POMmediated photocatalysis. Kim et al. (2004) demonstrated that the POM-mediated photooxidation is completely inhibited in the presence of tert-butanol (t-BuOH: OH radical scavenger) regardless of the type of substrates and concluded that the OH radical is the sole dominant photooxidant in PW12-mediated photocatalysis. In the similar context, the OH radical may be an important oxidant in the POM-PCI of microorganisms. The eects of the hydroxyl radical scavengers on the PCI of E. coli were compared between POMs and TiO2 (Fig. 6). MeOH was used as the primary scavenger and t-BuOH as the secondary scavenger for comparison. The eects of MeOH on POM-PCI (Fig. 6ac) were drastically dierent from those on TiO2-PCI (Fig. 6d). The eects of t-BuOH are also shown for SiW12 (Fig. 6b) and TiO2 (Fig. 6d). The inactivation of E. coli on TiO2 was signicantly reduced but not completely inhibited in the presence of MeOH (or t-BuOH). This is consistent with previous reports (Cho et al., 2004, 2005). Since the TiO2-PCI is mediated by not only OH radicals but also other reactive oxygen species such as O 2 and H2O2 that pass through the cell membrane (Kikuchi et al., 1997), the inactivation cannot be completely quenched by OH radical scavengers (Cho et al., 2005). Although the specic biocidal mechanisms seem to be diverse, the TiO2-PCI eciency is always reduced in the presence of OH radical scavengers. On the other hand, the POM-PCI was enhanced in the presence of MeOH (or t-BuOH) on the contrary (Fig. 6ac). The data do not support that OH radicals are the main oxidant in POM-PCI. In addition, even the dark control experiments in the MeOHPOM system also exhibited a signicant inactivation eect although the presence of MeOH alone did not induce any inactivation of E. coli. The dark disinfection eect of t-BuOHPOM system is even greater than MeOHPOM. Considering that the ground state POMs are known to be moderate oxidants with the one electron-reduction potentials of E0(SiW12) = 0.054 VNHE, E0(PW12) = 0.218 VNHE, and E0(PMo12) = 0.65 VNHE (Akid and Darwent, 1985; Hiskia et al., 2001b), the reaction (dark as well as photochemical) between POMs and alcohols seems to generate oxidized byproducts that are toxic to E. coli. The excited POMs are far stronger oxidants and can carry out the photochemical alcohol dehydrogenation (Yamase, 1998). The reaction of POM and MeOH results in the formation of formaldehyde (HCHO; reaction (4)), which is a toxin to microorganisms. It was reported that formaldehyde has a bactericidal property at low levels (LC50 for E. coli = 1 ppm or 33 lM) (Karel, 2001). 2POM MeOH ! 2POM HCHO 2H 4

Our study conrmed that formaldehyde was generated in situ from the reaction of POM and MeOH in the dark

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0
MeOH alone PW12 + MeOH (Dark)

0
MeOH alone SiW12 + MeOH (Dark) SiW12+ MeOH SiW12 SiW12 + t-BuOH (Dark) SiW12 + t-BuOH

-1

PW12 + MeOH PW12

-2

Log (N/N0)

-2

-4

-3

-6

-4 0 5 10 15 20 25

-8 0 5 10 15 20 25

-2

-1

Log (N/N0)

-4

-2
UV control TiO 2

-6

MeOH alone PMo12 + MeOH (Dark) PMo12 + MeOH PMo12

-3

TiO2 + MeOH TiO 2 + t-BuOH

-8 0 5 10 15 20 25

-4 0 10 20 30 40

Irradiation time (min)

Irradiation time (min)

Fig. 6. Eect of OH radical scavenger ([MeOH] = [t-BuOH] = 30 mM) on the inactivation of E. coli in illuminated POM [(a) PW12, (b) SiW12, and (c) PMo12] and (d) TiO2 suspensions. N, concentration of microorganisms (in cfu per milliliter); N0, initial N. [PW12] = 0.35 mM, [SiW12] = 0.1 mM, [PMo12] = 0.05 mM, [TiO2] = 1 g l1.

as well as under UV illumination. With 30 mM MeOH, dark POM reaction produced 515 lM HCHO while UV-POM reaction generated 20110 lM HCHO in 20 min. The formaldehyde concentration level was high enough to show toxicity. A dark control test with E. coli and 10 lM HCHO (in the absence of POM) showed $2 log decrease in 20 min. Therefore, the MeOH (or t-BuOH) eect on the POM-PCI is not related with its role as the OH radical scavenger but with the unexpected production of toxic byproducts (e.g. formaldehyde) from the oxidation of alcohols. We also investigated the MeOH eects on PCI using the PCR method. We found that the intensity of 16S rDNA band was highly reduced when MeOH (OH radical scavenger) was added in the TiO2/UV system (Fig. 4b, lane 1 vs lane 2). However, the cell lysis did not occur with POM either in the presence or absence of MeOH (Fig. 4b, lane 3 vs lane 4). Judging from this observation, TiO2-PCI is mediated by the action of hydroxyl radicals with the cell disruption accompanied but the OH radical-induced cell lysis does not seem to occur in POM-PCI. Although the eect of MeOH in POM-PCI is certainly unexpected and has little practical meaning for real PCI, it clearly shows that POM-PCI is very dierent from TiO2-PCI.

Although we do not have any more evidences to elaborate on the POM-PCI mechanism, a plausible hypothesis is that POM penetrates the cell membrane and the damaging oxidation reactions are initiated inside the cell. The size of POM is small enough (e.g. 0.8 nm for PW12) (Song and Barteau, 2004) to penetrate the cell membrane although the size is not the sole factor to determine the degree of penetration into the cell membrane. For example, silver nanoparticles in the size range of 110 nm are able to penetrate the bacterial cell membrane and cause the damage by possibly interacting with biomolecules such as DNA within the cell (Morones et al., 2005). The dark bactericidal activity of POMs shown in this work supports such mechanism because any reactive oxygen species cannot be generated in the dark/POM system. The UV irradiation excites the intracellular POM and the excited POM may react with a variety of intracellular components with enhancing the biocidal activity of POM. The fact that UV light penetrates into microbial cells to damage DNA structure is well established from the studies of UV disinfection technologies (Oppenla nder, 2003). Since the cell disruption in POM-PCI was not observed up to 20 min irradiation, the OH radical-induced cell lysis may take much longer time in POM-PCI.

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4. Conclusions This study demonstrates that POM has an interesting photochemical biocidal property that is dierent from TiO2 photocatalyst in many aspects. Although POM-PCI has many disadvantages such as the acidic working condition, the dicult recovery of POM from water, and the relatively high cost of POM, it could be a highly ecient disinfection method when a rapid biocidal action is required. Note that 99.9% of E. coli inactivation could be achieved with SiW12 in a minute. However, the POMPCI mechanism is unknown at this stage and needs to be investigated in further studies. Incidentally, POMs can be also immobilized on oxide supports to make their recovery easier in practical applications (Kim et al., 2004). Acknowledgements The authors would like to acknowledge the nancial supports from the ERC program (Grant No. R01-2006000-10055-0; to H.J. Cha) and the SRC program (Grant No. R11-2000-070-080010; to W. Choi) of MOST/KOSEF, KOSEF (Grant No. R01-2003-000-10053-0), and the Brain Korea 21 program. References
Akid, R., Darwent, J.R., 1985. Heteropolytungstates as catalysts for the photochemical reduction of oxygen and water. J. Chem. Soc. Dalton Trans., 395399. Androulaki, E., Hiskia, A., Dimotikali, D., Minero, C., Calza, P., Pelizzetti, E., Papaconstantinou, E., 2000. Light induced elimination of mono- and polychlorinated phenols from aqueous solutions by PW12 O3 40 . The case of 2,4,6-trichlorophenol. Environ. Sci. Technol. 34, 20242028. Bekbo let, M., 1997. Photocatalytic bactericidal activity of TiO2 in aqueous suspensions of E. coli. Water Sci. Technol. 35 (1112), 95100. Cho, M., Chung, H., Choi, W., Yoon, J., 2004. Linear correlation between inactivation of E. coli and OH radical concentration in TiO2 photocatalytic disinfection. Water Res. 38, 10691077. Cho, M., Chung, H., Choi, W., Yoon, J., 2005. Dierent inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection. Appl. Environ. Microbiol. 71, 270275. Choi, W., 2006. Pure and modied TiO2 photocatalysts and their environmental applications. Catal. Surv. Asia 10, 1628. Feigl, F., 1956. Spot Tests in Organic Analysis. Elsevier, Amsterdam. Hatchard, C.G., Parker, C.A., 1956. A new sensitive chemical actinometer. Proc. R. Soc. London A 235, 518536. Hiskia, A., Ecke, M., Troupis, A., Kokorakis, A., Hennig, H., Papaconstantinou, E., 2001a. Sonolytic, photolytic, and photocatalytic decomposition of atrazine in the presence of polyoxometalates. Environ. Sci. Technol. 35, 23582364. Hiskia, A., Mylonas, A., Papaconstantinou, E., 2001b. Comparison of the photoredox properties of polyoxometallates and semiconducting particles. Chem. Soc. Rev. 30, 6269. Homan, M.R., Martin, S.T., Choi, W., Bahnemann, D.W., 1995. Environmental applications of semiconductor photocatalysis. Chem. Rev. 95, 6996. Ireland, J.C., Klostermann, P., Rice, E.W., Clark, R.M., 1993. Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation. Appl. Environ. Microbiol. 59, 16681670.

Karel, V., 2001. Handbook of Environmental Data on Organic Chemicals. Wiley, New York. Kikuchi, Y., Sunada, K., Iyoda, T., Hashimoto, K., Fujishima, A., 1997. Photocatalytic bactericidal eect of TiO2 thin lms: dynamic view of the active oxygen species responsible for the eect. J. Photochem. Photobiol. A 106, 5156. Kim, S., Choi, W., 2002. Kinetics and mechanisms of photocatalytic degradation of (CH3)n NH 4n (0 6 n 6 4) in TiO2 suspension: the role of OH radicals. Environ. Sci. Technol. 36, 20192025. Kim, S., Park, H., Choi, W., 2004. Comparative study of homogeneous and heterogeneous photocatalytic redox reactions: PW12 O3 40 vs TiO2. J. Phys. Chem. B 108, 64026411. Lee, J., Choi, W., 2004. Eect of Pt deposits on TiO2 on the anoxic photocatalytic degradation pathways of alkylamines in water dealkylation and N-alkylation. Environ. Sci. Technol. 38, 40264033. Lee, S., Nishida, K., Otaki, M., Ohgaki, S., 1997. Photocatalytic inactivation of phage QB by immobilized titanium dioxide mediated photocatalyst. Water Sci. Technol. 35 (1112), 101106. Lv, K., Xu, Y., 2006. Eects of polyoxometalate and uoride on adsorption and photocatalytic degradation of organic dye X3B on TiO2: the dierence in the production of reactive species. J. Phys. Chem. B 110, 62046212. Maldotti, A., Amadelli, R., Varani, G., Tollari, S., Porta, F., 1994. Photocatalytic processes with polyoxotungsates: oxidation of cyclohexylamine. Inorg. Chem. 33, 29682973. Matsunaga, T., Tomoda, R., Nakajima, T., Wake, K., 1985. Photochemical sterilization of microbial cells by semiconductor powders. FEMS Microbiol. Lett. 29, 211214. Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., rez, J.T., Yacaman, M.J., 2005. The bactericidal eect of silver Ram nanoparticles. Nanotechnology 16, 23462353. Nadtochenko, V.A., Rincon, A.G., Stanca, S.E., Kiwi, J., 2005. Dynamics of E. coli membrane cell peroxidation during TiO2 photocatalysis studied by ATR-FTIR spectroscopy and AFM microscopy. J. Photochem. Photobiol. A 169, 131137. Ollis, D.F., Al-Ekabi, H., 1993. Photocatalytic Purication and Treatment of Water and Air. Elsevier, Amsterdam. Oppenla nder, T., 2003. Photochemical Purication of Water and Air. Wiley-VCH, Weinheim. Ozer, R.R., Ferry, J.L., 2002. Photocatalytic oxidation of aqueous 1,2dichlorobenzene by polyoxometalates supported on the NaY zeolite. J. Phys. Chem. B 106, 43364342. Park, H., Choi, W., 2005. Photocatalytic conversion of benzene to phenol using modied TiO2 and polyoxometalates. Catal. Today 101, 291 297. Satio, T., Iwase, T., Horie, J., Morioka, T., 1992. Mode of photocatalytic bactericidal action of powered semiconductor TiO2 on mutans streptococci. J. Photochem. Photobiol. B 14, 369379. Song, I.K., Barteau, M.A., 2004. Redox properties of Keggin-type heteropolyacid (HPA) catalysts: eect of counter-cation, heteroatom, and polyatom substitution. J. Mol. Catal. A-Chem. 212, 229 236. Sunada, K., Watanabe, T., Hashimoto, K., 2003. Studies on photokilling of bacteria on TiO2 thin lm. J. Photochem. Photobiol. A 156, 227 233. Wamer, W.G., Yin, J.-J., Wei, R.R., 1997. Oxidative damage to nucleic acids photosensitized by titanium dioxide. Free Radical Bio. Med. 23, 851858. Wei, C., Lin, W.-Y., Zainal, Z., Williams, N.E., Zhu, K., Kruzic, A.P., Smith, R.L., Rajeshwar, K., 1994. Bactericidal activity of TiO2 photocatalyst in aqueous media: toward a solar-assisted water disinfection system. Environ. Sci. Technol. 28, 934938. Weinstock, I.A., 1998. Homogeneous-phase electron-transfer reactions of polyoxometalates. Chem. Rev. 98, 113170. Yamase, T., 1998. Photo- and electrochromism of polyoxometalates and related materials. Chem. Rev. 98, 307325.