Evidence for the Mechanism of Photocatalytic Degradation of the

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Langmuir 2005, 21, 4631-4641

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Evidence for the Mechanism of Photocatalytic Degradation of the Bacterial Wall Membrane at the TiO2 Interface by ATR-FTIR and Laser Kinetic Spectroscopy J. Kiwi and V. Nadtochenko* Laboratory of Photonics and Interfaces, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology, Lausanne 1015, Switzerland Received December 8, 2004. In Final Form: February 20, 2005 The photocatalytic peroxidation of E. coli cell, lipo-polysaccharide (LPS), phosphatidyl-ethanolcholine (PE), and peptidoglycan (PGN) of the E. coli membrane wall has been investigated on TiO2 porous films by ATR-FTIR spectroscopy. The fast reactions of the photogenerated charge carriers in TiO2 with E. coli, LPS, and PE were monitored by laser kinetic spectroscopy. ATR-FTIR spectroscopy allowed the identification of E. coli, LPS, PE, and PGN as photocatalytic peroxidation products. The PGN was observed to be the most resistant membrane wall component. Shorter peroxidation times were observed for LPS and PE. Laser photolysis shows that E. coli, LPS, and PE compete in the scavenging of a surface trapped holes (h+) with the recombination reaction of h+ with the generated electrons (e-) within times >50 ns. This scavenging leads to the formation of organic radicals initiating the radical chain peroxidation of E. coli, LPS, PE, and PE.

Introduction The photocatalytic-induced destruction of the microbial cells with TiO2 has been documented,1-8 but the mechanism leading to the photocatalytic killing of the bacterial cell is still a subject of increasing research. Work in this area is important in the field of self-sterilizing TiO2 surfaces. This is important in medicine, the industrial food sector, pharmaceutical, and perfume products. TiO2 decontamination of surfaces for reusable devices such as catheters is recently gaining attention in the health-care industry. The current study focuses on the TiO2 photocatalytic damage on gram-negative bacteria (E. coli). Gram-negative bacteria wall presents a complex structure composed of two-membranes.9 Scheme 1 shows the structure of a membrane wall of the gram-negative bacteria. The main components of the cell wall are a nonsymmetrical outer lipopolysaccharide bilayer (LPS), a phospholipid layer, a peptidoglycan (PGN) layer, and the phospholipids bilayer of the cytoplasmic membrane. LPS is found exclusively in the outer membrane. The inner leaflet of the outer membrane contains phospholipids. The PGN is bound to the outer membrane lipoprotein via a peptide bond. Together, the peptidoglycan and the outer membrane provide the mechanical protection necessary to maintain an intact cell morphology. Phosphatidylethanolamine (PE) is the major phospholipid component (1) Matsunaga, T.; Tomoda, R.; Nakajima, T.; Wake, H. FEMS Microbiol. Lett. 1985, 29, 211. (2) Wolfrum, E. J.; Huang, J.; Blake, D. M.; Maness, P.-C.; Huang, Z.; Fiest, J.; Jacoby, W., A. Environ. Sci. Technnol. 2002, 36, 3412 and references therein. (3) Rincon, G. A.; Pulgarin, C. Appl. Catal., B 2003, 44, 263. (4) Sunada, K.; Watanabe, T.; Hashimoto, K. J. Photochem. Photobiol., A 2003, 156, 227 and references therein. (5) Tao, H.; Wei, W. Z.; Zhang, S. F. J. Photochem. Photobiol., A 2004, 161, 193. (6) Seven, O.; Dinider, B.; Aydemir, S.; Metin, D.; Ozinel, M. A.; Icli, S. J. Photochem. Photobiol., A 2004, 165, 103. (7) Ku¨hn, K. P.; Chaberny, I. F.; Massholder, K.; Stickler, M.; Benz, V. W.; Sonntag, H.-G.; Erdinger, L. Chemosphere 2003, 53, 71. (8) Chirstensen, P. A.; Curtis, T. P.; Kosa, S. A. M.; Tinlin, J. R. Appl. Catal., B 2003, 41, 371. (9) Huijbesgts, R. P. H.; de Kroon, A. I. P. M.; de Kuijff, B. Biochim. Biophys. Acta 2000, 1469, 43.

Scheme 1. Structure of the Wall of a Gram-Negative Bacteria

in E. coli constituting ∼70-80% of the total pool of phospholipids. The remaining phospholipids are phosphatidylglycerol (15-20%) and cardiolopin (CdO str. of ester >CdO str. of ester amide I amide I amide I aspartate or glutamate carboxylate stretching

1546

amide II

1517 1494 1470

1397

1290 1279 1245 1216 1085 1171 1151 1122 1060 1042 1026 997 966

LPS11,15,33-37 cm-1

amide II phenylalanine δ(CH2) scissor

-COO- str.

amide III amide III νa(PO2-) str. νa(PO2-) str. νs(PO2-) ν(C-O) ring, ν(C-O), ν(C-C), δ(COH) of carbohydrates, -C-O-C -P-O-C C-N stretching asymmetric

∼3320 ∼3090

νa(CH3) νa(CH2) νs(CH3) νs(CH2) >CdO str. of ester >CdO str. of ester amide I amide I amide I

1557

amide II

1537 1513

amide II amide II

1469

δ(CH2) scissor

971

-COO- str. δs(CH3) umbrella progression of the CH2 wagging modes

amide III amide III νa(PO2-) str. νa(PO2-) str. νs(PO2-) ν(C-O) ring, ν(C-O), ν(C-C), δ(COH) of carbohydrates, -C-O-C -P-O-C C-N stretching asymmetric

wide spectral region decay as a function of time. The spectra in Figure 1 were normalized to be able to compare the changes in the bands on the same scale. The spectra of every particular region were divided by the value of the spectral integral of the particular region. A similar normalization procedure was used for the spectra of LPS, PE, and PGN. Figure 1a reveals meaningful changes of the spectral profile of E. coli for the disappearance of amide A ∼3295 cm-1 and amide B ∼3060 cm-1, also for the disappearance of C-H bands at 2964, 2927, 2873, and 2853 cm-1 after 960 min. Concomitantly, the wide band of the OHvibrations is transformed in the skewed form with a maximum at 3480 cm-1. Figure 1b shows significant changes of the initial band shapes of oligosaccharide bands around 1087 cm-1 at 45 min, and profile changes of the PO2- bands near 1242 cm-1, as well as the decay of the amide I band near 1653 cm-1 and the amide II near 1545 cm-1. In parallel, the rise of the absorbance in the specific region related to CdO bonds of aldehydes and ketones between 1680 and 1750 cm-1 was observed. Beyond 120

PGN11,42 cm-1 ∼3277 ∼3070

amide A amide B

2962 2925 2879 2856 1733 1715 1662 1653 1635 1575

1408 1377 1343 1300 1270 1241 1224 1198 1168 1294 1283 1237 1207 1080 1150 1118 1057 1041 1023 997

SUV PE16-19,40,41 cm-1

3007 2959 2920 2873 2851 1744 1727

isolated cis CdC-H νa(CH3) νa(CH2) νs(CH3) νs(CH2) >CdO str. of ester >CdO str. of ester

1468 1457 1416

δ(CH2) scissor δa(CH3) δ(R-CH2) scissoring mode attached to CO or PO

1379 1342 1298 1266 1242 1198 1168

δs(CH3) umbrella progression of the CH2 wagging modes

1246

νa(PO2-)

1088 1064 1045

νs(PO2-) -C-O-C, -P-O-C

969

C-N stretching asymmetric

amide A amide B

2965 2925 2872 2856

νa(CH3) νa(CH2) νs(CH3) νs(CH2)

1692 1655 1623 1587

amide I amide I amide I

1544

amide II

1516

amide II

1449

1409 1376

-COO- str. δs(CH3) umbrella

1282 1228

amide III amide III

1160 1116 1065 1030 1011 970

C-N stretching asymmetric

min (Figure 1b), the most prominent peaks were seen at 1410 and 1370 cm-1, indicating an increase in the concentration of carboxylic groups. It is of significance to note that photocatalytic peroxidation-induced changes of the ATR-FTIR spectra of the E. coli occur due to two reasons: first, the decrease of the initial bio-molecule concentration and concomitant formation of peroxidation products and, second, the photocatalytic-induced structure modification of the wall membrane giving rise to the shift of the -CH2 bands of the fatty tails of lipid molecules and changes of the asymmetric phosphate ester (C-PO4--C) stretching vibration ν(PdO). Further, the analysis of the photocatalysis-induced changes of FTIR spectra will consider both effects. Figure 2 presents the Fourier deconvoluted spectra in the region 1800-940 cm-1. The E. coli cell-peroxidation as a function of time is shown by the peaks obtained by enhanced resolution of the experimental bands in Figure 2. First, the amide I band of 1653 cm-1 (in Figure 1b as one band) was resolved into separate peaks of 1655 cm-1, involving the helical content in proteins, and two peaks

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Figure 2. Deconvoluted ATR-FTIR spectra of E. coli in the fingerprint region 1800-950 cm-1. The deconvolution procedure used the parameters K ) 2.084 (K is defined as the effective increase in resolution), half-width ) 21.893 cm-1. Bessel apodization was applied.

Figure 3. Deconvoluted ATR-FTIR spectra of E. coli on Al2O3 porous films at different irradiation times using a BL light source. Dashed line for t ) 0 min, the E.coli ATR-FTIR spectra on TiO2 surface are shown. K ) 2.084, half-width ) 21.893 cm-1. Bessel apodization was applied.

for β-sheet and/or turn motifs at 1687 cm-1 and β-sheet peak of 1636 cm-1.13 The relative intensities of three amide I peak bands at 1687, 1655, and 1636 cm-1 as well as the intensities of the two amide II peaks at 1546 and 1517 cm-1 are seen to decrease within times >30 min. This indicates a secondary structure change of the proteins in the E. coli due to photocatalytic peroxidation.13 Second, the shapes of the oligosaccharide bands 1110-950 cm-1 were substantially modified during the 30-60 min irradiation period. This suggests an outer leaflet damage of E. coli due to the peroxidation occurring during photocatatysis. The amide-bands in Figure 1b and in Figure 2 show a higher resistance to peroxidation than the oligosaccharide bands. Third, changes in the profile of PO2- bands near 1245 and 1216 cm-1 are observed in Figure 2, involving the alteration of hydrogen bonds related to the phosphate group.14 The enhanced resolution (13) Vedantham, G.; Sparks, H. G.; Sane, S. U.; Tzannis, S.; Przybycien, T. M. Anal. Biochem. 2000, 285, 33. (14) Selle, C.; Pohle, W.; Fritzsche, H. J. Mol. Struct. 1999, 480-481, 401.

of the bands in Figure 2 also allows one to sort out the peroxidation products formed during photocatalysis. The main feature of the ATR-FTIR spectra of peroxidized products is the formation of IR bands in the CdO vibrational region. A tentative assignment of the CO bands related to the peroxidation products will be discussed below. Figure 3 shows the results of control experiments when E. coli is coated on Al2O3 porous films. Almost no changes were observed in the ATR-FTIR spectra of E. coli when Al2O3 after 840 min of irradiation. Very little variation in the band shapes of amide I and amide II was detected. This contrast with the results presented in Figures 1 and 2 when TiO2 films were used. This is a further proof of the photocatalytic nature of the E. coli peroxidation occurring on the TiO2 surface. ATR-FTIR Spectral Changes of LPS during Photocatalytic Peroxidation on TiO2. Figure 4 shows normalized ATR-FTIR spectra of LPS on TiO2 porous film during irradiation under BL light. Control experiments under the same experimental conditions but with LPS

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Langmuir, Vol. 21, No. 10, 2005 4635 Table 2. C-H Bands of LPS on the TiO2 Film, Dissolved in Water, and LPS Films on Glass C-H bandsb TiO2 film dissolved in water LPS films on glassa νs(CH2) νa(CH2) νa(CH3) a

Figure 4. Normalized ATR-FTIR spectra of LPS at the TiO2 film interface after different times of BL light irradiation. The absorbance of the net TiO2 film was subtracted for each spectrum. (a) Spectral region 3600-2600 cm-1. The integral absorbance in the region of 3600-2600 cm-1 was used as the normalization factor. (b) Spectral region 1850-950 cm-1. The integral absorbance in the region of 1850-950 cm-1 was used as the normalization factor. Scheme 2. Structure of Kdo2-Lipid A in E. coli K12 As Compared to Phosphatidyl-ethanolamine (PE)a

a Lipid A is glucosamine-based and lacks glycerol. Abbreviations: Kdo, 3-deoxy-D-manno-octulosonic acid.

films on Al2O3 porous membrane did not show meaningful changes in the ATR-FTIR spectra of LPS. This is a further evidence for photocatalytic peroxidation shown in Figure 4. The structure of LPS is shown in Scheme 2. Lipid A forms the hydrophobic anchor of LPS to which the core region is attached, containing phosphorylated nonrepeating oligosaccharides. In wild-type E. coli, the core sugars are usually followed by an O-antigen repeating oligosaccharide, which is absent in most E. coli K12 strains.9 We found that the positions of the C-H peaks of LPS at the TiO2 porous film at t ) 0 min were different from previously reported data for LPS existing in a different molecular surrounding.11 Becuse FTIR is a nondestructive technique, it is used for both structural and conformational studies in cell biology and medicine. The C-H band position in FTIR spectra of lipid layers indicates the changes in the fatty-tail structure15 related to the mo-

2856 2925 2962

2854 2922 2962

2850 2917 2957

∼100 µm thick. b νs(CH3) band is very weak.

lecular ordering of lipid A when LPS was adsorbed on TiO2. To clarify this point, the C-H peak position was compared for LPS in different molecular surroundings, and the data are summarized in Table 2. The data for νs(CH2) in the region 2850-2852 cm-1 depend on temperature between 20 and 45 °C.15 The blue shift of 3-5 cm-1 for LPS on TiO2 suggests the alteration of the fattytail structure due to increased structural disorder leading to increased fluidity.15 As can be seen from the Table 2, a shift of almost 5 cm-1 to the blue for νs(CH2) was observed between the LPS on TiO2 and on the glass plate. It suggests that the TiO2 affects the LPS fatty-lipid tail packing introducing disorder. The values found for the LPS film on glass are in good agreement with the reported data.15 The position and shape of the CO stretching vibrations of the acyl-band and the asymmetrical phosphate bands of LPS were observed to be sensitive to the interaction of LPS with TiO2. A decrease of the LPS bands in the spectra of Figure 4b was observed during the course of photocatalytic peroxidation. The sugar peaks around 1078 cm-1 decreased significantly after 27 min. A substantial decay of the amide A, amide B, and C-H bands were observed in Figure 4a at 60 min. Concomitantly to the decay of LPS bands, a modest rise of new bands with time was observed in the Figure 4b due to peroxidation products of LPS. Deconvoluted and second-derivative of the LPS bands in the range of 1800-900 cm-1 during photocatalysis are shown in Figure 5. The assignment of the LPS bands is presented in Table 1. The most prominent peroxidation peak relates to the carbonyl group in aldehydes, ketones, and carboxylic acids. The decay of the LPS features due to the peroxidation resembles the decay of the spectral features found for the E. coli samples. However, qualitatively the decay for LPS sample occurs faster than for E. coli. Taking into account the disorder of LPS layer introduced by the TiO2 film, it is suggested that the organized LPS outer leaflet in the E. coli wall is more resistant to peroxidation than the LPS having a disordered structure introduced by the presence of the TiO2 layer. ATR-FTIR Spectral Changes of SUV PE Due to Photocatalytic Peroxidation on TiO2. Figure 6 presents changes in spectra of SUV PE cast on TiO2 porous film during photocatalytic peroxidation in two different spectral regions. The chemical structure of PE has been shown in Scheme 2. PE lipids from bovine contain double bonds -CdC-H. Meaningful changes in the PE spectra as a function of BL irradiation time were not found with control experiments using Al2O3 instead of TiO2. The spectral changes related to the peroxidation shown in Figure 6a can be summarized as follows: a faster disappearance of the isolated cis CdC-H bands at 3009 cm-1 during 30-65 min as compared to the degradation of the C-H bands because the C-H bands at 2956, 2918, and 2851 cm-1 are detected even after 120-165 min of reaction. Figure 6b shows meaningful changes for the CdO acyl-bond profile near 1740 cm-1 up to 30 min: the (15) Brandenburg, K.; Lindner, B.; Schromm, A.; Koch, M. H. J.; Bauer, J.; Merkli, A.; Zbaeren, C.; Davies, J. G.; Seydel, U. Eur. J. Biochem. 2000, 267, 3370.

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Figure 5. Deconvoluted ATR-FTIR spectra of LPS at different irradiation times at the TiO2 film interface. K factor 1.674, halfwidth 11.193 cm-1. Bessel apodization was applied.

Figure 6. ATR-FTIR spectra of SUV PE after different irradiation times at the TiO2 interface in the region (a) 27003650 cm-1 and (b) 950-1850 cm-1. Spectra are presented in the normalized form. The absorbance of pure TiO2 from spectra of SUV-PE on TiO2 was not subtracted. TiO2 spectrum is the spectrum of TiO2 without PE.

appearance of new CdO bands at later irradiation stages, the decay of the >CH2 scissoring band mode at 1467 cm-1, the changes of the band shape near 1222 cm-1 up to 30 min, and the changes of -C-O-C, -P-O-C bands at 1100-1000 cm-1.16-18 PE deconvoluted spectra are shown in Figure 7. The band assignment for phospholipids has been carried out according to the literature references16-19 and is noted in Table 1. The enhanced resolution after the deconvolution of the band 1740 cm-1 (Figure 6b) leads to the appearance of two new bands for CH2-COOC ν(CdO) at 1744 and 1727 cm-1 shown in Figure 7. This spectral region of acylbonds was analyzed by using second derivative spectroscopy and fitted using the Levenberg-Marquardt algo(16) Kinder, R.; Ziegler, C.; Wessels, J. M. Int. Journal Radiat. Biol. 1997, 71, 561-571. (17) Lukes, P. J.; Yarwood, J.; Yarwood, J. Langmuir 2004, 8, 30433050. (18) Wolfangel, P.; Lehnert, R.; Meyer, H. H.; Muller, K. Phys. Chem. Chem. Phys. 1999, 1, 4833-4841. (19) Parker, F. S. Applications of Infrared, Raman, and Resonance Raman Spectroscopy in Biochemistry; Plenum Press: New York, 1983.

rithm. This analysis revealed the disappearance of the initial acyl-bands even at early stages of peroxidation CH2, PO2- and for bands corresponding to C-O-C and -P-O-C. At the same time, the >CH2 scissoring band of 1467 cm-1 and the umbrella mode of the -CH3 vibrations at 1379 cm-1 were observed up to 95 min. At 165 min, the initial PE bands disappear (Figure 7). Peroxidation peaks at 1738, 1722, 1702, 1645, and 1420 cm-1 were predominant in the spectra after 165 min of light irradiation. The spectra and nature of these peroxidation products are discussed below. ATR-FTIR Spectral Changes of PGN Due to the Photocatalytic Peroxidation on TiO2. The peptidoglycan structure is shown below in Scheme 3. The muramic acid is the subunit of the peptidoglycan found in E. coli. The glycan backbone is made out of a polymer composed by two amino sugars, N-acetyl-glucosamine (GlNAc) and N-acetylmuramic acid (MurNAc). The neighboring tetrapeptide side chains are linked by interpeptide bonds between DAP on one chain and D-ala on the other.9 Figure 8a demonstrates spectral changes due to the peroxidation of PGN on the TiO2 porous film. The assignment of the bands is found in Table 1. A specific feature of the ATR-FTIR spectral peaks as a function of time is the disappearance of the PGN bands after 44 h of photocatalysis. Figure 8a reveals that amide and C-H bands are still present after 5 h irradiation, while in the case of LPS or PE, the bands almost completely disappeared due to peroxidation. Amide A and B as well as the C-H bands dominate in the spectra at irradiation times of ∼5 h 40 min. Figure 8b shows in the spectral region 1800-900 cm-1 the ATR-FTIR spectra of the amide I band near 1628 cm-1, the amide II band near 1536 cm-1,11,13 and the carboxy band near 1395 cm-1 19,20 as the main bands after 5 h 40 min of irradiation. After 21 h of

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Figure 7. Fourier deconvoluted spectra of SUV PE during the photocatalytic peroxidation at the TiO2 porous film interface. Scheme 3. Muramic Acid Subunit of the Peptidoglycan of E. coli

Figure 8. PGN photocatalytic peroxidation at the TiO2 porous film interface: (a) 2700-3650 cm-1 and (b) 950-1850 cm-1. Spectra are presented in the normalized form. The absorbance of the TiO2 was subtracted.

irradiation, the peroxidation products are seen to grow near 1400-1340 cm-1. After 44 h of irradiation, the PGN amide bands almost disappear. Changes in the band-shape of the GlNAc and MurNAc moieties of PGN near 11001000 cm-1 are seen in Figure 8b. This observation suggests a slower peroxidation of the tetrapeptide side chains of PGN due to their higher resistance to peroxidation by TiO2 photocatalysis. Figure 9 shows the deconvoluted ATR-FTIR spectra of PGN on the TiO2 surface in the region 1800-900 cm-1. (20) Li-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: Boston, 1991.

Table 1 presents the assignment of bands in this region. Figure 9 shows that even relative intensities of the peaks in the amide I and amide II bands are conserved during peroxidation up to ∼5 h 40 min. Some changes observed in the GlNAc and MurNAc spectral moieties before 5 h 40 min suggest that peroxidation begins to be important for the amino-sugar of PGN. ATR-FTIR Spectra of Peroxidized Products of E. coli, LPS, PE, and PGN. The ATR-FTIR spectra as function of time for E. coli, LPS, PE, and PGN reveal the disappearance of the initial bands and the concomitant growth of new bands due to the peroxidation products. The spectral bands of peroxidation intermediates for E. coli, LPS, PE, and PGN are shown in Figures 2, 5, 7, and 9, respectively. A common feature of the studied ATRFTIR spectra as a function of time is the changes of CdO bands due to the formation of the aldehydes, ketones, and carboxylic acids observed during peroxidation processes.24,27 Concomitant to formation of new CdO bands (21) Dubois, J.; van de Voort, F. R.; Sedman, J.; Ismail, A. A.; Ramaswamy, H. R. J. Am. Oil Chem. Soc. 1996, 73, 787. (22) Primet, M.; Pichat, P.; Mathieu, M. V. J. Phys. Chem. 1971, 75, 1221. (23) Rotzinger, F. P.; Kesselman-Truttmann, J. M.; Hug, S. J.; Shklover, V.; Gratzel, M. J. Phys. Chem. B 2004, 108, 5004.

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Figure 9. Fourier deconvoluted spectra of PGN from 950 to 1750 cm-1 after different irradiation times on the TiO2 film interface. Spectra are shown without normalization. K factor ) 1.584, half width ) 15.893 cm-1. Bessel apodization was applied.

during peroxidation, the formation of new C-H bands can be resolved in the region of 2850-2700 cm-1, which can be tentatively assigned to peroxidation products. For example, the formation of R, β unsaturated aldehydes was detected by thio-barbituric acid in the TiO2 photocatalysis of phosphopholipid,24 the peroxidation of phospholipids under γ-irradiation,16 and during photocatalytic peroxidation of E. coli.2,14 In this study, the assignment focuses on the prominent CdO peaks observed during peroxidation of E. coli, LPS, PE, and PGN. The peaks at 1732 cm-1 of LPS at t ) 80 min (Figure 5), and of PE at 1738 cm-1 at t ) 165 min (Figure 7), can be ascribed to aliphatic aldehydes (R-CHO) corresponding to the bands at 17401730 cm-1.19,20 For the CdC-CHO aldehydes, the bands in the 1705-1640 cm-1 have been reported.21 The peak of E. coli at 1714 cm-1 after 185 min (Figure 2), of PE at 1717 cm-1 after 66 min (Figure 7), and for LPS at 1716 cm-1 after 80 min (Figure 5) represent CdO bands of R-COCH3 ketones.19,20 The peak at 1722 cm-1 is observed for LPS after 10 min, for E. coli after 90 min, and for PE after 165 min. This band is assigned to carboxylic acid dimers with frequencies of 1720-1680 cm-1.19 It should be taken into account that the origin of the primary carboxylic acids from lipids membranes is due to the scission of the acyl-bonds and the formation of fatty acids. New carboxylic groups can arise during the initial stages of peroxidation after 10-15 min reaction of E. coli, LPS, and PE. Bands near 1410, 1340, 1550-1590, and 1290 cm-1 are observed in the E. coli spectrum (Figures 1 and 2). This is also the case of LPS (Figures 4 and 5), of PE (Figures 6 and 7), and of PGN (Figures 8 and 9) during later reaction stages. These bands correspond to the vibrations of the carboxylic acid groups of the deep peroxidation products.19,20 The asymmetrical vibrations of COO- are associated with bands in the 1580-1550 cm-1 region, and the symmetric COOvibrations are associated with bands at 1410-1350 cm-1. (24) Kiwi, J.; Nadtochenko, V. J. Phys. Chem. B 2004, 108, 17675. (25) Cameron, D. G.; Dluhy, R. A. In Spectroscopy in the biomedical sciences; Gendreau, R. M., Ed.; CRC Press: Boca Raton, FL, 1986; Chapter 3, p 53. (26) Nadtochenko, V.; Rincon, G. A.; Stanca, E. S.; Kiwi, J. J. Photochem. Photobiol., A 2004, 169, 131. (27) Knorre, D. G.; Emanuel, N. M. Chemical Kinetics of Homogeneous Reactions; Wiley: New York, 1973.

The C-OH stretching vibration is associated with bands around 1280 cm-1. The δ(CH3) coupled with COOvibration of acetic acids appears at 1350 cm-1.19,20 The carboxylic acids at the TiO2 surface exist in a variety of states: as nonadsorbed species, as physisorbed molecules, in the form of complexes with Ti4+, in protonated and nonprotonated forms.22,23 Adsorption and formation of complexes between COO- and Ti4+-ion on the TiO2 surface shift the position of the bands several tens of reciprocal centimeters.22,23 The trends of the peroxidation band products during the final reaction stages were observed to be consistent with the IR spectra of simple carboxylic acids (formate, acetate) on the TiO2 surface.22,23 Consideration of the Kinetics of ATR-FTIR Spectra of E. coli, LPS, PE, and PGN during Photocatalytic Peroxidation. The intensity decay of ATR-FTIR spectral bands suggests the mineralization of organic material on the TiO2 surface during photocatalysis. To estimate the dynamics of the mineralization process, the integral absorbance of ATR-FTIR spectra as a function of time was evaluated. The kinetics of the integral absorbance is a rough approximation for the amount of the organic material on TiO2, because the extinction coefficient is different for the different peroxidation intermediates. Nevertheless, the integral absorbance in the wide spectral region represents the amount of organic material if it is assumed that the integral oscillator strength of the IR transitions does remain fairly constant. The integral absorbance was calculated in the 1800-900 cm-1 region. Figure 10 shows the dependence of this value for E. coli, LPS, SUV PE, and PGN. The trend of the decay traces shown in Figure 10 is consistent with the expected shape known for the kinetic plots observed for the radical oxidation process.27 First, the kinetic traces in Figure 10a, b, and d show lag periods for E. coli (∼20-30 min), LPS (∼20 min), and PGN (∼300 min). The lag period almost vanishes for SUV PE (Figure 10c). Second, after the lag period, the decay can be approximated by exponential curves. Figure 10d shows the significantly lower rate of the decay for the integral absorbance of PGN relative to E. coli, LPS, or PE. This suggests a higher resistance to photocatalytic peroxidation of PGN relative to E. coli, LPS, and PE. The exponential decay after the lag period follows the reciprocal time constant of (4 ( 0.6) × 10-2 min-1 for

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Figure 10. Kinetics of the integral absorbance decay in the region of 1800-900 cm-1 for (a) E. coli, (b) LPS, (c) SUV PE, and (d) PGN.

Figure 11. Kinetics of the decay ratio of the absorbance areas. (a) E. coli. A0 was determined as integral absorbance in the region of 1750-950 cm-1, and A-area of the band: (1) amide band region 1700-1586 cm-1, (2) phosphate band region 1272-1193 cm-1, (3) sugar band region of 1095-1075 cm-1 (it is scaled ×9), (4) COO- band region 1430-1363 cm-1. (b) LPS on TiO2. A0 was determined as the integral absorbance in the region of 1750-950 cm-1, and A-area of the band: (1) sugar band domain of 1102-1025 cm-1, (2) amide I peak of 1634-1689 cm-1 region, (3) the COO- spectral domain of 1440-1376 cm-1. (c) SUV-PE on TiO2: (1) the peak of isolated CdC-H bond at 3009 cm-1. It is normalized to the area of the CH2 and CH3 bands in the region of 3000-2850 cm-1. Areas of these peaks were determined by the decomposition in the separate Voigt peaks. A0 was determined as integral absorbance in the region of 1750-950 cm-1, and A-area of the band: (2) CH2 bond peak of scissoring vibrations at 1468 cm-1, (3) the COO- spectral domain of 1440-1376 cm-1. (d) PGN on TiO2. A0 was determined as integral absorbance in the region of 1750-950 cm-1, and A-area of the band: (1) the amide I domain 1590-1700 cm-1, (2) the COO- domain 1450-1390 cm-1.

E.coli, (9 ( 0.2) × 10-2 min-1 for LPS, and 0.005 ( 0.001 min-1 for PGN. The SUV-PE decay was a composite of the two exponential decay functions with a fast component of (8 ( 0.2) × 10-2 min-1 and a slow component of (4 ( 0.4) × 10-3 min-1. The fastest decay of the integral absorbance was observed for LPS, suggesting that LPS was the easiest wall component to mineralize. The decay time observed during the mineralization of PGN was more than 10 times slower than the times necessary to mineralize LPS. As it was indicated above, the rise of peroxidation bands was observed concomitant with the decrease of the initial bands of the membrane components in the spectra of E. coli, LPS, SUV PE, or PGN. Because of the overlapping

of various peroxidation species bands, the precise decomposition of spectra into separate peaks was not possible by using the χ2-minimization procedure. Nevertheless, it is possible to obtain a rough estimation of the integral absorption of some individual bands, as a function of time. The integral absorbance is presented in Figure 11, showing the integral absorbance of the chosen spectral region divided by the integral absorbance in the wide spectral region 1750-950 cm-1. An exception was made for the single CdC-H band at 3009 cm-1 for PE. Normalization was carried out for the area of the C-H bands in the 3010-2850 cm-1 wax region, because these bands do not overlap.

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The kinetics shown in Figure 11a shows that for E. coli the decay of the absorbance of the sugar ring bands at 1095-1075 cm-1 occurs faster than the decay in the region of the amide I band at 1700-1586 cm-1 for t < 60 min. The kinetic curve of absorbance of 1700-1586 cm-1 rises for t > 100 min, because the amide I band overlaps with the absorption of the peroxidation products. The trace of the absorbance at 1430-1363 cm-1 for the COO- group refers to the growth kinetics of the peroxidation products. During the initial reaction stage t < 60 min, the carboxylic absorption of the E. coli components was observed. Figure 11b shows kinetic curves for LPS samples in a way similar to the E. coli sample. The LPS absorption declines faster than amide I. The kinetics curve of the absorbance of the spectral domain of 1440-1376 cm-1 rises due to the accumulation of the peroxidation products having COO- groups. A comparison of kinetics for E. coli in Figure 11a and for LPS in Figure 11b shows that the bands of the sugar ring decay faster for LPS than for the E. coli. This suggests that an LPS with a disordered LPS structure on TiO2 peroxidizes faster than ordered LPS in the outer leaflet of E. coli. For the amide band region 1634-1689 cm-1 in Figure 11b, the distortion of the kinetic curve is observed at times >40 min, due to the overlapping of the absorption bands of the peroxidation products. Figure 11c shows the kinetics of the bands during SUV PE peroxidation. Figure 11c reveals that the fast decays observed are related to the isolated CdC-H bond. The >CH2 and -CH3 bands decay substantially slower than the CdC-H bond. Figure 11d shows the kinetics curves for the peroxidation of PGN. It can be seen that amide bonds of PGN and the carboxylic group formation occur in a slow process. The resistance of PGN to peroxidation has been reported by biochemical measurements.4 Our results for PGN peroxidation agree with the results reported in ref 4. Moreover, ATR-FTIR shows in Figure 2 that, during E. coli peroxidation, the amide band features at 1655, 1632, and 1544 cm-1 are detected at t ) 120 min, when other E. coli spectral features vanished. Figure 11d shows that, at long irradiation times, the carboxylic groups due to peroxidation disappear completely on the TiO2 surface. The kinetics shown in Figure 11 reveals that moieties with weak energy bonds as allyl CdC-H bonds (87.5 kcal/ mol28) or sugar cycles (as aldehydes have the C-H energy bond 86.5 kcal/mol28) correspond to more easily oxidizable (28) Benson, S. W. Thermochemical Kinetics; J. Wiley & Sons: New York, 1968. (29) Naumann, D.; Schultz, C. FEBS 1991, 294, 43. (30) Helm, D.; Naumann, D. FEBS Microbiol. Lett. 1995, 126, 75. (31) Zeroual, W.; Choisy, C.; Dolgia, S. M.; Bobichon, H.; Angibaust, J.-F.; Manfait, M. Biochim. Biophys. Acta 1994, 1222, 171. (32) Gue´, M.; Dupont, V.; Dufour, A.; Sire, O. Biochemistry 2001, 40, 11938. (33) Brandenburg, K.; Kusumoto, S.; Seydel, U. Biochim. Biophys. Acta 1997, 1329, 183. (34) Naumann, D.; Schultz, C.; Sabisch, A.; Kastowsky, M.; Labischinski, H. J. Mol. Struct. 1989, 214, 213. (35) Naumann, D.; Schultz, C.; Born, J.; Labischinski, H.; Brandenburg, K.; von Busse, G.; Brade, H.; Seysel, H. Eur. J. Biochem. 1987, 164, 159. (36) Kacurakova, M.; Mathlouthi, M. Carbohydr. Res. 1996, 284, 145. (37) Reiter, G.; Siam, M.; Falkenhagen, D.; Golneritsch, W.; Baurecht, D.; Fringeli, U. P. Langmuir 2002, 18, 5761. (38) Vernooij, E. A. A. M.; Kettenes-van den Bosch, J. J.; Crommelin, D. J. A. Langmuir 2002, 18, 3466. (39) Brandenburg, K.; Lindner, B.; Schromm, A.; Koch, M. H. J.; Bauer, J.; Merkli, A.; Zbaeren, C.; Gwynfor Davies, J.; Seydel, U. Eur. J. Biochem. 2000, 267, 3370. (40) Lozano, P.; Fernandez, A. J.; Ruiz, J. J.; Camacho, L.; Martin, M. T.; Munoz, E. J. Phys. Chem. B 2002, 106, 6507. (41) O’Leary, T.; Levin, I. W. J. Phys. Chem. 1984, 88, 1790. (42) Naumann, D.; Barnikel, G.; Bradaczek, H.; Labischinski, H.; Giesbrecht, P. Eur. J. Biochem. 1982, 125, 505.

Kiwi and Nadtochenko

Figure 12. Transients after laser pulse excitation. The probe wavelength was 820 nm. (a) E. coli. (1) TiO2 without E. coli. (2) TiO2 with E. coli. (b) LPS. (1) TiO2 without LPS. (2) TiO2 with LPS. (c) SUV-PE. (1) TiO2 without SUV-PE. (2) TiO2 with SUV-PE.

species in comparison with the C-H bonds of methylene (98 kcal/mol28) and methyl (104 kcal/mol28) groups. According to Polanyi-Semenov’s rule, the weaker bond should react faster in a radical oxidation process.27 This agrees with the radical peroxidation mechanism during TiO2 photocatalysis. Effect of the E. coli, LPS, and SUV PE on the Dynamics of Charge Carriers in TiO2 by Fast Kinetic Spectroscopy. An important question for the understanding of the primary steps of photocatalytic peroxidation of wall membrane on the TiO2 interface is: Do the photogenerated at 354 nm light charge carriers react with E. coli, LPS, and PE, or will the photogenerated charge carriers produce HO• and HO2•/O2-• radicals and will these radicals then react with RH (cell wall organics)? In both cases, the radicals (R•) produced by chain radical oxidation23 lead to aldehydes, ketones, and carboxylic acids. To answer this question, we carried out laser photolysis experiments. Figure 12 presents the transients observed in TiO2 colloids with and without addition of E. coli, LPS, and PE. The transient observed by the colloidal TiO2 Degussa P25 is due to the absorbance of the photogenerated e- by the TiO2 particle. The transient decay is determined by the recombination of photogenerated holes and electrons with some influence from the e- reacting with O2. The addition of E. coli, LPS, and PE leads to a significant decrease of the decay rate for the photogenerated electron as shown in Figure 12. This decrease indicates that the holes on the TiO2 surface are scavenged by RH. This scavenging of holes leads to the decrease of the observed electron decay. The scavenging of h+ by RH competes with e-/h+ recombination only at longer time scales beyond a few tens of nanoseconds. By femtosecond laser photolysis, it was found that in the time scale CH2, -CH3 bands, and amide bands on TiO2 films is reported in this study. The oligosaccharide bands of E. coli and LPS and the isolated CdC-H bands disappear during short irradiation times. The peroxidation rate of different moieties qualitatively correlates with the bond energies, in accordance with Polanyi-Semenov’s theory. The formation of peroxidation products such as aldehydes, ketones, and carboxylic acids is detected in parallel to the disappearance of the constituents of the cell wall membranes. The FTIR spectra found for the peroxidation products of E. coli, LPS, PE, and PGN reveal numerous features related to the appearance of oxidation products due to the TiO2 photocatalysis on sugar rings, lipid chains, and polypeptide molecules. These features were shown and discussed in Figures 2, 5, 7, and 9. The ATR-FTIR data about LPS on TiO2 surface suggest the introduction of disorder in the LPS lipid layer during

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the photocatalysis. The peroxidation of LPS on TiO2 is faster than that of E. coli, suggesting that the order of the LPS bilayer structure determines the rate of the photocatalytic peroxidation process. The comparison of the peroxidation of three main components of the wall LPS, PE, and PGN show that PGN is the most resistant toward peroxidation. A shift of the >CH2 stretching bonds was also observed during photocatalysis. This shift seems to be the precursor of the structural changes in the cell wall membranes during lipid peroxidation leading to bacterial lysis in the later stages of the photocatalysis. By laser photolysis, it is shown that the reaction of E. coli, LPS, or PE with the hole (h+) competes with the recombination reaction of the h+ with e-. This reaction is considered as the initial step in the chain-radical peroxidation process. Acknowledgment. We gratefully acknowledge the financial support of CTI/KTI TOP NANO 21 under Grant 5897.5 (Bern, Switzerland). Supporting Information Available: AMF image of TiO2 porous film. This material is available free of charge via the Internet at http://pubs.acs.org. LA046983L