Environ. Sci. Technol. 2003, 37, 4785-4789
Bactericidal Activity of Copper-Deposited TiO2 Thin Film under Weak UV Light Illumination K A Y A N O S U N A D A , †,§ TOSHIYA WATANABE,† AND K A Z U H I T O H A S H I M O T O * ,†,§ Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, and Kanagawa Academy of Science and Technology, KSP Building, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan
The bactericidal activity of copper-deposited titanium dioxide thin film (Cu/TiO2) was investigated under very weak ultraviolet (UV) light illumination. To elucidate the roles of the film photocatalyst and the deposited copper in the bactericidal activity, cells from a copper-resistant Escherichia coli (E. coli) strain were utilized. A decrease in survival rate was not observed with the copper-resistant cells under dark conditions, but when illuminated with a very weak UV intensity of 1 µW/cm2, the survival rate decreased, suggesting photocatalytic bactericidal activity. The decay curve of survival on the Cu/TiO2 film under very weak UV light illumination consisted of two steps, similar to the survival change of normal E. coli on TiO2 films under rather strong UV illumination. The first step is due to the partial decomposition of the outer membrane in the cell envelope by a photocatalytic process, followed by permeation of the copper ions into the cytoplasmic membrane. The second step is due to a disorder of the cytoplasmic membrane caused by the copper ions, which results in a loss of the cell’s integrity. These processes explain why the Cu/ TiO2 film system shows an effective bactericidal activity even under very weak UV light illumination.
Introduction Numerous studies have utilized a strong oxidizing power of TiO2 photocatalysts to purify water and air of environmentally toxic substances (1-7). TiO2 photocatalysts have also been applied to inactivate bacteria, viruses, and cancer cells (5, 8-23). When water containing toxic substances or bacteria was treated by photocatalysis, a fine TiO2 powder and a strong light, such as a mercury lamp, were utilized in many studies. However, this system requires the recovery of TiO2 powder and a high cost of light. Our research has focused on the photocatalytic reactions of TiO2 thin films coated on various substances. These films possessed deodorizing, self-cleaning, and bactericidal activities under weak intensities of ultraviolet (UV) light (6, 7, 20-35). The bactericidal process on the TiO2 films was investigated by generating decay curves of survival, * Corresponding author phone: 81-3-5452-5080; fax: 81-3-54525083; e-mail:
[email protected]. Corresponding author address: Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 1538904, Japan. † University of Tokyo. § Kanagawa Academy of Science and Technology. 10.1021/es034106g CCC: $25.00 Published on Web 09/18/2003
2003 American Chemical Society
examining changes in the concentrations of cell envelope components, and taking atomic force microscopy measurements. These data suggest that the bactericidal process consists of two steps (22, 23). First, disordering of the outer membrane in the cell envelope occurs; this is followed by the disordering of the inner membrane. However, these reactions were reported at a relatively strong UV light intensity of 1 mW/cm2. To obtain the bactericidal activity under dark conditions and very weak UV intensity such as indoor UV light, TiO2 films deposited with antibacterial metals such as copper and silver have been developed (34, 35). Although these bactericidal materials have already been commercialized, there has been no clear scientific explanation of their bactericidal activity. It has simply been suggested that the photocatalytic activity of TiO2 and the antibacterial activity of the deposited metal ions are combined. This paper explores the roles of TiO2 and copper in generating the bactericidal activity of copper photodeposited TiO2 film (Cu/TiO2) under weak UV illumination of 1 µW/cm2, which corresponds to the typical UV intensity of indoor light. [We use the words antibacterial and bactericidal distinctly. Antibacterial activity is used to inactive bacteria by simple bacteriostatic action by the copper ions. In contrast, bactericidal activity is used when decomposition of some cell components caused the cell inactivation.]
Experimental Section TiO2 thin films were prepared by a dip-coating technique. Silica-coated soda-lime glass plates were dipped in a commercially available titanium isopropoxide solution (NDH510C, Nippon Soda) and subsequently withdrawn from the solution at a fixed rate of 20 cm/min. The plates were then placed in a furnace and calcined at 500 °C for 1 h. This procedure was repeated four times to produce a TiO2 thin film with a thickness of ∼0.4 µm on both sides of the glass plate. Copper was deposited onto the TiO2 film by the following method. A hydrophilic treatment, UV light illumination by black light bulbs, was used to ensure that the copper was uniformly photodeposited onto the TiO2 films (36). Copper acetate solution (0.02%) was spread on the hydrophilic TiO2 film surface, and then the film was illuminated to deposit the copper overnight by black light bulbs with a UV intensity of 1.5 mW/cm2 (35). The light intensity was measured with a UV power meter (UVR-36, Topcon), which is sensitive to light of wavelengths between 300 and 400 nm. Figure 1 shows the emission spectrum of black fluorescent light bulb and the sensitivity spectrum of the UV power meter. After illumination, the Cu/TiO2 film was washed with distilled water and dried. The amount of copper deposited to the TiO2 film was determined to be 0.023 µg/cm2 by ICP-AES (inductively coupled plasma-atomic emission spectrometry) measurements of the solution after soaking the Cu/TiO2 film overnight in 1 N HNO3 under dark conditions. Bactericidal activity of the Cu/TiO2 film was evaluated using two types of Escherichia coli (E. coli) cells (IFO 3301 strain and 53TNE007 strain). Strain 53TNE007 (50, 52) is a mutant of E. coli K-12 and is resistant to copper ions. The IFO 3301 strain, derived from E. coli K-12, is normal and not resistant to copper ions. The cells of each strain were treated by the same methods. The cells were precultured in 2.5 mL of a nutrient broth (“Daigo”, Nippon Seiyaku) at 36 °C for 18 h and then washed by centrifuging at 4000 rpm. The cells were then resuspended and diluted to ∼2 × 105 colony forming units (CFU)/mL with 1/500 solution of the nutrient broth. The diluted cell suspension was pipetted onto either a Cu/TiO2 film or a TiO2 film and placed in an airtight VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Emission spectra of black and white fluorescent light bulbs, absorption spectrum of TiO2 film, and sensitivity spectrum of the UV power meter. illumination chamber to prevent drying (21, 23). The chambers were illuminated with various UV light intensities of 40 µW/cm2, 7 µW/cm2, or 1 µW/cm2 by changing the distance between white light bulbs and the chamber. The emission spectrum of white fluorescent light bulb and absorption spectrum of TiO2 film are shown in Figure 1. After being illuminated for a specified time, the cell suspension was collected into a 0.15 M saline solution. The collected cells were appropriately diluted and were incubated at 36 °C for 24 h on a nutrient agar medium (Standard Method Agar “Nissui”, Nissui Seiyaku) in order to determine the number of viable cells in terms of CFU. The amount of •OH produced in the system was measured using coumarin as the detection probe. Coumarin readily reacts with •OH to induce the highly fluorescent hydroxy product (7-hydroxycoumarin). Therefore, the amount of •OH produced could be determined by measuring the fluorescence intensity of the hydroxy product (31, 32). A 9 mm × 9 mm TiO2 or Cu/TiO2 film glass was placed at the bottom of a 1 cm × 1 cm fused silica standard spectrophotomeric cell with 1 mL of coumarin solution (1 mM), and the fluorescence spectrum in response to excitation by 332 nm light was measured on a Hitachi F-4500 fluorescence spectrometer. The spectrum was observed before and after illumination with a black light bulb with a UV light intensity of 1 mW/cm2 for 20 min. In addition, 1 × 10-3 mM hydrogen peroxide (H2O2) was added to the system for some experiments. The fluorescence intensities at 460 nm under various conditions were compared to that observed from the TiO2 film after UV light illumination.
Results and Discussion Figure 2 shows the survival of normal E. coli cells (IFO3301), which were not copper-resistant, dropped onto either a TiO2 film or a Cu/TiO2 film as a function of illumination time. Obvious changes in survival were not observed on the TiO2 film stored in the dark. In contrast, the survival decreased on the Cu/TiO2 film even in the dark, which demonstrated that copper deposited to a TiO2 film has antibacterial activity in the dark. It has been reported that the copper species photodeposited on TiO2 particles in the suspension or on the TiO2 film are a mixture of metallic copper (Cu(0)) and Cu2O, and these species are oxidized and dissolved under exposure to air in the dark (37-40). The copper ions taken into the cytoplasmic membrane disorder the function and structure of the membrane, extinguishing cellular activity (49). When the Cu/TiO2 film was illuminated by white fluorescent light bulbs with a UV intensity of 40 µW/cm2, fewer 4786
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FIGURE 2. Changes in the survival of normal E. coli cells (IFO3301) versus illumination time. A cell suspension (2 × 105 CFU/mL) was incubated on a TiO2 film (O) and on a Cu/TiO2 film (0) under dark conditions, respectively. The suspension was also incubated on a Cu/TiO2 film under UV illumination at a UV light intensity of 40 µW/cm2 (9) and 1 µW/cm2 (b). Error bars: standard deviations of three replicate experiments.
FIGURE 3. Changes in survival of copper-resistant E. coli cells (53TNE007) on a Cu/TiO2 film under dark condition (b) and under UV illumination at light intensity of 7 µW/cm2 (9) and 1 µW/cm2 (2). The cell concentration was 2 × 105 CFU/mL. Error bars: standard deviations of three replicate experiments. E. coli cells survived than under the dark condition. This is explained by the photocatalytic reaction of TiO2 under UV illumination (21-23). However, the survival rate on the Cu/ TiO2 film at a UV intensity of 1 µW/cm2 was almost the same as that on the film stored in the dark. This is probably because the decreased rate of survival due to the copper ion is higher than that of the photocatalytic reaction of TiO2 at a UV intensity of 1 µW/cm2. Therefore, the bactericidal activity of the TiO2 photocatalytic reaction is hidden by the activity of the copper ion. In light of this result, we used copper-resistant E. coli cells (53TNE007) to elucidate the photocatalytic bactericidal activity of the Cu/TiO2 film under very weak UV intensities. Figure 3 shows the changes in survival of the copperresistant cells dropped onto the Cu/TiO2 film. In this case, most E. coli cells survived under dark conditions. As was described previously, the antibacterial activity of the copper ions appears when they are taken into the cell. However, the copper-resistant E. coli has little porin protein in the outer membrane. Therefore, the outer membrane loses the ability to transport solutes such as copper ion from the extracellular environment into the cell (50-54). In other words, the outer membrane can serve as a defensive shield against the antibacterial ions. However, when the Cu/TiO2 film was illuminated with very weak UV light, the survival rate began to decrease. This demonstrates photocatalytic bactericidal activity even under 1 µW/cm2, which is almost the UV light intensity of indoor
FIGURE 4. A schematic illustration of the bactericidal process for the copper-resistant E. coli cell on a normal TiO2 film and on a Cu/TiO2 film: (a) illustration of E. coli cell and (b)-(e) enlarged illustration of cell envelope parts. lighting using white fluorescent light bulbs. The survival decay curves under illumination in Figure 3 were not simple exponential curves. Initial illumination had little effect on the survival rate, but after 2 h a dramatic decrease in survival was observed. We recently reported a similar two-step decay process for the survival rate of normal E. coli on TiO2 film (without copper deposition) under relatively strong UV illumination such as 1 mW/cm2 (23). This behavior may be explained as follows: UV illumination of TiO2 produces various reactive species (e.g., •OH, HO2•, H2O2) in the presence of water and air by the following reactions (3, 4, 30).
TiO2 + hν f e- + h+
(1)
O2 + e- f O2-
(2)
O2- + H+ f HO2•
(3)
HO2• + HO2• f H2O2 + O2
(4)
H2O2 + e- f OH- + •OH
(5)
H2O + h+ f •OH
(6)
OH + •OH f H2O2
(7)
H2O2 + 2h+ f 2H+ + O2
(8)
〈Reduction site〉
〈Oxidation site〉
•
These reactive oxygen species can decompose organic compounds and extinguish cellular activity. However, the
cells are protected by the outer membrane as shown in Figure 4(a), and these species cannot break into the cytoplasmic membrane. Therefore, the first step in the survival curve corresponds to the period when the outer membrane of the E. coli cell envelope is partially decomposed by these reactive species (Figure 4(b),(c)). After the outer membrane has been disordered and partially decomposed, the reactive species penetrate to the cytoplasmic membrane, causing the cell to die. During this period, the cell is efficiently killed by UV illumination (Figure 4(c),(d)). Because these species are so reactive and they are easily trapped at the outer membrane, relatively strong UV light is necessary to cause cell death. By analogy with the above photocatalytic bactericidal model for the TiO2 film under strong UV illumination, we can speculate as to the bactericidal process for the copperresistant E. coli cells on the Cu/TiO2 film under weak UV illumination. The first step is a similar period during which the outer membrane is attacked by the reactive species produced by TiO2 photocatalysis (Figure 4(b),(c)). The second step is a period during which copper ions are effectively taken into the cytoplasmic membrane (Figure 4(c),(e)). In this case, the photocatalytic reaction mainly plays a role in assisting the intrusion of copper ions into the cell. This is probably the reason the E. coli cells are effectively killed on Cu/TiO2 film even under very weak UV light. This speculation is supported by the following control experiments. Figure 5 shows the survival of copper-resistant E. coli under 1 µW/cm2 UV illumination. Open and closed squares represent the survivals on TiO2 and Cu/TiO2 films, respectively. However, the closed circles were obtained by changing the photocatalyst film during the experiments. In this case, the cell suspension was first dropped on the TiO2 film and illuminated for 2 h, and then the suspension was transferred to the Cu/TiO2 film, followed by another 2 h illumination. The survival decay process under these experimental conditions was almost the same as that observed VOL. 37, NO. 20, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Amount of •OH on TiO2 Film and Cu/TiO2 Film without and with Addition of External H2O2 under UV Illumination (1 mW/cm2) for 20 Minutes and Dark Conditiona thin film
amount of •OH without external H2O2
amount of •OH with external H2O2
TiO2 + dark TiO2 + UV Cu/TiO2 + dark Cu/TiO2 + UV
ndb 1.0 ( 0.04 ndb 1.4 ( 0.07
ndb 1.1 ( 0.13 ndb 2.4 ( 0.03
a The fluorescence intensity at 460 nm observed from TiO film after 2 UV illumination was counted as 1.0. b nd, not detected.
FIGURE 5. Change in survival of copper-resistant E. coli cells (2 × 105 CFU/mL) on a TiO2 film (0) and on a Cu/TiO2 film (9) under UV illumination at light intensity of 1 µW/cm2. The survival was also determined when the cell suspension was initially illuminated on a TiO2 film for 2 h and then transferred to a Cu/TiO2 film, which was illuminated for another 2 h (b). The UV intensity was 1 µW/ cm2. Error bars: standard deviations of three replicate experiments. on Cu/TiO2, indicating that the first step proceeds even on normal TiO2. In addition, Figure 5 suggests that the second step is mainly due to the deposited copper because the decrease in E. coli survival is observed only on copperdeposited TiO2 films but not on TiO2 films. Finally, let us consider the valence state of the copper playing the key role in the bactericidal process. As was described previously, copper ions, which have antibacterial activity, exist on the Cu/TiO2 system in the dark when it is exposed to air. When the system is illuminated, however, the copper ions can be reduced to metallic copper by photogenerated electrons (eq 9), and the metallic copper can be oxidized to ions by photogenerated holes (eq 10) (37, 4248).
Therefore, the valence of copper might change under UV illumination. To elucidate whether copper ions exist under UV illumination, we tried to determine the amount of •OH produced with and without the addition of H2O2 to the photocatalytic system. The addition of external H2O2 produces •OH in the presence of copper ions and illuminated TiO2 through so-called photo-Fenton reactions as follows (37, 42-45).
H2O2 + Cu+ f •OH + OH- + Cu2+
(11)
Cu2+ + e- (or O2-) f Cu+ (or + O2)
(12)
The results are listed in Table 1. Here, the amount of •OH produced by the illumination of TiO2 for 20 min with UV intensity of 1 mW/cm2 is normalized to one. The TiO2 illumination produces •OH photocatalytically at both reduction and oxidation sites according to eqs 2-5 and eq 6, respectively. In the dark, •OH formation was not detected in either the TiO2 or Cu/TiO2 system and the addition of H2O2 to these systems did not change the situation, showing that the above photo-Fenton reaction did not proceed in either system. In other words, the Cu+ quantity was insufficient to be detected by this reaction even in the Cu/TiO2 system in the dark. However, in the Cu/TiO2 system under UV illumination, the addition of H2O2 clearly increased •OH formation, although such a drastic change was not observed in the TiO2 system. These data demonstrate that the amount 4788
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of Cu+ in the Cu/TiO2 system is greater under illumination than in the dark, and thus efficient bactericidal function could appear on the Cu/TiO2 film even under weak UV illumination. In conclusion, the Cu/TiO2 film displays bactericidal activity even for copper-resistant E. coli cells under very weak UV illumination. This effect is due to the synergy of the photocatalytic decomposition activity of TiO2 and the antibacterial activity of copper ions. It is well-known that microorganisms have resistance to many environmental circumstances, and copper-resistant bacteria arise easily under copper antibacterial conditions. This is one reason antibacterial inorganic ions do not function well in some cases. However, our results show that the Cu/TiO2 system under illumination is effective even for copper-resistant bacteria. Therefore, this system may show much more efficient bactericidal function for various bacteria than the conventional copper system in an ordinary living space with very weak UV intensity.
Acknowledgments The authors thank Prof. T. Nakae (Graduate School of Medicine, Tokai University) for a gift of the bacteria strain (53TNE007) and for information on the mutant. We also thank Prof. A. Fujishima and Dr. A. Nakajima for their valuable discussions. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (Area No. 417 for K.H.) from the Ministry of Education, Science, Sports and Culture of Japan.
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Received for review February 6, 2003. Revised manuscript received July 12, 2003. Accepted August 11, 2003. ES034106G
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