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Photocatalytic inactivation of bacteria by photocatalyst Bi2WO6 under visible light
Catalysis Communications 10 (2009) 1940–1943
Contents lists available at ScienceDirect
Catalysis Communications journal homepage: www.elsevier.com/locate/catcom
Photocatalytic inactivation of bacteria by photocatalyst Bi2WO6 under visible light Jia Ren a, Wenzhong Wang a,*, Ling Zhang a, Jiang Chang b, Sheng Hu b a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, PR China b Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 1 July 2009 Accepted 7 July 2009 Available online 9 July 2009 Keywords: Photocatalysis Visible light Single phase oxide Escherichia coli K+ leakage
a b s t r a c t The photocatalytic inactivation of Escherichia coli under visible light irradiation (k > 420 nm) was performed with Bi2WO6 to investigate the photocatalytic bactericidal capability. Our work shows that the single phase oxide photocatalyst Bi2WO6 is effective in photocatalytic inactivation on E. coli. And the results revealed that the photocatalytic inactivation rate of E. coli with Bi2WO6 followed pseudo-firstorder kinetics. The bactericidal action was directly observed by TEM and further proved by the measurement of K+ leakage from the inactive E. coli through the ICP-OES analysis. The results demonstrated that the photocatalysis could cause drastic damage in E. coli cells. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the traditional disinfection processes such as chlorination led to the production of toxic by-products [1], there has been a growing interest in the development of photocatalysis for water disinfection in recent years. The earliest example of the semiconductor photocatalysis applied as a disinfection method was reported by Matsunaga et al. [2]. Since then, TiO2 photocatalytic disinfection has been studied extensively because of its safety, low-cost and efficiency [3]. However, its low quantum yields and the absorbance of only near UV-region (k < 400 nm) limit its practical application. From the viewpoint of utilizing solar irradiation, many works have been devoted to enhance the photocatalytic efficiency and visible light utilization of TiO2 by modification methods, including impurity doping, metallization, sensitization and coating, to extend the absorbance edge of TiO2 into visible light region [4–7]. However, the modifying processes are complex and the as-prepared samples are often suffered from the disadvantages such as low stability. Recently, the development of efficient visible light driven photocatalysts has been an important issue. The majority work on exploring new visible light driven photocatalysts are focused on the photocatalytic degradation of organic pollutants. However, similar work is rather few in photocatalytic destruction of microor-
* Corresponding author. Fax: +86 21 5241 3122. E-mail address: [email protected] (W. Wang). 1566-7367/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.catcom.2009.07.006
ganism [8,9]. Therefore, it is significative to develop efficient pure visible light driven bactericidal photocatalysts. Bi2WO6, as a typical Aurivillius oxide, obtained great interest of research due to its excellent intrinsic physical and chemical properties [10]. And it was confirmed that Bi2WO6 could perform well in photocatalytic degradation of organic substances under visible light irradiation [11]. Previously, our group had reported the preparation, photocatalytic activity, and stability of Bi2WO6 in the degradation of dyes [12]. Inspired by the disinfection effect of TiO2 photocatalyst, we conceive that Bi2WO6 might be a good candidate for water disinfection due to its narrow band gap and good stability. In the present study, the photocatalytic bactericidal capability of Bi2WO6 was studied through the inactivation of Escherichia coli. The target of the work is to provide a basic investigation on the bactericidal kinetics of the Bi2WO6 photocatalyst under visible light irradiation, which is a non-TiO2, single phase oxide photocatalyst system.
2. Experimental section 2.1. Preparation of photocatalysts The Bi2WO6 sample was prepared according to Ref. [12], except that ammonium bismuth citrate (Bi(NH3)2C6H7O7H2O) was replaced by Bi(NO3)35H2O as Bi source, which is much cheaper, with the assistance of citric acid.
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
1941
2.2. Characterization Transmission electron microscopy (TEM) was used to observe the morphology of E. coli on a Hitachi H-600 before and after the bactericidal experiment. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were performed on a Vista AX (end view pattern). 2.3. Preparation of the bacterial culture E. coli, a Gram-negative bacterium, was used as model bacteria in this study. It was incubated in Lysogeny Broth (LB) medium at 37 °C for 18 h. Cells were harvested from overnight culture by centrifugation at 4000 rpm for 5 min and then washed twice with 0.9% saline. The treated cells were then re-suspended and diluted to 2 107 colony-forming units (cfu/mL) with 0.9% saline. 2.4. Bactericidal activity All materials used in the experiments were autoclaved at 121 °C for 40 min before use to ensure sterility. The final photocatalyst concentration was adjusted to 0.2, 0.5 and 1.0 mg/mL, respectively. A 500 W Xe lamp was used as the light source with a UV cutoff filter (k > 420 nm) to provide visible light irradiation. The reaction mixture was stirred with a magnetic stirrer to prevent settling of the photocatalysts. The experiments were carried out at room temperature. Before and during the experiments, an aliquot of the reaction mixture was immediately diluted with 0.9% saline solution and plated on LB-agar plates. The colonies were counted after incubation at 37 oC for 24 h. The survival ratio of E. coli were determined by the ratio of Ct/C0, where C0 and Ct are the numbers of cfu at the initial and each following time interval, respectively. All of the above experiments were repeated three times and the average values were given.
3. Results and discussion 3.1. Bacterial inactivation under visible light irradiation The bacterial activity of the Bi2WO6 was exhibited by the killing effect of E. coli, which were evaluated through the decrease of the colony number formed on an agar plate. According to Fig. 1A, E. coli can be almost completely inactivated within 2 h with Bi2WO6 photocatalysts undervisiblelight irradiation.Neithervisiblelightwithoutthephotocatalyst nor Bi2WO6 in the dark showed any bactericidal effects on E.coli,indicatingthatthephotocatalystitselfisnottoxictoE.coli.Thus, the bactericidal effect on E. coli is surely ascribed to the photocatalytic reaction of the Bi2WO6 under visible light irradiation. When the concentration of the Bi2WO6 is 0.5 mg/mL, E. coli inactivation efficiency is up to 95% after 2 h visible light irradiation. The results show that thesinglephaseoxidephotocatalystBi2WO6 exhibitsexcellentphotocatalytic inactivation on E. coli under visible light irradiation. And the photocatalyst Bi2WO6 exhibited good stability in the photocatalytic process (Fig. S4A). An exponential decrease in the remaining E. coli cfu within 90 min is observed in Fig. 1A, suggesting that pseudo-first-order kinetics may be applicable in our experiment. The log transformation of the inactivation process of E. coli confirms that the pseudofirst-order kinetics is followed. The Langmuir–Hinshelwood model is well-established for photocatalysis experiments, and we use the equation (1) to attempt a description of the photocatalytic disinfection process.
dC ¼ kC ¼ r dt
ð1Þ
Fig. 1. (A) Survival ratio of E. coli (2 107 cfu/mL, 20 mL) in aqueous dispersions (a) Bi2WO6 in the dark (0.5 mg/mL); (b) No catalyst; and (c) Bi2WO6 (0.5 mg/mL) under visible light irradiation (BWO is short for Bi2WO6). (B) Kinetic linear simulation curve of E. coli photocatalytic inactivation with Bi2WO6.
Integration of the above equation will obtain the following equation
ln
C0 C
¼ kt
ð2Þ
where C is the E. coli concentration in the bulk solution, C 0 is the initial concentration in the bulk solution, k is the apparent pseudofirst-order rate constant and t is the reaction time, with the restriction of C = C0 at t = 0. A kinetic curve of E. coli inactivation based on the data plotted in Fig. 1A is shown in Fig. 1B. The value of k for the inactivation process is 0.0157 with an average correlation coefficient of 0.996. The photocatalytic inactivation process within initial 90 min followed a good linear relationship with the reaction time. The simple linear model has been widely used in the literature to compare the efficiency of different inactivation processes through the values of the kinetic constant, k, and the points within the time of initial reaction process fit a good linear relationship [13,14]. The data at the final point when t = 120 min deviated from the linear relationship, which could be relevant to the self-defense and autorepairing mechanisms of bacteria. The response to oxidative stress has been well characterized for some microorganisms and especially for the facultative aerobic Gram-negative bacteria as E. coli [15]. The capacity of self-defense and autorepairing mechanisms of bacteria, which is unfavorable for the inactivation process, is des-
1942
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
Fig. 2. The antibacterial efficiency of E. coli with 0.2, 0.5 and 1.0 mg/mL Bi2WO6.
tructed badly and cannot work efficiently with continuous visible light irradiation at the terminal stage of the process. Thus, a positive deviation appeared at the final data point in Fig. 1B. 3.2. Effect of Bi2WO6 concentration on E. coli inactivation As is well known, the concentration of the photocatalyst has a significant influence on the bactericidal effects. In our experiments, a higher efficiency of E. coli inactivation was obtained when the concentration of Bi2WO6 was increased from 0.2 to 0.5 mg/mL (Fig. 2). More photocatalysts generate more reactive sites for E. coli inactivation. However, the bactericidal effect was not significantly enhanced when the concentration of photocatalyst was increased from 0.5 to 1.0 mg/mL. The saturating photoactivity with increasing Bi2WO6 concentration could be understood as the competition between the surface area and the light scattering loss. While Bi2WO6 with higher concentration provides more active catalytic sites, the light penetration depth into the suspension decreases due to the increased light scattering, which reduces the efficiency of the photocatalytic inactivation of E. coli [16]. 3.3. Evidence of cell damage To study the bactericidal action of the Bi2WO6 photocatalyst, the morphology of the bacteria was investigated by TEM before and after the bactericidal experiment. The specimens were colored
by mixing with 2% phosphotungstic acid followed by observing. As shown in Fig. 3, the living E. coli showed a regular intact cellular structure clearly with an evenly rendered interior of the cell, and its flagellum could also be observed (Fig. 3A). However, the morphology of the E. coli had changed greatly after the bactericidal experiment. The damage-free cellular structure disappeared, while white/empty regions were observed. As shown in Fig. 3B and the inset, the cells of E. coli treated for 2 h were damaged with inhomogeneous ruinate structures. The cells were not intact anymore, and the flagella also disappeared. The results suggested that the E. coli cells were damaged in the presence of Bi2WO6 under visible light irradiation, which was confirmed by the FT-IR measurements on the bacterial cells before and after photocatalytic process (Fig. S4B). The bactericidal action was further confirmed by the measurement of K+ leakage, which was used to examine the permeability of the cell membrane. In this work, the measurement of K+ leakage from the inactive E. coli was carried out under variant conditions by ICP-OES before and after every experiment respectively (Fig. 4). With Bi2WO6 in the dark, the K+ leakage from E. coli cells was nearly the same when the time prolonged. Without catalyst under visible light irradiation only, there was a slight increase in the K+ leakage. Contrarily, K+ leakage increased notably with the Bi2WO6 under visible light irradiation. However, the detected K+ concentrations are not proportional to the number of dead bacteria. The membrane of partial bacteria may not be damaged badly, especially the cytoplasmic membrane was still unspoiled and did not leak out the K+, but the cells had already lost their viability, which cannot propagate into a visible colony anymore [14]. Therefore, the K+ concentrations are less proportional to the number of dead bacteria. The bactericidal mechanisms are still in research. So far, there are two main mechanisms presented to explain the photocatalytic inactivation of pathogenic bacteria. The first putative killing mechanism proposed by Matsunaga et al. implies an oxidation of the intracellular coenzyme A (CoA), which inhibits the cell respiration and subsequently causes cell death as a result of a direct contact between the photocatalysts and the target cells [2]. The second killing mode suggests that bacterial death is caused by a significant disorder in the cell permeability and by the decomposition of the cell walls [17]. Photocatalytic treatment progressively increases the cell permeability and subsequently allows free efflux of intracellular constituents, which eventually lead to the cell death. Considering the K+ measurements combined with TEM images, the photocatalytic action brought a certain level of damage to the membrane and a resultant leakage of intracellular substances. The photocatalytic bactericidal mechanism over Bi2WO6 needs to be further studied in subsequent work.
Fig. 3. TEM images of E. coli irradiated by visible light with Bi2WO6 (A) E. coli before reaction; (B) E. coli treated for 2 h.
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
1943
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 50672117 and 50732004) and the Nanotechnology Programs of Science and Technology Commission of Shanghai Municipality (0852nm00500). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.catcom.2009.07.006. References
Fig. 4. The content of K+ ion in aqueous dispersions before and after experiments (treated for 2 h) under different conditions: (1) E. coli suspension without Bi2WO6 under visible light irradiation; (2) E. coli suspension containing 0.5 mg/mL Bi2WO6 in the dark; (3) E. coli suspension containing 0.5 mg/mL Bi2WO6 under visible light irradiation.
4. Conclusions Bi2WO6 was found to be a novel and promising visible light driven bactericidal catalyst. It exhibited a notable visible light induced photocatalytic antibacterial capability due to its appropriate band gap and the adsorption of bacteria. The concentration of photocatalyst has a significant impact on the efficiency of bacteria inactivation. Experimental results suggested that the photocatalytic inactivation rate could be characterized by pseudo-first-order kinetic behavior. In addition, the results of antibacterial experiments under visible light irradiation verified that the E. coli cells were damaged by the photocatalysis, and the permeability of the cell membrane disordered leading to the leakage of intracellular substances.
[1] C.E. Lawrance, P.R. Taylor, B.J. Trock, A.A. Reilly, Nat. Cancer Inst. 72 (1984) 563–568. [2] T. Matsunaga, R. Tomada, T. Nakajima, H. Wake, FEMS Microbiol. Lett. 29 (1985) 211–214. [3] J.Y. Choi, K.H. Kim, K.C. Choy, K.T. Oh, K.N. Kim, J. Biomed. Mater. Res. – A 2 (2007) 353–359. [4] J.C. Yu, W.K. Ho, J.G. Yu, H.Y. Yip, P. Wong, J.C. Zhao, Environ. Sci. Technol. 39 (2005) 1175–1179. [5] M.R. Elahifard, S. Rahimnejad, S. Haghighi, M.R. Gholami, J. Am. Chem. Soc. 129 (2007) 9552–9553. [6] T. Ohno, T. Mitsui, M. Matsumura, Chem. Lett. 32 (2003) 364–365. [7] R.W. Fessenden, P.V. Kamat, J. Phys. Chem. 99 (1995) 12902–12906. [8] C. Hu, X. Hu, J. Guo, J. Qu, Environ. Sci. Technol. 40 (2006) 5508–5513. [9] H.T. Yu, X. Quan, Y.B. Zhang, N. Ma, S. Chen, H.M. Zhao, Langmuir 24 (2008) 7599–7604. [10] C. Zhang, Y. Zhu, Chem. Mater. 17 (2005) 3537–3545. [11] J.G. Yu, J.F. Xiong, B. Cheng, Y. Yu, J. Wang, J. Solid State Chem. 178 (2005) 1968–1972. [12] M. Shang, W. Wang, S. Sun, L. Zhou, L. Zhang, J. Phys. Chem. C 112 (2008) 10407–10411. [13] D.D. Sun, J.H. Tay, K.M. Tan, Water Res. 37 (2003) 3452–3462. [14] Y. Li, M. Ma, X. Wang, X. Wang, J. Environ. Sci. 20 (2008) 1527–1533. [15] M. Gourmelon, J. Cillard, M. Pommepuy, J. Appl. Bacteriol. 77 (1994) 105–112. [16] M. Bekbolet, Water Sci. Technol. 35 (1997) 95–100. [17] T. Saito, T. Iwase, T. Morioka, J. Photochem. Photobiol. B 14 (1992) 369–379.
Contents lists available at ScienceDirect
Catalysis Communications journal homepage: www.elsevier.com/locate/catcom
Photocatalytic inactivation of bacteria by photocatalyst Bi2WO6 under visible light Jia Ren a, Wenzhong Wang a,*, Ling Zhang a, Jiang Chang b, Sheng Hu b a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, PR China b Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 1 July 2009 Accepted 7 July 2009 Available online 9 July 2009 Keywords: Photocatalysis Visible light Single phase oxide Escherichia coli K+ leakage
a b s t r a c t The photocatalytic inactivation of Escherichia coli under visible light irradiation (k > 420 nm) was performed with Bi2WO6 to investigate the photocatalytic bactericidal capability. Our work shows that the single phase oxide photocatalyst Bi2WO6 is effective in photocatalytic inactivation on E. coli. And the results revealed that the photocatalytic inactivation rate of E. coli with Bi2WO6 followed pseudo-firstorder kinetics. The bactericidal action was directly observed by TEM and further proved by the measurement of K+ leakage from the inactive E. coli through the ICP-OES analysis. The results demonstrated that the photocatalysis could cause drastic damage in E. coli cells. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the traditional disinfection processes such as chlorination led to the production of toxic by-products [1], there has been a growing interest in the development of photocatalysis for water disinfection in recent years. The earliest example of the semiconductor photocatalysis applied as a disinfection method was reported by Matsunaga et al. [2]. Since then, TiO2 photocatalytic disinfection has been studied extensively because of its safety, low-cost and efficiency [3]. However, its low quantum yields and the absorbance of only near UV-region (k < 400 nm) limit its practical application. From the viewpoint of utilizing solar irradiation, many works have been devoted to enhance the photocatalytic efficiency and visible light utilization of TiO2 by modification methods, including impurity doping, metallization, sensitization and coating, to extend the absorbance edge of TiO2 into visible light region [4–7]. However, the modifying processes are complex and the as-prepared samples are often suffered from the disadvantages such as low stability. Recently, the development of efficient visible light driven photocatalysts has been an important issue. The majority work on exploring new visible light driven photocatalysts are focused on the photocatalytic degradation of organic pollutants. However, similar work is rather few in photocatalytic destruction of microor-
* Corresponding author. Fax: +86 21 5241 3122. E-mail address: [email protected] (W. Wang). 1566-7367/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.catcom.2009.07.006
ganism [8,9]. Therefore, it is significative to develop efficient pure visible light driven bactericidal photocatalysts. Bi2WO6, as a typical Aurivillius oxide, obtained great interest of research due to its excellent intrinsic physical and chemical properties [10]. And it was confirmed that Bi2WO6 could perform well in photocatalytic degradation of organic substances under visible light irradiation [11]. Previously, our group had reported the preparation, photocatalytic activity, and stability of Bi2WO6 in the degradation of dyes [12]. Inspired by the disinfection effect of TiO2 photocatalyst, we conceive that Bi2WO6 might be a good candidate for water disinfection due to its narrow band gap and good stability. In the present study, the photocatalytic bactericidal capability of Bi2WO6 was studied through the inactivation of Escherichia coli. The target of the work is to provide a basic investigation on the bactericidal kinetics of the Bi2WO6 photocatalyst under visible light irradiation, which is a non-TiO2, single phase oxide photocatalyst system.
2. Experimental section 2.1. Preparation of photocatalysts The Bi2WO6 sample was prepared according to Ref. [12], except that ammonium bismuth citrate (Bi(NH3)2C6H7O7H2O) was replaced by Bi(NO3)35H2O as Bi source, which is much cheaper, with the assistance of citric acid.
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
1941
2.2. Characterization Transmission electron microscopy (TEM) was used to observe the morphology of E. coli on a Hitachi H-600 before and after the bactericidal experiment. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were performed on a Vista AX (end view pattern). 2.3. Preparation of the bacterial culture E. coli, a Gram-negative bacterium, was used as model bacteria in this study. It was incubated in Lysogeny Broth (LB) medium at 37 °C for 18 h. Cells were harvested from overnight culture by centrifugation at 4000 rpm for 5 min and then washed twice with 0.9% saline. The treated cells were then re-suspended and diluted to 2 107 colony-forming units (cfu/mL) with 0.9% saline. 2.4. Bactericidal activity All materials used in the experiments were autoclaved at 121 °C for 40 min before use to ensure sterility. The final photocatalyst concentration was adjusted to 0.2, 0.5 and 1.0 mg/mL, respectively. A 500 W Xe lamp was used as the light source with a UV cutoff filter (k > 420 nm) to provide visible light irradiation. The reaction mixture was stirred with a magnetic stirrer to prevent settling of the photocatalysts. The experiments were carried out at room temperature. Before and during the experiments, an aliquot of the reaction mixture was immediately diluted with 0.9% saline solution and plated on LB-agar plates. The colonies were counted after incubation at 37 oC for 24 h. The survival ratio of E. coli were determined by the ratio of Ct/C0, where C0 and Ct are the numbers of cfu at the initial and each following time interval, respectively. All of the above experiments were repeated three times and the average values were given.
3. Results and discussion 3.1. Bacterial inactivation under visible light irradiation The bacterial activity of the Bi2WO6 was exhibited by the killing effect of E. coli, which were evaluated through the decrease of the colony number formed on an agar plate. According to Fig. 1A, E. coli can be almost completely inactivated within 2 h with Bi2WO6 photocatalysts undervisiblelight irradiation.Neithervisiblelightwithoutthephotocatalyst nor Bi2WO6 in the dark showed any bactericidal effects on E.coli,indicatingthatthephotocatalystitselfisnottoxictoE.coli.Thus, the bactericidal effect on E. coli is surely ascribed to the photocatalytic reaction of the Bi2WO6 under visible light irradiation. When the concentration of the Bi2WO6 is 0.5 mg/mL, E. coli inactivation efficiency is up to 95% after 2 h visible light irradiation. The results show that thesinglephaseoxidephotocatalystBi2WO6 exhibitsexcellentphotocatalytic inactivation on E. coli under visible light irradiation. And the photocatalyst Bi2WO6 exhibited good stability in the photocatalytic process (Fig. S4A). An exponential decrease in the remaining E. coli cfu within 90 min is observed in Fig. 1A, suggesting that pseudo-first-order kinetics may be applicable in our experiment. The log transformation of the inactivation process of E. coli confirms that the pseudofirst-order kinetics is followed. The Langmuir–Hinshelwood model is well-established for photocatalysis experiments, and we use the equation (1) to attempt a description of the photocatalytic disinfection process.
dC ¼ kC ¼ r dt
ð1Þ
Fig. 1. (A) Survival ratio of E. coli (2 107 cfu/mL, 20 mL) in aqueous dispersions (a) Bi2WO6 in the dark (0.5 mg/mL); (b) No catalyst; and (c) Bi2WO6 (0.5 mg/mL) under visible light irradiation (BWO is short for Bi2WO6). (B) Kinetic linear simulation curve of E. coli photocatalytic inactivation with Bi2WO6.
Integration of the above equation will obtain the following equation
ln
C0 C
¼ kt
ð2Þ
where C is the E. coli concentration in the bulk solution, C 0 is the initial concentration in the bulk solution, k is the apparent pseudofirst-order rate constant and t is the reaction time, with the restriction of C = C0 at t = 0. A kinetic curve of E. coli inactivation based on the data plotted in Fig. 1A is shown in Fig. 1B. The value of k for the inactivation process is 0.0157 with an average correlation coefficient of 0.996. The photocatalytic inactivation process within initial 90 min followed a good linear relationship with the reaction time. The simple linear model has been widely used in the literature to compare the efficiency of different inactivation processes through the values of the kinetic constant, k, and the points within the time of initial reaction process fit a good linear relationship [13,14]. The data at the final point when t = 120 min deviated from the linear relationship, which could be relevant to the self-defense and autorepairing mechanisms of bacteria. The response to oxidative stress has been well characterized for some microorganisms and especially for the facultative aerobic Gram-negative bacteria as E. coli [15]. The capacity of self-defense and autorepairing mechanisms of bacteria, which is unfavorable for the inactivation process, is des-
1942
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
Fig. 2. The antibacterial efficiency of E. coli with 0.2, 0.5 and 1.0 mg/mL Bi2WO6.
tructed badly and cannot work efficiently with continuous visible light irradiation at the terminal stage of the process. Thus, a positive deviation appeared at the final data point in Fig. 1B. 3.2. Effect of Bi2WO6 concentration on E. coli inactivation As is well known, the concentration of the photocatalyst has a significant influence on the bactericidal effects. In our experiments, a higher efficiency of E. coli inactivation was obtained when the concentration of Bi2WO6 was increased from 0.2 to 0.5 mg/mL (Fig. 2). More photocatalysts generate more reactive sites for E. coli inactivation. However, the bactericidal effect was not significantly enhanced when the concentration of photocatalyst was increased from 0.5 to 1.0 mg/mL. The saturating photoactivity with increasing Bi2WO6 concentration could be understood as the competition between the surface area and the light scattering loss. While Bi2WO6 with higher concentration provides more active catalytic sites, the light penetration depth into the suspension decreases due to the increased light scattering, which reduces the efficiency of the photocatalytic inactivation of E. coli [16]. 3.3. Evidence of cell damage To study the bactericidal action of the Bi2WO6 photocatalyst, the morphology of the bacteria was investigated by TEM before and after the bactericidal experiment. The specimens were colored
by mixing with 2% phosphotungstic acid followed by observing. As shown in Fig. 3, the living E. coli showed a regular intact cellular structure clearly with an evenly rendered interior of the cell, and its flagellum could also be observed (Fig. 3A). However, the morphology of the E. coli had changed greatly after the bactericidal experiment. The damage-free cellular structure disappeared, while white/empty regions were observed. As shown in Fig. 3B and the inset, the cells of E. coli treated for 2 h were damaged with inhomogeneous ruinate structures. The cells were not intact anymore, and the flagella also disappeared. The results suggested that the E. coli cells were damaged in the presence of Bi2WO6 under visible light irradiation, which was confirmed by the FT-IR measurements on the bacterial cells before and after photocatalytic process (Fig. S4B). The bactericidal action was further confirmed by the measurement of K+ leakage, which was used to examine the permeability of the cell membrane. In this work, the measurement of K+ leakage from the inactive E. coli was carried out under variant conditions by ICP-OES before and after every experiment respectively (Fig. 4). With Bi2WO6 in the dark, the K+ leakage from E. coli cells was nearly the same when the time prolonged. Without catalyst under visible light irradiation only, there was a slight increase in the K+ leakage. Contrarily, K+ leakage increased notably with the Bi2WO6 under visible light irradiation. However, the detected K+ concentrations are not proportional to the number of dead bacteria. The membrane of partial bacteria may not be damaged badly, especially the cytoplasmic membrane was still unspoiled and did not leak out the K+, but the cells had already lost their viability, which cannot propagate into a visible colony anymore [14]. Therefore, the K+ concentrations are less proportional to the number of dead bacteria. The bactericidal mechanisms are still in research. So far, there are two main mechanisms presented to explain the photocatalytic inactivation of pathogenic bacteria. The first putative killing mechanism proposed by Matsunaga et al. implies an oxidation of the intracellular coenzyme A (CoA), which inhibits the cell respiration and subsequently causes cell death as a result of a direct contact between the photocatalysts and the target cells [2]. The second killing mode suggests that bacterial death is caused by a significant disorder in the cell permeability and by the decomposition of the cell walls [17]. Photocatalytic treatment progressively increases the cell permeability and subsequently allows free efflux of intracellular constituents, which eventually lead to the cell death. Considering the K+ measurements combined with TEM images, the photocatalytic action brought a certain level of damage to the membrane and a resultant leakage of intracellular substances. The photocatalytic bactericidal mechanism over Bi2WO6 needs to be further studied in subsequent work.
Fig. 3. TEM images of E. coli irradiated by visible light with Bi2WO6 (A) E. coli before reaction; (B) E. coli treated for 2 h.
J. Ren et al. / Catalysis Communications 10 (2009) 1940–1943
1943
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos. 50672117 and 50732004) and the Nanotechnology Programs of Science and Technology Commission of Shanghai Municipality (0852nm00500). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.catcom.2009.07.006. References
Fig. 4. The content of K+ ion in aqueous dispersions before and after experiments (treated for 2 h) under different conditions: (1) E. coli suspension without Bi2WO6 under visible light irradiation; (2) E. coli suspension containing 0.5 mg/mL Bi2WO6 in the dark; (3) E. coli suspension containing 0.5 mg/mL Bi2WO6 under visible light irradiation.
4. Conclusions Bi2WO6 was found to be a novel and promising visible light driven bactericidal catalyst. It exhibited a notable visible light induced photocatalytic antibacterial capability due to its appropriate band gap and the adsorption of bacteria. The concentration of photocatalyst has a significant impact on the efficiency of bacteria inactivation. Experimental results suggested that the photocatalytic inactivation rate could be characterized by pseudo-first-order kinetic behavior. In addition, the results of antibacterial experiments under visible light irradiation verified that the E. coli cells were damaged by the photocatalysis, and the permeability of the cell membrane disordered leading to the leakage of intracellular substances.
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