Studies on characteristics of nanostructure of N-TiO₂ thin films and photo-bactericidal action

Studies on characteristics of nanostructure of N-TiO 2 thin films and photo-bactericidal action 1 first-author †E-mail:xuxuxu20042005@126.com †E-mail:xuxuxu20042005@126.com xuxuxu20042005@126.com 1 2 2 Department of Bioengineering, Jinan University, Guangzhou 510632, China Department of Bioengineering, Jinan University, Guangzhou 510632, China Institute of Aquatic Ecoscience, Jinan University, Guangzhou 510632, China Institute of Aquatic Ecoscience, Jinan University, Guangzhou 510632, China Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) from Ming lake were decomposed by photocatalytic nanostructure N-TiO 2 thin films in a photo-reactor under UV irradiation. The different thickness nanostructure N-TiO 2 thin films coated on mesh grid were prepared by sol-gel method and immobilized at 500 °C (films A) or 350 °C (films B) for 1 h in a muffle furnace. The results showed that N-TiO 2 thin film B (8.18 nm thickness, 2.760 nm height and 25.15 nm diameter) has more uniform granular nanostructure and thinner flat texture than N-TiO 2 thin film A (12.17 nm thickness, 3.578 nm height and 27.50 nm diameter). The bactericidal action of N-TiO 2 thin film A and film B for Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis varniger strain (AS1.439) were investigated in this work. More than 95% of photocatalytic bactericidal efficiency for Pseudomonas aeruginosa strain (AS1.50) and 75% for Bacillus subtilis strain (AS1.439) were achieved by using N-TiO 2 thin films-B for 70~80 min of irradiation during the photo-bactericidal experimental process. The results indicated that the photo-induced bactericidal efficiency of N-TiO 2 thin films probably depended on the characteristics of the films. Photocatalytic degradation Photo-bactericidal action N-TiO 2 thin film INTRODUCTION 1 Matsunaga et al.( 1985 1985 ) first discovered the bactericidal activity of photocatalytic TiO 2 reactions. Using TiO 2 powder particles in the bactericidal action and photocatalytic degradation for the oxidation of organic and inorganic water pollutants were extensively studied (Michael et al., 1999 1999 ). However, conventional TiO 2 powder catalysts were characterized by difficult separation and reproduction after reaction, so the application of titanium dioxide thin film has attracted much attention in recently years. We investigated the relationship between the antibacterial activity and the properties of the N-TiO 2 thin films, and found that the crystal structure (anatase, rutile) and morphology of particles size are important factors affecting the photocatalytic bactericidal action. The viability of Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) during the N-TiO 2 photocatalytic reactions are also discussed. MATERIALS AND METHODS 1 Preparation of nanostructure N-TiO 2 thin film 2 The nanostructure N-TiO 2 thin film was prepared by using the sol-gel method (Fig. 1 1 ). A certain amount of tetrabutylorthotitanate and acetylacetone were mixed together first, then ethanol was added with vigorous stirring for 1 h at room temperature. After that, distilled water and nitric acid were added under stirring into the above solution. The resultant alkoxide solution was further mixed with carbamide/chloroethylamine solution for 1 h and preserved at room temperature for full hydrolysis to form the N-TiO 2 colloidal sol. The mesh grid (156.2 mm×25.4 mm×1.5 mm), which was pretreated as the loaded basis for thin films, was dipped into the N-TiO 2 colloidal sol solution for 2 min, then withdraw from the solution at rate of 1 mm/s. The film was formed on mesh grid and dried at ambient temperature for 24 h. The mesh grid coated with gel films was treated at different temperature (film A at 500 °C and film B at 350 °C/500 °C) for 1 h in a muffle furnace. For film A, the mesh grid coated with the gel was sintered at 500 °C for 1 h with temperature raising rate of 3 °C/min in the muffle furnace, then repeatedly coated and calcined several times and finally sintered at 500 °C. For film B, the mesh grid coated with the gel films was sintered at 350 °C for 1 h, then repeatedly coated and calcined several times, with the last coating film being sintered at 500 °C for 1 h. Different thickness films were obtained by repeating the above process several times. Characteristics of N-TiO 2 films 2 The surface morphology of the films and nanoparticles were determined by atomic force microscope (AFM, Dimension 3100, American DI). The crystallite size and phase of the N-TiO 2 thin films were determined by BDX3300 X-ray diffraction (XRD) measurements, carried out with Cu K α radiation ( λ =1.54 nm). The accelerating voltage and the applied current were 40 kV and 40 mA, respectively. Setup of photocatalytic reactor system 2 Fig. 2 2 is a schematic diagram of the experimental apparatus. The photo-reactor with cooling jacket was assembled with UV light (mercury lamp 20 W, 30 cm long at wavelength 365 nm), covered with 3 mm thick transparent quartz glass cannula and immersed in the reaction solution at a constant temperature, used as a source of UV radiation. Sterile compressed air was bubbled into the reactor through air line. The incident UV radiation for the reactor was measured by ultraviolet luminometer. Cultivation of microbial strain 2 Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) were isolated from lake water and grown aerobically in 500 ml liquid nutrient broth at 37 °C on a rotary shaker (120 r/min) for 16 h. The medium was autoclaved at 121 °C for 30 min to ensure the sterility for testing. Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) cells were cultivated for 2 d and harvested by centrifugation at 8000×g for 10 min, washed and resuspended in the 1000 ml phosphate-buffer solution (0.2 mol/L, pH=7.0). The number of viable cells in the suspensions samples was counted by using the spread plate colony-counting method. Serial dilution of the cells was performed to obtain initial concentration of 10 6 colonies forming units per milliliter (CFU/ml) for photocatalytic bactericidal experiment. Photocatalytic bactericidal action procedure and cell viability assay 2 The 750 ml cells-suspended solution with initial concentration of 10 6 CFU/ml in 1 L reactor was illuminated by irradiation of 20 WUV-Hg lamps (GGZ-20 W) with emitting spectral maximum at 365 nm peak wavelength. The light intensity reaching the surface of the films fixed at the glass reactor was approximately 18 W/m 2 , which was measured by UV-meter with the peak intensity at 365 nm (model J-1221, UVP Inc. USA). N-TiO 2 film A or N-TiO 2 film B was placed into the reactor. Aliquots of cells solution were taken at 15 min intervals after irradiation with continuous stirring magnetically for uniform distribution, were added into the tube containing phosphate buffer solution for CFU testing. The CFU were determined by plating aliquots of serially diluted suspensions on duplicate nutrient agar plates. All plates were incubated for 24 h at 30 °C, the viable number of cells were counted. The loss of cell viability was examined by the viable count procedure. All experiments were performed at least in triplicate. RESULTS AND DISCUSSION 1 Characteristics of the nanostructure N-TiO 2 thin films 2 It is well known that the films surface morphology is dependent on the calcining treatment temperature. Different size particles on the N-TiO 2 films will be obtained at different sintering temperature for the immobilized films treatment. The film styles formed were divided into film A and film B based on a different sintering temperature. The results are shown in Fig. 3 3 . Fig. 3 3 is the AFM images of the N-TiO 2 thin films with five layers in film A and film B. N-TiO 2 film B has more uniform granular microstructure and thinner flat texture than N-TiO 2 film A (Fig. 3 3 ). The thickness and the average diameter of the particle size of the film were 12.17 nm and 27.50 nm for film A, 8.18 nm and 25.15 nm for film B respectively determined by AFM and XRD technique. The granular microstructure is the key factor influencing the photocatalytic activity (Jang et al., 2001 2001 ). In the N-TiO 2 film B, the mass fraction of crystallitic anatase present in granular nanostructure are mostly up to 94.8% higher than 65.2% of mass fraction in the N-TiO 2 film A. Zarzychi et al.( 1982 1982 ) explained the mechanism of the particles growth of gel films in the calcining process. From Fig. 4 4 shows that during calcining treatment for the film A process, there are many H 2 O moleculars and hydroxyl groups forming hydrogen bonds on the sol-gel surface of the N-TiO 2 film coated on mesh grid, when N-TiO 2 gel films were treated at 500 °C five times, hydrogen bonds between the two molecules would turn into chemical covalent bond during sintering process, then link close N-TiO 2 crystallitic particles tightly, result in the particles growing and solid cluster conglomeration, consequently, lead to the rapidly increasing number of larger diameter particles. In film B, N-TiO 2 thin film (8.18 nm thickness, 2.760 nm height and 25.15 nm average nanoparticle diameter) has more uniform granular microstructure and thinner flat texture than N-TiO 2 thin film A (12.17 nm thickness, 3.578 nm height and 27.50 nm average nanoparticle diameter) because low sintering temperature decreases the coagulation and growth rate of particles, hence, the mass fraction of N-TiO 2 anatase nanostructure has more reactive center in N-TiO 2 thin film B than in N-TiO 2 thin film A, which is probably due to the smaller size of anatase nanoparticle. Effect of N-TiO 2 nanoparticles of thin film on photocatalytic bactericidal activity 2 Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) cells inactivation was found to be rapid in the presence of nanostructured N-TiO 2 films during UV irradiation. Fig. 5 5 shows the effect of illuminated N-TiO 2 thin films B (8.18 nm thickness) on the sterilization of Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439) in comparison with N-TiO 2 thin films A (12.17 nm thickness) under the conditions of initial concentration of 10 6 CFU/ml, 18 W/m 2 of UV intensity. The viability of cells is determined by colonies counting. The survival curves are shown in Fig. 5 5 for Pseudomonas aeruginosa strain (AS1.50) and Bacillus subtilis strain (AS1.439), respectively. More than 95% photocatalytic sterilization rate for Pseudomonas aeruginosa strain (AS1.50) and 75% for Bacillus subtilis strain (AS1.43) were achieved by using N-TiO 2 thin films B for 70~80 min of irradiation during the photo-bactericidal experimental process. The results indicated that the antibactericidal effect of N-TiO 2 film was associated directly with the thickness of films (Trapalis et al., 2003 2003 ). The photo-bactericidal action no Bacillus subtilis strain (AS1.439) and Pseudomonas aeruginosa strain (AS1.50) treated by N-TiO 2 thin films B was shown to be more effectively than N-TiO 2 thin films A. Bacillus subtilis strain (AS1.439) showed stronger resistance to photocatalytic sterilization than the Pseudomonas aeruginosa strain (AS1.50) (Fig. 5 5 ). The photocatalytic chemical reaction mainly occurs on the surface of the films, indicating that the photocatalytic reaction mainly occurs on the specific particles size, the photocatalytic activity is enhanced with decreasing nanoparticles diameter and increasing contact area between the photocatalyst and target material (Maness et al., 1999 1999 ). In addition, the higher temperature treatment could not only cause the aggregation of N-TiO 2 particles, but also partially change crystalline phase from the anatase to rutile which probably result in losing some active sites of the films for photocatalytic reaction during different thermal treatment (Trapalis et al., 2003 2003 ). The experimental result suggested photo-induced bactericidal efficiency of N-TiO 2 was affected by the morphology and thickness of N-TiO 2 films, the grain size of nanoparticles, surface area, mass anatase composition of N-TiO 2 . References HD Jang , SK Kim , SJ Kim . Effect of particle size and phase composition of titanium dioxide nanoparticles on the photocatalytic properties . J Nano Res , 2001 . 3 ( 2/3 ): 141 - 147 . DOI: 10.1023/A:1017948330363 http://doi.org/10.1023/A:1017948330363 . Effect of particle size and phase composition of titanium dioxide nanoparticles on the photocatalytic properties PC Maness , S Smolinski , DM Blake , Z Huang , EJ Wolfrum , WA Jacoby . Bactericidal activity of photocatalytic TiO 2 reaction: toward an understanding of its killing mechanism . Appl Environ Microbio , 1999 . 65 ( 9 ): 4094 - 4098 . . Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing mechanism T Matsunaga , R Tomoda , T Nakajima , H Wake . Photochemical sterilization of microbial cells by semiconductor powders . FEMS Microbiology Letters , 1985 . 29 ( 1-2 ): 211 - 214 . DOI: 10.1111/j.1574-6968.1985.tb00864.x http://doi.org/10.1111/j.1574-6968.1985.tb00864.x . Photochemical sterilization of microbial cells by semiconductor powders RP Michael , RE Lindsey , MS Bertha , M Prassas . An investigation of TiO 2 photocatalysis for the treatment of water contaminated with metals and organic chemicals . Environ Sci Technol , 1999 . 27 1776 - 1782 . . An investigation of TiO2 photocatalysis for the treatment of water contaminated with metals and organic chemicals CC Trapalis , P Keivanidis , G Kordas , M Zaharescu , M Crisan , A Szatvanyi , M Gartner . TiO 2 (Fe 3+ ) nanostructured thin films with antibacterial properties . Thin Solid Films , 2003 . 433 ( 1-2 ): 186 - 190 . DOI: 10.1016/S0040-6090(03)00331-6 http://doi.org/10.1016/S0040-6090(03)00331-6 . TiO2 (Fe3+) nanostructured thin films with antibacterial properties J Zarzychi , M Prassas , J Phalippou . Study on drying stage of the mamoscale powder prepartion . J Mater Science , 1982 . 17 3370 - 3375 . . Study on drying stage of the mamoscale powder prepartion 10.1631/jzus.2006.B0586 Project (No. 2KM02501G) supported by the Program of Science and Technology Fund of Guangdong Province, China 2005-11-29 2006-05-06 2006-06-14 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 07