World J Microbiol Biotechnol DOI 10.1007/s11274-015-1855-9
ORIGINAL PAPER
Photocatalytic degradation of methylene blue and inactivation of pathogenic bacteria using silver nanoparticles modified titanium dioxide thin films Haytham M. M. Ibrahim1
Received: 23 December 2014 / Accepted: 4 April 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Titanium dioxide (TiO2) is a well-studied photocatalyst that is known to break down organic molecules upon ultraviolet irradiation. TiO2 thin films were fabricated on glass substrates using the doctor-blade procedure, the film surface was modified with silver nanoparticles to increase its visible light response. The Ag– TiO2 films were characterized by transmission electron microscopy, scanning electron microscopy equipped with energy dispersive spectrometry and X-ray diffraction. The photocatalytic degradation of methylene blue (MB) and inactivation of Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were studied. The modified films presented enhanced photocatalytic efficiency and can decompose MB solution twotimes faster than the unmodified TiO2 films, under illumination of sunlight. A nominal degradation (15 %) was observed in control MB under sunlight. The degradation efficiency of Ag–TiO2 films slightly decreased after five consecutive experiments. Ag–TiO2 films revealed very effective bactericidal activity against both E. coli and S. aureus. The photocatalytic inactivation toward E. coli and S. aureus showed a similar trend with much higher effectiveness toward E. coli under the same experimental conditions. The inactivation efficiency was maximized and reached 95 % for S. aureus and 97 % for E. coli, after 180 min incubation. These results demonstrate the
& Haytham M. M. Ibrahim [email protected] 1
Department of Radiation Microbiology, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, P.O. Box 29, Nasr City, Cairo 11731, Egypt
potential of application of Ag–TiO2 photocatalysis as a method for treatment of diluted waste waters in textile industries. Keywords Titanium dioxide Thin film Silver deposition Photocatalysis Microbial inactivation
Introduction Photocatalysis using titanium dioxide (TiO2) and solar or artificial light has been the subject of numerous investigations in recent years as it is an attractive, low-energy, water treatment method for many organic contaminants (Lu et al. 2011). TiO2 is a well-studied, chemically inert, corrosion resistant and stable photocatalyst commonly used in photocatalysis. TiO2 works under mild operating conditions (e.g., ambient temperature and pH, with no chemical additives required), and is environmentally safe (Arabatzis et al. 2002). The photocatalytic properties of TiO2 arise upon illumination with a light of energy equal or higher than its band gap energy (3.2 eV). The photon energy causes an electron to jump from the valance band to the conduction band, producing electrons (e-) and positively charged holes (h?) (Herrmann et al. 1998). These electron–hole pairs induce a series of reactions which involves the formation of sufficient concentrations of highly reactive oxygen species (ROS) like hydroxyl radicals, peroxyl radicals, superoxides, and hydrogen peroxide. Particularly the hydroxyl radical (OH-) is considered to be the dominant species contributing to degradation of various organic pollutants (Xiong et al. 2011). Photocatalysis has a wide variety of applications, including: hydrogen generation by solar water splitting (Tachibana et al. 2012), environmental
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remediation and purification of contaminated air, water, and soil (Fujishima et al. 2000), self-cleaning applications (Parkin and Palgrave 2005) in addition to photocatalysis assisted organic chemical synthesis (Yoon et al. 2010). Moreover, TiO2 has been intensively studied for its antimicrobial activity, and used as an antibacterial agent in waste water treatment (Alrousan et al. 2009). Despite the many benefits of using TiO2 as a photocatalyst to treat water, if the goal is to develop a solarpowered treatment technology, there are some drawbacks of the technology that hinder commercialization. One of the main drawbacks is the requirement for UV light to excite the electrons. Because of the relatively high band energy (3.1–3.4 eV), TiO2 is activated in the near UV light range (\400 nm) only (Kubacka et al. 2012). The UV portion of sunlight is only about 4–5 %, which for practical purposes, may be inadequate to initiate the photocatalytic reaction (Han et al. 2011). An approach to enhance the photocatalytic efficiency is adding transition metal to the TiO2 semiconductor. Decorating TiO2 with other metals (Ag, Cu, Co, Fe, Ce, Al, Cr, Nd) (Choi et al. 2010; Li et al. 2003), non-metals (C, N, B, F, S) (Han et al. 2011; Serpone 2006; Wang et al. 2007) or metal-nonmetal combinations (Hamadanian et al. 2013) can reduce its band-gap energy, and allow for activation by longer wavelengths of visible light (Malati and Wong 1984). For this reason, solar energy can be utilized more effectively for the photocatalytic process. It has been hypothesized that the advantages of doping of the transition metal into TiO2 lattice are the temporary rapping of the photogenerated charge carriers by the dopants and the inhibition of charge recombination during electron migration from inside of TiO2 to the surface, or the promoted adsorption of the pollutants onto the doped surface sites (Khang et al. 2011). Among metals, Ag? has been found to be more effective than Cu2?, Co2?, Fe3? and Ce4?, because it traps the photo-generated electrons, avoiding the recombination of electrons and holes due to the formation of Schottky barrier (Herrmann et al. 1997). Ag introduces the intermediate band energy inside the TiO2 band-gap and facilitates the photoactivity in the visible light region. This will enable better solar harvesting which could improve the overall effectiveness of the photocatalyst, rendering it more practical and sustainable. In addition, the strong bactericidal power of silver compounds makes Ag–TiO2 materials very attractive to explore the potential in photocatalytic disinfection applications, as confirmed by the enhancement for the photocatalytic inactivation of microorganisms (Kubacka et al. 2008). Water treatment using TiO2 is usually performed with the TiO2 as a slurry. Using a TiO2 slurry has the added disadvantage of the need to recover TiO2 particles from solution after the reaction. The TiO2 particles used are so small (15–30 nm) that they cannot be easily filtered or settled, which makes
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recovery difficult (Swarnakar et al. 2013). Therefore, the recent investigations of TiO2 photocatalysis have been focused on using TiO2 immobilized as a thin film. This precludes the need to recover TiO2 nanoparticles (Arabatzis et al. 2003). This work aim to prepare silver modified TiO2 thin films of increased efficiency and to investigate the photocatalytic activity of silver coated TiO2 films compared with uncoated films using natural sunlight for degradation of methylene blue (MB), as a model of organic compound (Snyder et al. 2013). Furthermore, the antimicrobial activity of Ag–TiO2 films against representative pathogenic bacteria was evaluated.
Materials and methods Microscope slides (7.62 9 2.54 cm) were used as glass substrates to prepare silver coated and uncoated TiO2 films. Titanium (IV) Oxide; a mixture of anatase and rutile (80:20 %) was procured from Sigma-Aldrich, Saint Louis, USA. Acetylacetone, triton X-100, polyethylene glycol 20000 and AgNO3 were purchased from Sigma-Aldrich. Pure Methylene blue (MB) (C16H18N3SCl) (Hoechest, Germany) was used in photodegradation experiments; all solutions were prepared using deionized distilled water. Microorganisms The Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and Gram-negative bacteria Escherichia coli (ATCC 8739) were obtained from Microbiologics, Inc. Minnesota USA, and used as model bacteria to investigate the bactericidal activities of Ag–TiO2 films. Preparation of TiO2 thin films Opaque TiO2 thin films were prepared on optically transparent microscopy glass substrates using the doctor-blade procedure (Arabatzis et al. 2003; Wang and Kerr 2012). Three grams of commercial nano-sized TiO2 were ground in a porcelain mortar with 1 ml of water and 0.1 ml of acetylacetone to prevent aggregation of the particles. The dispersed powder was further diluted by continuously adding up to 4 ml water during grinding. A 0.05 ml aliquot of triton X-100 was also added to spread the colloid on the glass substrates. At the end, 2 ml of ethanol and 3 % of polyethylene glycol by weight were added into the mixture and ground well. The glass slides were ultrasonically cleaned in ethanol prior to use and dried in an oven at temperatures 80–100 °C for 1 h before laying the TiO2 films. The colloidal TiO2 was spread over the glass substrate with a glass rod. The width and thickness of the TiO2
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film was controlled by application of adhesive tape to the glass slides. Equal thickness strips of tape were placed lengthwise along their sides creating a central trough into which the colloid was added. After air drying for 10–15 min, the films were heated at 100 °C for 15 min and annealed in an oven at 450 °C for 30 min. They were then cooled gradually inside the oven for 24 h (Wang and Kerr 2012). The film thickness was measured using a DigitrixMark II thickness gauge (precision ± 1 lm) Germany, at five random positions around the film. Silver deposition A simple, low cost, single dip-and-pull step photochemical method was used to deposit silver ions. The TiO2 thin films were dipped in an aqueous solution of AgNO3 (1 mM) for 5 s. Then, the films were thoroughly washed with deionized distilled water and dried under a stream of N2; nitrogen is an inert gas and it is better than using air contains oxygen which could react with the compounds of interest. The Ag? ions were attached on TiO2 surface. TiO2 as well as Ag–TiO2 films were illuminated for 2 h under UV-light (at 254 nm) from an UV lamp (SVL Vilbet Lourmate) (Arabatzis et al. 2003). Characterization of the photocatalyst The shape and size of TiO2 and silver nanoparticles (AgNPs) were determined using transmission electron microscopy (TEM) (JEOL-JEM-1200 EX, Japan), operating at an accelerating voltage of 80 kV. For TEM analyses the Ag–TiO2 films were scratched from glass substrate and mounted onto a carbon-coated copper grid (200–300 mesh, Ted Pella). Scanning electron microscopy (SEM) was employed to characterize the surface of the prepared films. Images of unmodified TiO2 films and Ag–TiO2 films were taken on (JEOL-JSM-5400, Japan), operating at voltage of 30 kV. The elemental composition of films was determined by energy dispersive spectrometer (EDX). The phase and crystallinity of Ag–TiO2 films were characterized using X-ray diffractometer (LabX-XRD-6000, Shimadzu, Japan) (Swarnakar et al. 2013). Photocatalytic degradation of MB The experiments were conducted under natural sunlight using the following: (1) MB solution without films as a control, (2) unmodified TiO2 thin films and (3) Ag–TiO2 thin films. Ag–TiO2 films in dark was carried out simultaneously. Under all previously mentioned conditions, two films were dipped into glass beaker containing 50 ml of MB solution (10 mg/l). The beakers were placed outside in direct sunlight between the hours of 10 and 12 a.m. The
surface area of film to volume (MB) ratio was 234 cm2/l. The light intensity was measured with a digital light meter, model LX-101A (Lutron Electronic Enterprise Co., Ltd., Taiwan), equipped with exclusive photo diode and color corrective filter and it was 0.01 W/cm2. Even though the solutions were not stirred, the mixing may have occurred due to diffusion (Swarnakar et al. 2013). Every 10 min 3 ml of MB solution was removed from each beaker and the spectra from 200 to 800 nm were acquired using UV– Vis spectrophotometer (Helios Gamma, Thermo Corporation, England). The absorption at 664 nm was used to calculate the degradation (%) of MB as follows: MB degradation ð%Þ ¼
A0 At 100 A0
where, A0 is the initial absorption of MB and At is the absorption of MB at sampling time during the processing (Wang et al. 2013). All experiments were performed in triplicate and the mean value was reported. Effect of repeated use of Ag–TiO2 films The effectiveness of Ag–TiO2 films for MB degradation was tested after multiple uses and compared to the effectiveness of new films. After the initial solar irradiation in MB, the films were dried overnight and the process was repeated. The films can be cleansed of any adsorbed dye on the surface by placing them in water under UV light or sunlight for 10 min, which allows for more versatility in their use without worry of cross contamination (Snyder et al. 2013). Photocatalytic inactivation of bacterial strains The bacterial strains were inoculated into 100 ml of nutrient broth and incubated on a rotary shaker (Gallen Kamp, UK) at 32 °C and 150 rpm overnight. About 100 ll of the broth culture was transferred into another 100 ml fresh medium and incubated as aforementioned. To prepare the reaction suspensions, 50 ml of the bacterial suspension (containing *108 CFU/ml) was centrifuged at 4000g at ambient temperature for 10 min. The supernatant was discarded, and the pellet was washed twice with phosphate buffered saline, and resuspended in 50 ml sterile deionized water. Bacterial suspensions were transferred to sterile Petri dishes each containing two films and irradiated under direct sunlight. The sunlight intensity was 0.01 W/cm2. One ml of the suspension was taken at 0, 10, 20, 30, 60, 90, 120, 150 and 180 min and diluted with sterile 0.9 % NaCl solution. Then, 100 ll of appropriate dilutions were spread on nutrient agar medium plates. After being incubated at 32 °C for 48 h, the colony forming unites were enumerated. Controls, including TiO2 films and bacterial
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suspensions under solar irradiation, bacterial suspensions without films under solar irradiation and Ag–TiO2 films and bacterial suspensions in dark were carried out simultaneously. Each data point was the mean of three replicates. Bacteria inactivation efficiency was calculated using the following equation: C0 Ct Inactivation efficiency ð%Þ ¼ 100 C0 where C0 and Ct represent the initial concentration of bacteria and those at sampling time t during process, respectively (Wang et al. 2013). Statistical analysis The data from three independent experiments were presented as the mean ± standard deviation of the mean. Statistical comparisons were carried out by analysis of variance (ANOVA) test to determine the level of significance (p \ 0.05) using IBM SPSS statistics 19.0 software.
Results Preparation of TiO2 thin films The doctor-blade technique can be easily employed as a fast and non-energy consuming procedure to mass
Fig. 1 Digital images showing the dried rectangular coatings of TiO2 colloid on the microscopic slides (a), color of films before and after 2 h irradiation under UV light: TiO2 film before UV irradiation (b), Ag–TiO2 film before UV irradiation (c), TiO2 film after UV irradiation (d) and Ag–TiO2 film after UV irradiation (e), TEM of TiO2 film (f) and TEM of Ag–TiO2 film (g)
production of TiO2 thin films with good uniformity and reproducible properties (Fig. 1a). The dimensions of the films were length 6.5 cm, width 1.8 cm, surface area of 11.7 cm2 and thickness of 8 lm. Response of TiO2 and Ag–TiO2 films under UV light To detect the presence of Ag on the TiO2 films after dipping in the silver nitrate solution (1 mM), the Ag–TiO2 and unmodified TiO2 thin films were exposed to UV irradiation (at 254 nm) for 2 h (Fig. 1). Before UV exposure the films did not show any color change as illustrated in Fig. 1b, c. The exposure did not change the color of unmodified TiO2 films (Fig. 1d), while Ag–TiO2 films changed into a dark brown color (Fig. 1e) as previously reported by Arabatzis et al. (2003). Characterization of Ag–TiO2 films TEM analysis TEM analysis was performed to investigate the morphology of TiO2 and Ag–TiO2 films (Fig. 1). TiO2 particles were spherical shaped with an average diameter of 19.8 nm (Fig. 1f). Many particles are stacked on top of one another. In Ag–TiO2 film (Fig. 1g), spherical AgNPs were clearly seen on top of TiO2 particles as shown by arrows in Fig. 1g. The AgNPs appear darker than TiO2 particles.
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SEM–EDX analysis Macroscopic observations and SEM analysis of the films suggested that AgNPs are uniformly spread on the surface without the formation of islands. SEM images of TiO2 films show that films are rough, forming a sponge like structure, and particles were spherical in shape (Fig. 2a). Silver deposition seems to affect the surface characteristics of the resulting Ag–TiO2 films. Figure 2b depicts a slight decrease on the roughness and height of surface features. Ag nanoparticles appear as bright dots dispersed in the TiO2 amorphous matrix. The magnified image (Fig. 2c) clearly shows the bright AgNP clusters. The films heated at 400 °C and underwent slow cooling have AgNPs smaller than those observed after deposition (Fig. 2d). The EDX
Fig. 2 SEM images of unmodified TiO2 film (a), Ag– TiO2 film after deposition of AgNPs (b), magnified image showing the AgNPs cluster (c), Ag–TiO2 film after annealing at 400 °C (d), EDX of TiO2 film (e) and EDX of Ag–TiO2 film (f)
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analysis of unmodified TiO2 films confirmed the chemical composition as 100 % TiO2 (Fig. 2e), while EDX analysis of Ag–TiO2 films confirmed 0.50 % Ag presence on the TiO2 (99.5 %) film (Fig. 2f). X-ray diffraction analysis The Ag–TiO2 films prepared with the doctor-blade technique were examined by XRD. The characteristic reflections of both anatase and rutile TiO2 phases were easily detected (Fig. 3). The distinct peaks showed that the TiO2 particles are crystalline. The peaks appearing at 2h = 25.2°, 37.8° and 48° elucidate the diffractions of the (1 0 1), (0 0 4) and (2 0 0) (JCPDS File No. 83-2243) planes of anatase-type TiO2 (Naeem and Ouyang 2010), by
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Photocatalytic inactivation of bacterial strains
Fig. 3 X-ray diffraction of Ag–TiO2 films (A anatase and R rutile reflections)
applying the Scherrer’s formula (D = 0.9k/b1/2cosh), the TiO2 crystallites size can be estimated as 17.32 nm. This value is consistent with the particle size obtained in TEM analysis (19.8 nm). No reflection peak owing to metallic Ag can be seen. Photocatalytic degradation of MB The photocatalytic activity of TiO2 and Ag–TiO2 films under direct sunlight was investigated, using MB as a model conjugated organic molecule. The MB spectrum obtained from UV–Vis spectrophotometer revealed a strong absorbance peak (kmax) around 664 nm (Fig. 4a). The rate of decolorization was recorded with respect to the change in intensity of absorption peak at 664 nm. Control MB showed only 15 % degradation (Fig. 4a). For MB– TiO2 and MB–Ag–TiO2 films the absorption peaks successively decreased with time indicating the degradation of MB dye as represented in Figs. 4b and 5a. MB–TiO2 films resulted in 50 % degradation of the original concentration (Fig. 4b). MB–Ag–TiO2 films performed better, resulting in *100 % degradation of MB (Fig. 5a). The absorption peak of MB blue shifted from 664 to 658 nm during illumination as illustrated in the inset of Fig. 5a. For MB–Ag– TiO2 system in dark almost no change in peak intensity was observed (Fig. 5b). Percentage of residual MB concentration during the different treatments was illustrated in Fig. 6. The digital images in Fig. 7a, b revealed the complete degradation of MB dye after 120 min of solar irradiation in the presence of Ag–TiO2 films. Effect of repeated use of Ag–TiO2 films The effectiveness of Ag–TiO2 films to degrade MB was tested for five consecutive cycles and compared to that of the new films. The degradation efficiency of the films was slightly decreased by repeated use, i.e., only small losses (10 %) of their catalytic activity were observed after the fifth cycle as presented in Fig. 7c, d.
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As indicated in Fig. 8 Ag–TiO2 thin films had a similar inactivation effect on either S. aureus (Fig. 8a) or E. coli (Fig. 8b) under natural sunlight. The overall counts of both live bacteria in the treated suspensions decreased as illumination time increased. The counts of either S. aureus or E. coli slightly changed during the first 30 min, the inactivation efficiency was 14 and 16 %, respectively. Then they started decreasing significantly (p \ 0.05), with decreases in S. aureus count being slightly slower than those in E. coli. The inactivation efficiency was as high as 55 % for S. aureus and 67 % for E. coli after 90 min. By 120 min, the inactivation efficiency of Ag–TiO2 films continuously increased to over 80 % for both strains. After 180 min, the inactivation efficiency was maximal, it reached 95 and 97 % for S. aureus and E. coli, respectively. These results indicate that S. aureus and E. coli might have different responses to Ag–TiO2 under the previously mentioned experimental conditions. The inactivation efficiency of both bacteria was very low in the presence of unmodified TiO2 films, as represented in Fig. 8a, b. It was 20 % for S. aureus and 23 % for E. coli after 180 min under direct sunlight. Control experiment conducted under sunlight without films did not reveal any change in the bacterial count of both strains as illustrated in Fig. 8a, b. For Ag–TiO2 films in dark, insignificant decrease in bacterial count was obtained, 11 % for S. aureus and 16 % for E. coli after 180 min (Fig. 8a, b).
Discussion The mixture of anatase and rutile has been considered the most active formulation of TiO2. Presence of rutile phase in adjacent to anatase phase in appropriate ratio causes a synergistic effect. Bickley et al. (1991) attributed the high photocatalytic activity of the mixture to the interaction between different phases of rutile and anatase. Accordingly, the photogenerated electrons move from anatase to a lower electron trapping site in rutile. It was proposed that this electron transfer reduces the recombination of anatase and leads to more efficient electron–hole separation as well as higher photoactivity. The combined action of anatase and rutile phases was further studied by Ohno et al. (2003), who examined commercial titanium oxide samples containing only either anatase or rutile or mixtures of anatase and rutile in various ratios. They found that mixtures of anatase and rutile were the most active for the studied reaction, which means that a synergistic effect may take place. The main issue arising in photocatalysis lies in the inability of TiO2 to efficiently use solar light, which is
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composed of only 4–5 % UV. However, solar irradiation consists of approximately 43 % visible light, so more efficient utilization of this portion is desirable. Efforts to address this issue by increasing visible light absorption have been made through a number of catalyst modifications such as metals deposition (Arabatzis et al. 2003; Swarnakar et al. 2013). In addition, the rate of recombination of the photoexcited electrons and holes is a major factor limiting the efficiency of photocatalytic processes, and as such research in photocatalyst development has also been focused on the design and fabrication of photocatalysts possessing reduced recombination rates (Swarnakar et al. 2013). Photoreduction of Ag? under UV light is a simple and efficient method for deposition of AgNPs and nanoclusters. The primary mechanism for Ag? reduction in this system is believed to be the adsorption of Ag? ions onto the surface
of TiO2 films, where Ag? can be reduced by excited TiO2 photoelectrons that are transferred to the Ag? ions (Sahyun and Serpone 1997). Macroscopic observation of the prepared films suggests that AgNPs are widely spread on the surface without the formation of islands and that the resulting surface modified photocatalysts possess a solid structure on the glass substrate with more than satisfactory scratch resistance and adherence. Darkens of Ag–TiO2 films upon UV irradiation means that adsorbed Ag? ions were reduced and converted to zero valence silver (Ag0), this color is attributed to the surface plasmon resonance characteristic of AgNPs (Arabatzis et al. 2003). The deposition of AgNPs on TiO2 surface was further confirmed using transmission and scanning electron microscopy. The AgNPs appear darker in the TEM image than the TiO2 particles due to their higher electron density.
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Fig. 5 UV–Vis spectra of MB solution treated with Ag–TiO2 thin films under natural sunlight irradiation; inset showing the absorption peak shift of MB (a) and in dark (b)
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In XRD pattern the anatase reflections are dominating but rutile is also present, as the original material (TiO2) contains both phases (80 % anatase and 20 % rutile). No reflection peak owing to metallic Ag can be seen, suggests that silver particles are not crystallized on TiO2 surface. Similar XRD results were reported by Arabatzis et al. (2003) and Swarnakar et al. (2013). The control MB hardly degraded after illumination under sunlight for 120 min, which indicates that MB could not be decomposed without the photocatalyst. Cationic MB molecules are known to strongly adsorb on the surface of TiO2 via electrostatic attraction as well as interaction with surface hydroxyl groups (El-Sharkawy et al. 2007). The enhanced photocatalytic degradation of MB in the presence of Ag–TiO2 films compared with TiO2 films could be
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ascribed to the combined effect of enhanced photocatalytic activity of Ag–TiO2 films and the fact that more MB molecules are adsorbed on the Ag–TiO2 films than TiO2 films as a result of increased surface area, roughness, and negative charges provided by the presence of Ag nanoclusters (Rodrı´guez et al. 2009). The fast preferential MB adsorption on AgNPs will allow more dye molecules to be in contact with the catalyst over the entire irradiation period and will contribute to its degradation (Yogi et al. 2008). Nevertheless, introduction of the intermediate band energy inside the TiO2 band gap majorly contributes to improvement of photocatalytic efficiency, since without creation of sufficient OH radicals, the accumulation of large amount of MB molecules will be useless. In addition, in the presence of TiO2 films the ROS concentration was
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much lower than that in Ag–TiO2 system, as it generated by the UV portion of sunlight (4–5 %). The blue shift from 664 to 658 nm during the illumination of MB dye could be due to the N-demethylation of MB by TiO2 nanoparticles in a stepwise manner under solar irradiation or the formation of N-demethylated intermediates (Zhang et al. 2001; Wang et al. 2013). Ag–TiO2 films in dark did not
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Fig. 7 Digital images of MB solution under solar irradiation in presence of Ag–TiO2 films: at zero time (a) and after 120 min, showing complete decolorization of MB solution (b) and effect of repeated use of Ag–TiO2 thin films on MB degradation (%) under direct sunlight irradiation (c, d). Shown is the mean value of 3 readings ± standard deviation. Values followed by different superscript letters are significantly different at p \ 0.05
result in any MB degradation because dark conditions did not allow a direct titania excitation (Mitoraj et al. 2007). These results are in agreement with that reported by Snyder et al. (2013). The fact that with repeated use the degradation efficiency of Ag–TiO2 films was slightly decreased supports the practical use of doctor-blade technique, as the films produced by this technique can be used multiple times with only small losses of their catalytic properties. This result is in accordance with previously published reports (Swarnakar et al. 2013). The photocatalytic inactivation is based on redox properties of surface trapped photogenerated charges and 1 formed ROS (OH, O2 , H2O2, O2 etc.) (Ishibashi et al. 2000). One of the most toxic for microorganisms is the OH radical because of its ability to oxidise many organic substrates like carbohydrates, lipids, proteins and nucleic acids (Srinivasan and Somasundaram 2003). It promotes peroxidation of polyunsaturated phospholipid components of the lipid membrane and induces disorders in the cell membrane. Maness et al. (1999) reported that ROS causes major disorders in the E. coli cell membrane leading to inhibition of fundamental vital processes of the cell and in consequence to its death. Cho et al. (2004) found a linear correlation between the amount of photogenerated OH radicals and the extent of E. coli inactivation in TiO2 photocatalytic disinfection processes. Moreover, ROS
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might oxidize the surface of AgNPs, the oxidized AgNPs have the ability to kill bacteria because of the extensive and persistent release of Ag? from oxidized AgNPs (Zhao et al. 2013). Inactivation of bacteria requires a certain amount of cumulative damage, a process to prevent bacteria from growing or to kill them. The damage cannot be completed in short time even though there are enough radicals produced by photocatalytic nano-TiO2 (Maruga´n et al. 2008). The delay in the inactivation during the first 30 min could be due to the concentration of photogenerated ROS, it increases until reaches a level which is harmful to bacteria. Below this concentration the self-defence mechanisms of bacteria involving enzymes, catalase and superoxide dismutase protect the cell from the oxidative stress and it can grow again after being injured (Mitoraj et al. 2007). When the concentration of ROS is higher (after 90 min), the defense mechanisms of bacteria involving enzymes are insufficient. Therefore, the oxidation of cell wall and membrane components leads to bacteria death. The decreasing inactivation rate in the last step is probably caused by the ROS consumption not only by living cells, but also by products of bacterial lysis (Rincon and Pulgarin 2005). The obtained results reveal that Ag–TiO2 thin films are more effective antibacterial materials than TiO2 thin films under sunlight. The significant difference in antibacterial effects between TiO2 thin films and Ag–TiO2 thin films may be attributed to the difference in antibacterial mechanisms between both films. It is the photocatalytic property of the TiO2 thin films that makes it to have an antimicrobial capability. But the percent of ultraviolet light from natural light is only 4–5 % which results in the lower antibacterial activity. On the other hand, the antibacterial activity of Ag–TiO2 thin films is related to the enhanced photocatalytic property of the TiO2 as well as the antimicrobial activity of AgNPs. Ag–TiO2 thin films showed a stronger effect on E. coli than S. aureus under the same conditions perhaps due to the
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Survival fraction C/Co (%)
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Fig. 8 Survival fraction of S. aureus (a) and E. coli (b) treated with Ag–TiO2 films under different experimental conditions. Shown is the mean value of 3 readings ± standard deviation
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difference in cell wall properties (its structure, thickness, etc.) between Gram-positive and Gram-negative bacteria. Gram-positive bacteria require a longer treatment time than Gram-negative bacteria (Lydakis-Simantiris et al. 2010). This is because Gram-positive bacteria have a very thick cell wall consisting of many layers of peptidoglycan and teichoic acids, while the cell wall of Gram-negative bacteria is very thin consisting of only a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins (Robertson et al. 2012). The obtained results are in agreement with the reported order of bacterial resistance in UV photoinactivation processes in the presence of titania: E. coli \ S. aureus & Enterococcus sp. (Kuhn et al. 2003; Seven et al. 2004; Rincon and Pulgarin 2005). The dark conditions did not allow either a direct titania excitation or a direct bacteria inactivation (Mitoraj et al. 2007). The insignificant inactivation obtained by Ag–TiO2 films in dark may be due to adsorption of bacteria onto the surface of the TiO2, inducing cell stress which could lead to mortality, the direct contact with AgNPs on the film surface as well as influence of Ag? release (Li et al. 2011).
Conclusions A very simple and cost effective method of film making, the doctor-blade procedure was adopted. Degradation of MB under different experimental conditions was evaluated. MB in solutions exposed to natural sunlight with the silver modified TiO2 films degraded at a rate two-times faster than those of unmodified TiO2 films. These results suggest that Ag–TiO2 photocatalysis may be envisaged as a method to clean colored waste waters in textile industries. Ag–TiO2 also effectively inactivated E. coli and S. aureus as representatives of Gram-negative and Gram-positive bacteria. It took 30 min before the inactivation can be measured and 180 min to reach the maximal inactivation rate. The
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photocatalytic bacteria reducing process induced by sunlight would be useful in hospitals, microbiological laboratories, food processing plants, pharmaceutical industry, pharmacies, and wherever there is a strong requirement for clean and sterile surfaces.
References Alrousan DMA, Dunlop PSM, McMurray TA, Byrne JA (2009) Photocatalytic inactivation of E. coli in surface water using immobilized nanoparticle TiO2 films. Water Res 43:47–54 Arabatzis IM, Antonaraki S, Stergiopoulos T, Hiskia A, Papaconstantinou E, Bernard MC, Falaras P (2002) Preparation, characterization and photocatalytic activity of nanocrystalline thin film TiO2 catalysts towards 3,5-dichlorophenol degradation. J Photochem Photobiol A Chem 149:237–245 Arabatzis IM, Stergiopoulos T, Bernard MC, Labou D, Neophytides SG, Falaras P (2003) Silver-modified titanium dioxide thin films for efficient photodegradation of methyl orange. Appl Catal B Environ 42:187–201 Bickley RI, Gonzalez-Carreno T, Lees JS, Palmisano L, Tilley RJD (1991) A structural investigation of titanium dioxide photocatalysts. J Solid State Chem 92:178–190 Cho M, Chung H, Choi W, Yoon J (2004) Linear correlation between inactivation of E. coli and OH radical concentration in TiO2 photocatalytic disinfection. Water Res 38:1069–1077 Choi J, Park H, Hoffmann MR (2010) Effects of single metal-ion doping on the visible-light photoreactivity of TiO2. J Phys Chem C 114:783–792 El-Sharkawy EA, Soliman AY, Al-Amer KM (2007) Comparative study for the removal of methylene blue via adsorption and photocatalytic degradation. J Colloid Interface Sci 310:498–508 Fujishima A, Rao TN, Tryk DA (2000) Titanium dioxide photocatalysis. J Photochem Photobiol C 1:1–21 Hamadanian M, Reisi-Vanani A, Razi P, Hoseinifard S, Jabbari V (2013) Photodeposition-assisted synthesis of novel nanoparticulate In, S-codoped TiO2 powders with high visible lightdriven photocatalytic activity. Appl Surf Sci 285P:121–129 Han C, Pelaez M, Likodimos V, Kontos AG, Falaras P, O’Shea K, Dionysiou DD (2011) Innovative visible light-activated sulfur doped TiO2 films for water treatment. Appl Catal B Environ 107:77–87 Herrmann J-M, Tahiri H, Ait-Ichou Y, Lassaletta G, Gonza´lez-Elipe AR, Ferna´ndez A (1997) Characterization and photocatalytic activity in aqueous medium of TiO2 and Ag–TiO2 coatings on quartz. Appl Catal B Environ 13:219–228 Herrmann J-M, Disdier J, Pichat P, Malato S, Blanco J (1998) TiO(2)based solar photocatalytic detoxification of water containing organic pollutants. Case studies of 2,4-dichlorophenoxyaceticacid (2,4-D) and of benzofuran. Appl Catal B Environ 17:15–23 Ishibashi K, Fujishima A, Watanabe T, Hashimoto K (2000) Quantum yields of active oxidative species formed on TiO2 photocatalyst. J Photochem Photobiol A 134:139–142 Khang NC, Khanh NV, Anh NH, Nga DT, Minh NV (2011) The origin of visible light photocatalytic activity of N-doped and weak ferromagnetism of Fe-doped TiO2 anatase. Adv Nat Sci Nanosci Nanotechnol 2:015008 Kubacka A, Ferrer M, Martı´nez-Arias A, Ferna´ndez-Garcı´a A (2008) Ag promotion of TiO2-anatase disinfection capability: study of Escherichia coli inactivation. Appl Cata B Environ 84:87–93 Kubacka A, Ferna´ndez-Garcı´a M, Colon G (2012) Advanced nanoarchitectures for solar photocatalytic applications. Chem Rev 112:1555–1614
Kuhn KP, Chaberny I, Massholder K, Stickler M, Benz VW, Sonntag H-G, Erdinger L (2003) Disinfection of surfaces by photocatalytic oxidation with titanium dioxide and UVA light. Chemosphere 53:71–77 Li W, Wang Y, Lin H, Shah SI, Huang CP, Doren DJ, Rykov SA, Chen JG, Barteau MA (2003) Band gap tailoring of Nd3?-doped TiO2 nanoparticles. Appl Phys Lett 83:4143–4145 Li M, Noriega-Trevino ME, Nino-Martinez N, Marambio-Jones C, Wang J, Damoiseaux R, Ruiz F, Hoek EMV (2011) Synergistic bactericidal activity of Ag–TiO2 nanoparticles in both light and dark conditions. Environ Sci Technol 45:8989–8995 Lu SY, Wu D, Wang QL, Yan JH, Buekens AG, Cen KF (2011) Photocatalytic decomposition on nano-TiO2: destruction of chloroaromatic compounds. Chemosphere 82:1215–1224 Lydakis-Simantiris N, Riga D, Katsivela E, Mantzavinos D, XekoukOulotakis NP (2010) Disinfection of spring water and secondary treated municipal wastewater by TiO2 photocatalysis. Desalination 250:351–355 Malati MA, Wong WK (1984) Doping TiO2 for solar-energy applications. Surf Technol 22:305–322 Maness P-C, Smolinski S, Blake D, Huang Z, Wolfrum AJ, Jacoby WA (1999) Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing mechanism. Appl Environ Microbiol 65:4094–4098 Maruga´n J, van Grieken R, Sordo C, Cruz C (2008) Kinetics of the photocatalytic disinfection of Escherichia coli suspensions. Appl Catal B Environ 82:27–36 Mitoraj D, Jan´czyk A, Strus M, Kisch H, Stochel G, Heczko PB, Macyk W (2007) Visible light inactivation of bacteria and fungi by modified titanium dioxide. Photochem Photobiol Sci 6:642–648 Naeem K, Ouyang F (2010) Preparation of Fe3?-doped TiO2 nanoparticles and its photocatalytic activity under UV light. Phys B Condens Matter 405:221–226 Ohno T, Tokieda K, Higashida S, Matsumura M (2003) Synergism between rutile and anatase TiO2 particles in photocatalytic oxidation of naphthalene. Appl Catal A Gen 244:383–391 Parkin IP, Palgrave RG (2005) Self-cleaning coatings. J Mater Chem 15:1689–1695 Rincon AG, Pulgarin C (2005) Use of coaxial photocatalytic reactor (CAPHORE) in the TiO2 photo-assisted treatment of mixed E. coli and Bacillus sp. and bacterial community present in wastewater. Catal Today 101:331–344 Robertson PKJ, Robertson JMC, Bahnemann DW (2012) Removal of microorganisms and their chemical metabolites from water using semiconductor photocatalysis. J Hazard Mater 211–212:161–171 Rodrı´guez A, Garcı´a J, Ovejero G, Mestanza M (2009) Adsorption of anionic and cationic dyes on activated carbon from aqueous solutions: equilibrium and kinetics. J Hazard Mater 172:1311–1320 Sahyun MRV, Serpone N (1997) Primary events in the photocatalytic deposition of silver on nanoparticulate TiO2. Langmuir 13:5082–5088 Serpone N (2006) Is the band gap of pristine TiO2 narrowed by anionand cation-doping of titanium dioxide in second-generation photocatalysts? J Phys Chem B 110:24287–24293 Seven O, Dindar B, Aydemir S, Metin D, Ozinel MA, Icli S (2004) Solar photocatalytic disinfection of a group bacteria and fungi aqueous suspensions with TiO2, ZnO and Sahara desert dust. J Photochem Photobiol A 165:103–107 Snyder A, Bo Z, Moon R, Rochet J-C, Stanciu L (2013) Reusable photocatalytic titanium dioxide–cellulose nanofiber films. J Colloid Interface Sci 399:92–98 Srinivasan C, Somasundaram N (2003) Bactericidal and detoxification effects of irradiated semiconductor catalyst, TiO2. Curr Sci 85:1431–1438 Swarnakar P, Kanel SR, Nepal D, Jiang Y, Jia H, Kerr L, Goltz MN, Levy J, Rakovan J (2013) Silver deposited titanium dioxide thin
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World J Microbiol Biotechnol film for photocatalysis of organic compounds using natural light. Sol Energy 88:242–249 Tachibana Y, Vayssieres L, Durrant JR (2012) Artificial photosynthesis for solar water-splitting. Nat Photonics 6:511–518 Wang B, Kerr LL (2012) Stability of CdS-coated TiO2 solar cells. J Solid State Electrochem 16:1091–1097 Wang YQ, Yu XJ, Sun DZ (2007) Synthesis, characterization, and photocatalytic activity of TiO2–xNx nanocatalyst. J Hazard Mater 144:328–333 Wang J, Li C, Zhuang H, Zhang J (2013) Photocatalytic degradation of methylene blue and inactivation of Gram-negative bacteria by TiO2 nanoparticles in aqueous suspension. Food Control 34:372–377 Xiong ZG, Ma JZ, Ng WJ, Waite TD, Zhao XS (2011) Silver modified mesoporous TiO2 photocatalyst for water purification. Water Res 45:2095–2103
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Yogi C, Kojima K, Wada N, Tokumoto H, Takai T, Mizoguchi T, Tamiaki H (2008) Photocatalytic degradation of methylene blue by TiO2 film and Au particles-TiO2 composite film. Thin Solid Films 516:5881–5884 Yoon TP, Ischay MA, Du J (2010) Visible light photocatalysis as a greener approach to photochemical synthesis. Nat Chem 2:527–532 Zhang TY, Oyama T, Aoshima A, Hidaka H, Zhao JC, Serpone N (2001) Photooxidative N-demethylation of methylene blue in aqueous TiO2 dispersions under UV irradiation. J Photochem Photobiol A Chem 140:163–172 Zhao X, Toyooka T, Ibuki Y (2013) Synergistic bactericidal effect by combined exposure to Ag nanoparticles and UVA. Sci Total Environ 458–460:54–62
ORIGINAL PAPER
Photocatalytic degradation of methylene blue and inactivation of pathogenic bacteria using silver nanoparticles modified titanium dioxide thin films Haytham M. M. Ibrahim1
Received: 23 December 2014 / Accepted: 4 April 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Titanium dioxide (TiO2) is a well-studied photocatalyst that is known to break down organic molecules upon ultraviolet irradiation. TiO2 thin films were fabricated on glass substrates using the doctor-blade procedure, the film surface was modified with silver nanoparticles to increase its visible light response. The Ag– TiO2 films were characterized by transmission electron microscopy, scanning electron microscopy equipped with energy dispersive spectrometry and X-ray diffraction. The photocatalytic degradation of methylene blue (MB) and inactivation of Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were studied. The modified films presented enhanced photocatalytic efficiency and can decompose MB solution twotimes faster than the unmodified TiO2 films, under illumination of sunlight. A nominal degradation (15 %) was observed in control MB under sunlight. The degradation efficiency of Ag–TiO2 films slightly decreased after five consecutive experiments. Ag–TiO2 films revealed very effective bactericidal activity against both E. coli and S. aureus. The photocatalytic inactivation toward E. coli and S. aureus showed a similar trend with much higher effectiveness toward E. coli under the same experimental conditions. The inactivation efficiency was maximized and reached 95 % for S. aureus and 97 % for E. coli, after 180 min incubation. These results demonstrate the
& Haytham M. M. Ibrahim [email protected] 1
Department of Radiation Microbiology, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, P.O. Box 29, Nasr City, Cairo 11731, Egypt
potential of application of Ag–TiO2 photocatalysis as a method for treatment of diluted waste waters in textile industries. Keywords Titanium dioxide Thin film Silver deposition Photocatalysis Microbial inactivation
Introduction Photocatalysis using titanium dioxide (TiO2) and solar or artificial light has been the subject of numerous investigations in recent years as it is an attractive, low-energy, water treatment method for many organic contaminants (Lu et al. 2011). TiO2 is a well-studied, chemically inert, corrosion resistant and stable photocatalyst commonly used in photocatalysis. TiO2 works under mild operating conditions (e.g., ambient temperature and pH, with no chemical additives required), and is environmentally safe (Arabatzis et al. 2002). The photocatalytic properties of TiO2 arise upon illumination with a light of energy equal or higher than its band gap energy (3.2 eV). The photon energy causes an electron to jump from the valance band to the conduction band, producing electrons (e-) and positively charged holes (h?) (Herrmann et al. 1998). These electron–hole pairs induce a series of reactions which involves the formation of sufficient concentrations of highly reactive oxygen species (ROS) like hydroxyl radicals, peroxyl radicals, superoxides, and hydrogen peroxide. Particularly the hydroxyl radical (OH-) is considered to be the dominant species contributing to degradation of various organic pollutants (Xiong et al. 2011). Photocatalysis has a wide variety of applications, including: hydrogen generation by solar water splitting (Tachibana et al. 2012), environmental
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remediation and purification of contaminated air, water, and soil (Fujishima et al. 2000), self-cleaning applications (Parkin and Palgrave 2005) in addition to photocatalysis assisted organic chemical synthesis (Yoon et al. 2010). Moreover, TiO2 has been intensively studied for its antimicrobial activity, and used as an antibacterial agent in waste water treatment (Alrousan et al. 2009). Despite the many benefits of using TiO2 as a photocatalyst to treat water, if the goal is to develop a solarpowered treatment technology, there are some drawbacks of the technology that hinder commercialization. One of the main drawbacks is the requirement for UV light to excite the electrons. Because of the relatively high band energy (3.1–3.4 eV), TiO2 is activated in the near UV light range (\400 nm) only (Kubacka et al. 2012). The UV portion of sunlight is only about 4–5 %, which for practical purposes, may be inadequate to initiate the photocatalytic reaction (Han et al. 2011). An approach to enhance the photocatalytic efficiency is adding transition metal to the TiO2 semiconductor. Decorating TiO2 with other metals (Ag, Cu, Co, Fe, Ce, Al, Cr, Nd) (Choi et al. 2010; Li et al. 2003), non-metals (C, N, B, F, S) (Han et al. 2011; Serpone 2006; Wang et al. 2007) or metal-nonmetal combinations (Hamadanian et al. 2013) can reduce its band-gap energy, and allow for activation by longer wavelengths of visible light (Malati and Wong 1984). For this reason, solar energy can be utilized more effectively for the photocatalytic process. It has been hypothesized that the advantages of doping of the transition metal into TiO2 lattice are the temporary rapping of the photogenerated charge carriers by the dopants and the inhibition of charge recombination during electron migration from inside of TiO2 to the surface, or the promoted adsorption of the pollutants onto the doped surface sites (Khang et al. 2011). Among metals, Ag? has been found to be more effective than Cu2?, Co2?, Fe3? and Ce4?, because it traps the photo-generated electrons, avoiding the recombination of electrons and holes due to the formation of Schottky barrier (Herrmann et al. 1997). Ag introduces the intermediate band energy inside the TiO2 band-gap and facilitates the photoactivity in the visible light region. This will enable better solar harvesting which could improve the overall effectiveness of the photocatalyst, rendering it more practical and sustainable. In addition, the strong bactericidal power of silver compounds makes Ag–TiO2 materials very attractive to explore the potential in photocatalytic disinfection applications, as confirmed by the enhancement for the photocatalytic inactivation of microorganisms (Kubacka et al. 2008). Water treatment using TiO2 is usually performed with the TiO2 as a slurry. Using a TiO2 slurry has the added disadvantage of the need to recover TiO2 particles from solution after the reaction. The TiO2 particles used are so small (15–30 nm) that they cannot be easily filtered or settled, which makes
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recovery difficult (Swarnakar et al. 2013). Therefore, the recent investigations of TiO2 photocatalysis have been focused on using TiO2 immobilized as a thin film. This precludes the need to recover TiO2 nanoparticles (Arabatzis et al. 2003). This work aim to prepare silver modified TiO2 thin films of increased efficiency and to investigate the photocatalytic activity of silver coated TiO2 films compared with uncoated films using natural sunlight for degradation of methylene blue (MB), as a model of organic compound (Snyder et al. 2013). Furthermore, the antimicrobial activity of Ag–TiO2 films against representative pathogenic bacteria was evaluated.
Materials and methods Microscope slides (7.62 9 2.54 cm) were used as glass substrates to prepare silver coated and uncoated TiO2 films. Titanium (IV) Oxide; a mixture of anatase and rutile (80:20 %) was procured from Sigma-Aldrich, Saint Louis, USA. Acetylacetone, triton X-100, polyethylene glycol 20000 and AgNO3 were purchased from Sigma-Aldrich. Pure Methylene blue (MB) (C16H18N3SCl) (Hoechest, Germany) was used in photodegradation experiments; all solutions were prepared using deionized distilled water. Microorganisms The Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and Gram-negative bacteria Escherichia coli (ATCC 8739) were obtained from Microbiologics, Inc. Minnesota USA, and used as model bacteria to investigate the bactericidal activities of Ag–TiO2 films. Preparation of TiO2 thin films Opaque TiO2 thin films were prepared on optically transparent microscopy glass substrates using the doctor-blade procedure (Arabatzis et al. 2003; Wang and Kerr 2012). Three grams of commercial nano-sized TiO2 were ground in a porcelain mortar with 1 ml of water and 0.1 ml of acetylacetone to prevent aggregation of the particles. The dispersed powder was further diluted by continuously adding up to 4 ml water during grinding. A 0.05 ml aliquot of triton X-100 was also added to spread the colloid on the glass substrates. At the end, 2 ml of ethanol and 3 % of polyethylene glycol by weight were added into the mixture and ground well. The glass slides were ultrasonically cleaned in ethanol prior to use and dried in an oven at temperatures 80–100 °C for 1 h before laying the TiO2 films. The colloidal TiO2 was spread over the glass substrate with a glass rod. The width and thickness of the TiO2
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film was controlled by application of adhesive tape to the glass slides. Equal thickness strips of tape were placed lengthwise along their sides creating a central trough into which the colloid was added. After air drying for 10–15 min, the films were heated at 100 °C for 15 min and annealed in an oven at 450 °C for 30 min. They were then cooled gradually inside the oven for 24 h (Wang and Kerr 2012). The film thickness was measured using a DigitrixMark II thickness gauge (precision ± 1 lm) Germany, at five random positions around the film. Silver deposition A simple, low cost, single dip-and-pull step photochemical method was used to deposit silver ions. The TiO2 thin films were dipped in an aqueous solution of AgNO3 (1 mM) for 5 s. Then, the films were thoroughly washed with deionized distilled water and dried under a stream of N2; nitrogen is an inert gas and it is better than using air contains oxygen which could react with the compounds of interest. The Ag? ions were attached on TiO2 surface. TiO2 as well as Ag–TiO2 films were illuminated for 2 h under UV-light (at 254 nm) from an UV lamp (SVL Vilbet Lourmate) (Arabatzis et al. 2003). Characterization of the photocatalyst The shape and size of TiO2 and silver nanoparticles (AgNPs) were determined using transmission electron microscopy (TEM) (JEOL-JEM-1200 EX, Japan), operating at an accelerating voltage of 80 kV. For TEM analyses the Ag–TiO2 films were scratched from glass substrate and mounted onto a carbon-coated copper grid (200–300 mesh, Ted Pella). Scanning electron microscopy (SEM) was employed to characterize the surface of the prepared films. Images of unmodified TiO2 films and Ag–TiO2 films were taken on (JEOL-JSM-5400, Japan), operating at voltage of 30 kV. The elemental composition of films was determined by energy dispersive spectrometer (EDX). The phase and crystallinity of Ag–TiO2 films were characterized using X-ray diffractometer (LabX-XRD-6000, Shimadzu, Japan) (Swarnakar et al. 2013). Photocatalytic degradation of MB The experiments were conducted under natural sunlight using the following: (1) MB solution without films as a control, (2) unmodified TiO2 thin films and (3) Ag–TiO2 thin films. Ag–TiO2 films in dark was carried out simultaneously. Under all previously mentioned conditions, two films were dipped into glass beaker containing 50 ml of MB solution (10 mg/l). The beakers were placed outside in direct sunlight between the hours of 10 and 12 a.m. The
surface area of film to volume (MB) ratio was 234 cm2/l. The light intensity was measured with a digital light meter, model LX-101A (Lutron Electronic Enterprise Co., Ltd., Taiwan), equipped with exclusive photo diode and color corrective filter and it was 0.01 W/cm2. Even though the solutions were not stirred, the mixing may have occurred due to diffusion (Swarnakar et al. 2013). Every 10 min 3 ml of MB solution was removed from each beaker and the spectra from 200 to 800 nm were acquired using UV– Vis spectrophotometer (Helios Gamma, Thermo Corporation, England). The absorption at 664 nm was used to calculate the degradation (%) of MB as follows: MB degradation ð%Þ ¼
A0 At 100 A0
where, A0 is the initial absorption of MB and At is the absorption of MB at sampling time during the processing (Wang et al. 2013). All experiments were performed in triplicate and the mean value was reported. Effect of repeated use of Ag–TiO2 films The effectiveness of Ag–TiO2 films for MB degradation was tested after multiple uses and compared to the effectiveness of new films. After the initial solar irradiation in MB, the films were dried overnight and the process was repeated. The films can be cleansed of any adsorbed dye on the surface by placing them in water under UV light or sunlight for 10 min, which allows for more versatility in their use without worry of cross contamination (Snyder et al. 2013). Photocatalytic inactivation of bacterial strains The bacterial strains were inoculated into 100 ml of nutrient broth and incubated on a rotary shaker (Gallen Kamp, UK) at 32 °C and 150 rpm overnight. About 100 ll of the broth culture was transferred into another 100 ml fresh medium and incubated as aforementioned. To prepare the reaction suspensions, 50 ml of the bacterial suspension (containing *108 CFU/ml) was centrifuged at 4000g at ambient temperature for 10 min. The supernatant was discarded, and the pellet was washed twice with phosphate buffered saline, and resuspended in 50 ml sterile deionized water. Bacterial suspensions were transferred to sterile Petri dishes each containing two films and irradiated under direct sunlight. The sunlight intensity was 0.01 W/cm2. One ml of the suspension was taken at 0, 10, 20, 30, 60, 90, 120, 150 and 180 min and diluted with sterile 0.9 % NaCl solution. Then, 100 ll of appropriate dilutions were spread on nutrient agar medium plates. After being incubated at 32 °C for 48 h, the colony forming unites were enumerated. Controls, including TiO2 films and bacterial
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suspensions under solar irradiation, bacterial suspensions without films under solar irradiation and Ag–TiO2 films and bacterial suspensions in dark were carried out simultaneously. Each data point was the mean of three replicates. Bacteria inactivation efficiency was calculated using the following equation: C0 Ct Inactivation efficiency ð%Þ ¼ 100 C0 where C0 and Ct represent the initial concentration of bacteria and those at sampling time t during process, respectively (Wang et al. 2013). Statistical analysis The data from three independent experiments were presented as the mean ± standard deviation of the mean. Statistical comparisons were carried out by analysis of variance (ANOVA) test to determine the level of significance (p \ 0.05) using IBM SPSS statistics 19.0 software.
Results Preparation of TiO2 thin films The doctor-blade technique can be easily employed as a fast and non-energy consuming procedure to mass
Fig. 1 Digital images showing the dried rectangular coatings of TiO2 colloid on the microscopic slides (a), color of films before and after 2 h irradiation under UV light: TiO2 film before UV irradiation (b), Ag–TiO2 film before UV irradiation (c), TiO2 film after UV irradiation (d) and Ag–TiO2 film after UV irradiation (e), TEM of TiO2 film (f) and TEM of Ag–TiO2 film (g)
production of TiO2 thin films with good uniformity and reproducible properties (Fig. 1a). The dimensions of the films were length 6.5 cm, width 1.8 cm, surface area of 11.7 cm2 and thickness of 8 lm. Response of TiO2 and Ag–TiO2 films under UV light To detect the presence of Ag on the TiO2 films after dipping in the silver nitrate solution (1 mM), the Ag–TiO2 and unmodified TiO2 thin films were exposed to UV irradiation (at 254 nm) for 2 h (Fig. 1). Before UV exposure the films did not show any color change as illustrated in Fig. 1b, c. The exposure did not change the color of unmodified TiO2 films (Fig. 1d), while Ag–TiO2 films changed into a dark brown color (Fig. 1e) as previously reported by Arabatzis et al. (2003). Characterization of Ag–TiO2 films TEM analysis TEM analysis was performed to investigate the morphology of TiO2 and Ag–TiO2 films (Fig. 1). TiO2 particles were spherical shaped with an average diameter of 19.8 nm (Fig. 1f). Many particles are stacked on top of one another. In Ag–TiO2 film (Fig. 1g), spherical AgNPs were clearly seen on top of TiO2 particles as shown by arrows in Fig. 1g. The AgNPs appear darker than TiO2 particles.
(a)
(b)
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(g)
(c)
(d)
AgNPs
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SEM–EDX analysis Macroscopic observations and SEM analysis of the films suggested that AgNPs are uniformly spread on the surface without the formation of islands. SEM images of TiO2 films show that films are rough, forming a sponge like structure, and particles were spherical in shape (Fig. 2a). Silver deposition seems to affect the surface characteristics of the resulting Ag–TiO2 films. Figure 2b depicts a slight decrease on the roughness and height of surface features. Ag nanoparticles appear as bright dots dispersed in the TiO2 amorphous matrix. The magnified image (Fig. 2c) clearly shows the bright AgNP clusters. The films heated at 400 °C and underwent slow cooling have AgNPs smaller than those observed after deposition (Fig. 2d). The EDX
Fig. 2 SEM images of unmodified TiO2 film (a), Ag– TiO2 film after deposition of AgNPs (b), magnified image showing the AgNPs cluster (c), Ag–TiO2 film after annealing at 400 °C (d), EDX of TiO2 film (e) and EDX of Ag–TiO2 film (f)
(a)
analysis of unmodified TiO2 films confirmed the chemical composition as 100 % TiO2 (Fig. 2e), while EDX analysis of Ag–TiO2 films confirmed 0.50 % Ag presence on the TiO2 (99.5 %) film (Fig. 2f). X-ray diffraction analysis The Ag–TiO2 films prepared with the doctor-blade technique were examined by XRD. The characteristic reflections of both anatase and rutile TiO2 phases were easily detected (Fig. 3). The distinct peaks showed that the TiO2 particles are crystalline. The peaks appearing at 2h = 25.2°, 37.8° and 48° elucidate the diffractions of the (1 0 1), (0 0 4) and (2 0 0) (JCPDS File No. 83-2243) planes of anatase-type TiO2 (Naeem and Ouyang 2010), by
(b)
AgNPs
(c)
(d) AgNPs
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Photocatalytic inactivation of bacterial strains
Fig. 3 X-ray diffraction of Ag–TiO2 films (A anatase and R rutile reflections)
applying the Scherrer’s formula (D = 0.9k/b1/2cosh), the TiO2 crystallites size can be estimated as 17.32 nm. This value is consistent with the particle size obtained in TEM analysis (19.8 nm). No reflection peak owing to metallic Ag can be seen. Photocatalytic degradation of MB The photocatalytic activity of TiO2 and Ag–TiO2 films under direct sunlight was investigated, using MB as a model conjugated organic molecule. The MB spectrum obtained from UV–Vis spectrophotometer revealed a strong absorbance peak (kmax) around 664 nm (Fig. 4a). The rate of decolorization was recorded with respect to the change in intensity of absorption peak at 664 nm. Control MB showed only 15 % degradation (Fig. 4a). For MB– TiO2 and MB–Ag–TiO2 films the absorption peaks successively decreased with time indicating the degradation of MB dye as represented in Figs. 4b and 5a. MB–TiO2 films resulted in 50 % degradation of the original concentration (Fig. 4b). MB–Ag–TiO2 films performed better, resulting in *100 % degradation of MB (Fig. 5a). The absorption peak of MB blue shifted from 664 to 658 nm during illumination as illustrated in the inset of Fig. 5a. For MB–Ag– TiO2 system in dark almost no change in peak intensity was observed (Fig. 5b). Percentage of residual MB concentration during the different treatments was illustrated in Fig. 6. The digital images in Fig. 7a, b revealed the complete degradation of MB dye after 120 min of solar irradiation in the presence of Ag–TiO2 films. Effect of repeated use of Ag–TiO2 films The effectiveness of Ag–TiO2 films to degrade MB was tested for five consecutive cycles and compared to that of the new films. The degradation efficiency of the films was slightly decreased by repeated use, i.e., only small losses (10 %) of their catalytic activity were observed after the fifth cycle as presented in Fig. 7c, d.
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As indicated in Fig. 8 Ag–TiO2 thin films had a similar inactivation effect on either S. aureus (Fig. 8a) or E. coli (Fig. 8b) under natural sunlight. The overall counts of both live bacteria in the treated suspensions decreased as illumination time increased. The counts of either S. aureus or E. coli slightly changed during the first 30 min, the inactivation efficiency was 14 and 16 %, respectively. Then they started decreasing significantly (p \ 0.05), with decreases in S. aureus count being slightly slower than those in E. coli. The inactivation efficiency was as high as 55 % for S. aureus and 67 % for E. coli after 90 min. By 120 min, the inactivation efficiency of Ag–TiO2 films continuously increased to over 80 % for both strains. After 180 min, the inactivation efficiency was maximal, it reached 95 and 97 % for S. aureus and E. coli, respectively. These results indicate that S. aureus and E. coli might have different responses to Ag–TiO2 under the previously mentioned experimental conditions. The inactivation efficiency of both bacteria was very low in the presence of unmodified TiO2 films, as represented in Fig. 8a, b. It was 20 % for S. aureus and 23 % for E. coli after 180 min under direct sunlight. Control experiment conducted under sunlight without films did not reveal any change in the bacterial count of both strains as illustrated in Fig. 8a, b. For Ag–TiO2 films in dark, insignificant decrease in bacterial count was obtained, 11 % for S. aureus and 16 % for E. coli after 180 min (Fig. 8a, b).
Discussion The mixture of anatase and rutile has been considered the most active formulation of TiO2. Presence of rutile phase in adjacent to anatase phase in appropriate ratio causes a synergistic effect. Bickley et al. (1991) attributed the high photocatalytic activity of the mixture to the interaction between different phases of rutile and anatase. Accordingly, the photogenerated electrons move from anatase to a lower electron trapping site in rutile. It was proposed that this electron transfer reduces the recombination of anatase and leads to more efficient electron–hole separation as well as higher photoactivity. The combined action of anatase and rutile phases was further studied by Ohno et al. (2003), who examined commercial titanium oxide samples containing only either anatase or rutile or mixtures of anatase and rutile in various ratios. They found that mixtures of anatase and rutile were the most active for the studied reaction, which means that a synergistic effect may take place. The main issue arising in photocatalysis lies in the inability of TiO2 to efficiently use solar light, which is
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Fig. 4 UV–Vis spectra of control MB under natural sunlight irradiation; inset showing the chemical structure of MB (a) and spectra of MB solution treated with unmodified TiO2 films under natural sunlight irradiation (b)
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composed of only 4–5 % UV. However, solar irradiation consists of approximately 43 % visible light, so more efficient utilization of this portion is desirable. Efforts to address this issue by increasing visible light absorption have been made through a number of catalyst modifications such as metals deposition (Arabatzis et al. 2003; Swarnakar et al. 2013). In addition, the rate of recombination of the photoexcited electrons and holes is a major factor limiting the efficiency of photocatalytic processes, and as such research in photocatalyst development has also been focused on the design and fabrication of photocatalysts possessing reduced recombination rates (Swarnakar et al. 2013). Photoreduction of Ag? under UV light is a simple and efficient method for deposition of AgNPs and nanoclusters. The primary mechanism for Ag? reduction in this system is believed to be the adsorption of Ag? ions onto the surface
of TiO2 films, where Ag? can be reduced by excited TiO2 photoelectrons that are transferred to the Ag? ions (Sahyun and Serpone 1997). Macroscopic observation of the prepared films suggests that AgNPs are widely spread on the surface without the formation of islands and that the resulting surface modified photocatalysts possess a solid structure on the glass substrate with more than satisfactory scratch resistance and adherence. Darkens of Ag–TiO2 films upon UV irradiation means that adsorbed Ag? ions were reduced and converted to zero valence silver (Ag0), this color is attributed to the surface plasmon resonance characteristic of AgNPs (Arabatzis et al. 2003). The deposition of AgNPs on TiO2 surface was further confirmed using transmission and scanning electron microscopy. The AgNPs appear darker in the TEM image than the TiO2 particles due to their higher electron density.
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Fig. 5 UV–Vis spectra of MB solution treated with Ag–TiO2 thin films under natural sunlight irradiation; inset showing the absorption peak shift of MB (a) and in dark (b)
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In XRD pattern the anatase reflections are dominating but rutile is also present, as the original material (TiO2) contains both phases (80 % anatase and 20 % rutile). No reflection peak owing to metallic Ag can be seen, suggests that silver particles are not crystallized on TiO2 surface. Similar XRD results were reported by Arabatzis et al. (2003) and Swarnakar et al. (2013). The control MB hardly degraded after illumination under sunlight for 120 min, which indicates that MB could not be decomposed without the photocatalyst. Cationic MB molecules are known to strongly adsorb on the surface of TiO2 via electrostatic attraction as well as interaction with surface hydroxyl groups (El-Sharkawy et al. 2007). The enhanced photocatalytic degradation of MB in the presence of Ag–TiO2 films compared with TiO2 films could be
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ascribed to the combined effect of enhanced photocatalytic activity of Ag–TiO2 films and the fact that more MB molecules are adsorbed on the Ag–TiO2 films than TiO2 films as a result of increased surface area, roughness, and negative charges provided by the presence of Ag nanoclusters (Rodrı´guez et al. 2009). The fast preferential MB adsorption on AgNPs will allow more dye molecules to be in contact with the catalyst over the entire irradiation period and will contribute to its degradation (Yogi et al. 2008). Nevertheless, introduction of the intermediate band energy inside the TiO2 band gap majorly contributes to improvement of photocatalytic efficiency, since without creation of sufficient OH radicals, the accumulation of large amount of MB molecules will be useless. In addition, in the presence of TiO2 films the ROS concentration was
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much lower than that in Ag–TiO2 system, as it generated by the UV portion of sunlight (4–5 %). The blue shift from 664 to 658 nm during the illumination of MB dye could be due to the N-demethylation of MB by TiO2 nanoparticles in a stepwise manner under solar irradiation or the formation of N-demethylated intermediates (Zhang et al. 2001; Wang et al. 2013). Ag–TiO2 films in dark did not
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Fig. 7 Digital images of MB solution under solar irradiation in presence of Ag–TiO2 films: at zero time (a) and after 120 min, showing complete decolorization of MB solution (b) and effect of repeated use of Ag–TiO2 thin films on MB degradation (%) under direct sunlight irradiation (c, d). Shown is the mean value of 3 readings ± standard deviation. Values followed by different superscript letters are significantly different at p \ 0.05
result in any MB degradation because dark conditions did not allow a direct titania excitation (Mitoraj et al. 2007). These results are in agreement with that reported by Snyder et al. (2013). The fact that with repeated use the degradation efficiency of Ag–TiO2 films was slightly decreased supports the practical use of doctor-blade technique, as the films produced by this technique can be used multiple times with only small losses of their catalytic properties. This result is in accordance with previously published reports (Swarnakar et al. 2013). The photocatalytic inactivation is based on redox properties of surface trapped photogenerated charges and 1 formed ROS (OH, O2 , H2O2, O2 etc.) (Ishibashi et al. 2000). One of the most toxic for microorganisms is the OH radical because of its ability to oxidise many organic substrates like carbohydrates, lipids, proteins and nucleic acids (Srinivasan and Somasundaram 2003). It promotes peroxidation of polyunsaturated phospholipid components of the lipid membrane and induces disorders in the cell membrane. Maness et al. (1999) reported that ROS causes major disorders in the E. coli cell membrane leading to inhibition of fundamental vital processes of the cell and in consequence to its death. Cho et al. (2004) found a linear correlation between the amount of photogenerated OH radicals and the extent of E. coli inactivation in TiO2 photocatalytic disinfection processes. Moreover, ROS
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might oxidize the surface of AgNPs, the oxidized AgNPs have the ability to kill bacteria because of the extensive and persistent release of Ag? from oxidized AgNPs (Zhao et al. 2013). Inactivation of bacteria requires a certain amount of cumulative damage, a process to prevent bacteria from growing or to kill them. The damage cannot be completed in short time even though there are enough radicals produced by photocatalytic nano-TiO2 (Maruga´n et al. 2008). The delay in the inactivation during the first 30 min could be due to the concentration of photogenerated ROS, it increases until reaches a level which is harmful to bacteria. Below this concentration the self-defence mechanisms of bacteria involving enzymes, catalase and superoxide dismutase protect the cell from the oxidative stress and it can grow again after being injured (Mitoraj et al. 2007). When the concentration of ROS is higher (after 90 min), the defense mechanisms of bacteria involving enzymes are insufficient. Therefore, the oxidation of cell wall and membrane components leads to bacteria death. The decreasing inactivation rate in the last step is probably caused by the ROS consumption not only by living cells, but also by products of bacterial lysis (Rincon and Pulgarin 2005). The obtained results reveal that Ag–TiO2 thin films are more effective antibacterial materials than TiO2 thin films under sunlight. The significant difference in antibacterial effects between TiO2 thin films and Ag–TiO2 thin films may be attributed to the difference in antibacterial mechanisms between both films. It is the photocatalytic property of the TiO2 thin films that makes it to have an antimicrobial capability. But the percent of ultraviolet light from natural light is only 4–5 % which results in the lower antibacterial activity. On the other hand, the antibacterial activity of Ag–TiO2 thin films is related to the enhanced photocatalytic property of the TiO2 as well as the antimicrobial activity of AgNPs. Ag–TiO2 thin films showed a stronger effect on E. coli than S. aureus under the same conditions perhaps due to the
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Survival fraction C/Co (%)
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Fig. 8 Survival fraction of S. aureus (a) and E. coli (b) treated with Ag–TiO2 films under different experimental conditions. Shown is the mean value of 3 readings ± standard deviation
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difference in cell wall properties (its structure, thickness, etc.) between Gram-positive and Gram-negative bacteria. Gram-positive bacteria require a longer treatment time than Gram-negative bacteria (Lydakis-Simantiris et al. 2010). This is because Gram-positive bacteria have a very thick cell wall consisting of many layers of peptidoglycan and teichoic acids, while the cell wall of Gram-negative bacteria is very thin consisting of only a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins (Robertson et al. 2012). The obtained results are in agreement with the reported order of bacterial resistance in UV photoinactivation processes in the presence of titania: E. coli \ S. aureus & Enterococcus sp. (Kuhn et al. 2003; Seven et al. 2004; Rincon and Pulgarin 2005). The dark conditions did not allow either a direct titania excitation or a direct bacteria inactivation (Mitoraj et al. 2007). The insignificant inactivation obtained by Ag–TiO2 films in dark may be due to adsorption of bacteria onto the surface of the TiO2, inducing cell stress which could lead to mortality, the direct contact with AgNPs on the film surface as well as influence of Ag? release (Li et al. 2011).
Conclusions A very simple and cost effective method of film making, the doctor-blade procedure was adopted. Degradation of MB under different experimental conditions was evaluated. MB in solutions exposed to natural sunlight with the silver modified TiO2 films degraded at a rate two-times faster than those of unmodified TiO2 films. These results suggest that Ag–TiO2 photocatalysis may be envisaged as a method to clean colored waste waters in textile industries. Ag–TiO2 also effectively inactivated E. coli and S. aureus as representatives of Gram-negative and Gram-positive bacteria. It took 30 min before the inactivation can be measured and 180 min to reach the maximal inactivation rate. The
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photocatalytic bacteria reducing process induced by sunlight would be useful in hospitals, microbiological laboratories, food processing plants, pharmaceutical industry, pharmacies, and wherever there is a strong requirement for clean and sterile surfaces.
References Alrousan DMA, Dunlop PSM, McMurray TA, Byrne JA (2009) Photocatalytic inactivation of E. coli in surface water using immobilized nanoparticle TiO2 films. Water Res 43:47–54 Arabatzis IM, Antonaraki S, Stergiopoulos T, Hiskia A, Papaconstantinou E, Bernard MC, Falaras P (2002) Preparation, characterization and photocatalytic activity of nanocrystalline thin film TiO2 catalysts towards 3,5-dichlorophenol degradation. J Photochem Photobiol A Chem 149:237–245 Arabatzis IM, Stergiopoulos T, Bernard MC, Labou D, Neophytides SG, Falaras P (2003) Silver-modified titanium dioxide thin films for efficient photodegradation of methyl orange. Appl Catal B Environ 42:187–201 Bickley RI, Gonzalez-Carreno T, Lees JS, Palmisano L, Tilley RJD (1991) A structural investigation of titanium dioxide photocatalysts. J Solid State Chem 92:178–190 Cho M, Chung H, Choi W, Yoon J (2004) Linear correlation between inactivation of E. coli and OH radical concentration in TiO2 photocatalytic disinfection. Water Res 38:1069–1077 Choi J, Park H, Hoffmann MR (2010) Effects of single metal-ion doping on the visible-light photoreactivity of TiO2. J Phys Chem C 114:783–792 El-Sharkawy EA, Soliman AY, Al-Amer KM (2007) Comparative study for the removal of methylene blue via adsorption and photocatalytic degradation. J Colloid Interface Sci 310:498–508 Fujishima A, Rao TN, Tryk DA (2000) Titanium dioxide photocatalysis. J Photochem Photobiol C 1:1–21 Hamadanian M, Reisi-Vanani A, Razi P, Hoseinifard S, Jabbari V (2013) Photodeposition-assisted synthesis of novel nanoparticulate In, S-codoped TiO2 powders with high visible lightdriven photocatalytic activity. Appl Surf Sci 285P:121–129 Han C, Pelaez M, Likodimos V, Kontos AG, Falaras P, O’Shea K, Dionysiou DD (2011) Innovative visible light-activated sulfur doped TiO2 films for water treatment. Appl Catal B Environ 107:77–87 Herrmann J-M, Tahiri H, Ait-Ichou Y, Lassaletta G, Gonza´lez-Elipe AR, Ferna´ndez A (1997) Characterization and photocatalytic activity in aqueous medium of TiO2 and Ag–TiO2 coatings on quartz. Appl Catal B Environ 13:219–228 Herrmann J-M, Disdier J, Pichat P, Malato S, Blanco J (1998) TiO(2)based solar photocatalytic detoxification of water containing organic pollutants. Case studies of 2,4-dichlorophenoxyaceticacid (2,4-D) and of benzofuran. Appl Catal B Environ 17:15–23 Ishibashi K, Fujishima A, Watanabe T, Hashimoto K (2000) Quantum yields of active oxidative species formed on TiO2 photocatalyst. J Photochem Photobiol A 134:139–142 Khang NC, Khanh NV, Anh NH, Nga DT, Minh NV (2011) The origin of visible light photocatalytic activity of N-doped and weak ferromagnetism of Fe-doped TiO2 anatase. Adv Nat Sci Nanosci Nanotechnol 2:015008 Kubacka A, Ferrer M, Martı´nez-Arias A, Ferna´ndez-Garcı´a A (2008) Ag promotion of TiO2-anatase disinfection capability: study of Escherichia coli inactivation. Appl Cata B Environ 84:87–93 Kubacka A, Ferna´ndez-Garcı´a M, Colon G (2012) Advanced nanoarchitectures for solar photocatalytic applications. Chem Rev 112:1555–1614
Kuhn KP, Chaberny I, Massholder K, Stickler M, Benz VW, Sonntag H-G, Erdinger L (2003) Disinfection of surfaces by photocatalytic oxidation with titanium dioxide and UVA light. Chemosphere 53:71–77 Li W, Wang Y, Lin H, Shah SI, Huang CP, Doren DJ, Rykov SA, Chen JG, Barteau MA (2003) Band gap tailoring of Nd3?-doped TiO2 nanoparticles. Appl Phys Lett 83:4143–4145 Li M, Noriega-Trevino ME, Nino-Martinez N, Marambio-Jones C, Wang J, Damoiseaux R, Ruiz F, Hoek EMV (2011) Synergistic bactericidal activity of Ag–TiO2 nanoparticles in both light and dark conditions. Environ Sci Technol 45:8989–8995 Lu SY, Wu D, Wang QL, Yan JH, Buekens AG, Cen KF (2011) Photocatalytic decomposition on nano-TiO2: destruction of chloroaromatic compounds. Chemosphere 82:1215–1224 Lydakis-Simantiris N, Riga D, Katsivela E, Mantzavinos D, XekoukOulotakis NP (2010) Disinfection of spring water and secondary treated municipal wastewater by TiO2 photocatalysis. Desalination 250:351–355 Malati MA, Wong WK (1984) Doping TiO2 for solar-energy applications. Surf Technol 22:305–322 Maness P-C, Smolinski S, Blake D, Huang Z, Wolfrum AJ, Jacoby WA (1999) Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing mechanism. Appl Environ Microbiol 65:4094–4098 Maruga´n J, van Grieken R, Sordo C, Cruz C (2008) Kinetics of the photocatalytic disinfection of Escherichia coli suspensions. Appl Catal B Environ 82:27–36 Mitoraj D, Jan´czyk A, Strus M, Kisch H, Stochel G, Heczko PB, Macyk W (2007) Visible light inactivation of bacteria and fungi by modified titanium dioxide. Photochem Photobiol Sci 6:642–648 Naeem K, Ouyang F (2010) Preparation of Fe3?-doped TiO2 nanoparticles and its photocatalytic activity under UV light. Phys B Condens Matter 405:221–226 Ohno T, Tokieda K, Higashida S, Matsumura M (2003) Synergism between rutile and anatase TiO2 particles in photocatalytic oxidation of naphthalene. Appl Catal A Gen 244:383–391 Parkin IP, Palgrave RG (2005) Self-cleaning coatings. J Mater Chem 15:1689–1695 Rincon AG, Pulgarin C (2005) Use of coaxial photocatalytic reactor (CAPHORE) in the TiO2 photo-assisted treatment of mixed E. coli and Bacillus sp. and bacterial community present in wastewater. Catal Today 101:331–344 Robertson PKJ, Robertson JMC, Bahnemann DW (2012) Removal of microorganisms and their chemical metabolites from water using semiconductor photocatalysis. J Hazard Mater 211–212:161–171 Rodrı´guez A, Garcı´a J, Ovejero G, Mestanza M (2009) Adsorption of anionic and cationic dyes on activated carbon from aqueous solutions: equilibrium and kinetics. J Hazard Mater 172:1311–1320 Sahyun MRV, Serpone N (1997) Primary events in the photocatalytic deposition of silver on nanoparticulate TiO2. Langmuir 13:5082–5088 Serpone N (2006) Is the band gap of pristine TiO2 narrowed by anionand cation-doping of titanium dioxide in second-generation photocatalysts? J Phys Chem B 110:24287–24293 Seven O, Dindar B, Aydemir S, Metin D, Ozinel MA, Icli S (2004) Solar photocatalytic disinfection of a group bacteria and fungi aqueous suspensions with TiO2, ZnO and Sahara desert dust. J Photochem Photobiol A 165:103–107 Snyder A, Bo Z, Moon R, Rochet J-C, Stanciu L (2013) Reusable photocatalytic titanium dioxide–cellulose nanofiber films. J Colloid Interface Sci 399:92–98 Srinivasan C, Somasundaram N (2003) Bactericidal and detoxification effects of irradiated semiconductor catalyst, TiO2. Curr Sci 85:1431–1438 Swarnakar P, Kanel SR, Nepal D, Jiang Y, Jia H, Kerr L, Goltz MN, Levy J, Rakovan J (2013) Silver deposited titanium dioxide thin
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World J Microbiol Biotechnol film for photocatalysis of organic compounds using natural light. Sol Energy 88:242–249 Tachibana Y, Vayssieres L, Durrant JR (2012) Artificial photosynthesis for solar water-splitting. Nat Photonics 6:511–518 Wang B, Kerr LL (2012) Stability of CdS-coated TiO2 solar cells. J Solid State Electrochem 16:1091–1097 Wang YQ, Yu XJ, Sun DZ (2007) Synthesis, characterization, and photocatalytic activity of TiO2–xNx nanocatalyst. J Hazard Mater 144:328–333 Wang J, Li C, Zhuang H, Zhang J (2013) Photocatalytic degradation of methylene blue and inactivation of Gram-negative bacteria by TiO2 nanoparticles in aqueous suspension. Food Control 34:372–377 Xiong ZG, Ma JZ, Ng WJ, Waite TD, Zhao XS (2011) Silver modified mesoporous TiO2 photocatalyst for water purification. Water Res 45:2095–2103
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Yogi C, Kojima K, Wada N, Tokumoto H, Takai T, Mizoguchi T, Tamiaki H (2008) Photocatalytic degradation of methylene blue by TiO2 film and Au particles-TiO2 composite film. Thin Solid Films 516:5881–5884 Yoon TP, Ischay MA, Du J (2010) Visible light photocatalysis as a greener approach to photochemical synthesis. Nat Chem 2:527–532 Zhang TY, Oyama T, Aoshima A, Hidaka H, Zhao JC, Serpone N (2001) Photooxidative N-demethylation of methylene blue in aqueous TiO2 dispersions under UV irradiation. J Photochem Photobiol A Chem 140:163–172 Zhao X, Toyooka T, Ibuki Y (2013) Synergistic bactericidal effect by combined exposure to Ag nanoparticles and UVA. Sci Total Environ 458–460:54–62
