Inactivation and Mineralization of Aerosol Deposited Model

Jun 3, 2010 - (35) Bratbak, G.; Dundas, I. Bacterial dry matter content and biomass estimation. Appl. Environ. Microbiol. 1984, 48 (4), 755–757. ES1...
0 downloads 0 Views 469KB Size
Download PDF
Environ. Sci. Technol. 2010, 44, 5121–5126

Inactivation and Mineralization of Aerosol Deposited Model Pathogenic Microorganisms over TiO2 and Pt/TiO2 E. A. KOZLOVA,† A. S. SAFATOV,‡ S. A. KISELEV,‡ V. YU. MARCHENKO,‡ A. A. SERGEEV,‡ M. O. SKARNOVICH,‡ E. K. EMELYANOVA,‡ M. A. SMETANNIKOVA,‡ G. A. BURYAK,‡ A N D A . V . V O R O N T S O V * ,† Boreskov Institute of Catalysis, Novosibirsk 630090, Russian Federation, and Federal State Research Institution State Research Center of Virology and Biotechnology, Vector, Koltsovo, Novosibirsk region, 630559, Russian Federation

Received January 28, 2010. Revised manuscript received April 5, 2010. Accepted May 7, 2010.

Air disinfection from bacteria and viruses by means of photocatalytic oxidation is investigated with microorganisms loaded over photocatalysts’ films from aerosols. Deposition method and equipment have been developed to load Mycobacterium smegmatis, Bacillus thuringiensis, vaccinia virus, and influenza A (H3N2) virus on slides with undoped TiO2 and platinized sulfated TiO2 (Pt/TiO2). Inactivation dynamics was measured under UVA irradiation and in the dark. About 90% inactivation is reached in 30 min irradiation on TiO2 and from 90 to 99.8% on Pt/TiO2. The first-order inactivation rate coefficient ranged from 0.18 to 0.03 min-1, over Pt/TiO2 being higher than on TiO2 for all microorganisms except Bacillus thuringiensis. The photocatalytic mineralization of Bacillus thuringiensis was performed on TiO2 and Pt/TiO2 with different photocatalyst and microorganism loadings. Completeness of mineralization depended on the TiO2 to bacteria mass ratio. The rate of the photocatalytic carbon dioxide production grows with both the cell mass increase and the photocatalyst mass increase. Pt/ TiO2 showed increased rate of mineralization as well as of the inactivation likely due to a better charge carrier separation inthedopedsemiconductorphotocatalyst.Theresultsdemonstrate that photocatalytic filters with deposited TiO2 or Pt/TiO2 are able to inactivate aerosol microorganisms and completely decompose them into inorganic products and Pt/TiO2 provides higher disinfection and mineralization rates.

Introduction Biological contamination of water and air poses significant risks to the population in the context of emerging viral epidemics, increased threats of terrorism, and poor water quality in many regions. Photocatalytic oxidation over semiconductor oxides has been demonstrated to be applicable to air and water purification from both molecular pollutants and harmful microorganisms (1-3). * Corresponding author tel.: 7-383-326-9447; fax: 7-383-333-1617; e-mail: [email protected]. † Boreskov Institute of Catalysis. ‡ Federal State Research Institution State Research Center of Virology and Biotechnology. 10.1021/es100156p

 2010 American Chemical Society

Published on Web 06/03/2010

Photocatalytic inactivation of microorganisms over TiO2 photocatalyst was initially studied for Escherichia coli bacteria in aqueous systems in 1993 (4) and 1994 (5) and led to a conclusion that solar TiO2 disinfection could be a viable alternative to chlorination. Later a large number of studies appeared dealing with photocatalytic water purification from bacteria (e.g. refs 6-10), fungi (e.g., refs 11 and 12), and viruses (e.g., ref 13). Until recently, photocatalytic disinfection of air received smaller attention due to experimental difficulties of work with microorganisms at the dry semiconductor-air interface. The increased interest in photocatalytic air disinfection was induced by the ideas that this method needs UVA light only and does not require UVC light that generates toxic ozone, which possibly leads not only to permanent microorganism inactivation (14) but also to transformation of organic biological material into inorganic products, i.e. mineralization (15). Practical application of photocatalytic air purification needs reliable confirmation for inactivation of pathogenic microorganisms deposited from the air stream passing through the support with irradiated photocatalyst. Presently, this was done for Bacillus cereus (14), Staphylococcus aureus, Escherichia coli, Aspergillus niger, MS2 bacteriophage (16), Bacillus subtilis, Pseudomonas fluorescens, and Microbacterium sp (17). Another problem with gas-phase photocatalysis is cleaning the photocatalyst surface from biological matter since, unlike aqueous systems, there is no wash-out of the inactivation products. Partial mineralization over irradiated TiO2 was confirmed for E. coli only (15). In the present study, a special technique has been developed for uniform deposition of bacterial and viral microorganisms over photocatalyst-coated slides from aerosol state to closely simulate the conditions in photocatalytic air filters. Mycobacterium smegmatis, Bacillus thuringiensis, and vaccinia and influenza A (H3N2) viruses undergo inactivation after deposition over TiO2 and Pt/TiO2 photocatalyst films. These microorganisms can serve as simulants for highly pathogenic Mycobacterium tuberculosis, Bacillus anthracis (anthrax), Variola major (smallpox), and other strains of influenza virus, respectively. The platinized sulfated photocatalyst is applied because it was demonstrated to possess increased activity in acetone mineralization (18), and photocatalytic activity in organic compounds oxidation correlates with inactivation of bacteria (19). The increased rate of both inactivation and mineralization is observed over Pt/TiO2.

Experimental Section Photocatalysts Preparation. Rectangular 2.5 × 3.5 cm glass plates were washed with concentrated NaHCO3 for better catalyst adhesion. Undoped and platinized sulfated commercial TiO2 Hombifine N (100% anatase, SSA 336 m2/g, Sachtleben Chemie, GmbH) was used. The content of Pt in Pt/TiO2 was 1% wt, platinization and sulfation was described in refs 18 and 20. The deposition of the photocatalysts on glass plates was done from suspension with the concentration 10 (I) or 50 (II) g/L. A 0.4 mL portion of suspension was poured by pipet on the surface of slides, the size of the spot was 2.5 × 2.5 cm. Then it was dried at ambient conditions for 6 h. For type I, the deposition was performed once; for type II, it was done twice. After applying to glasses, the coatings were subjected to sterilization at 110 °C for 1 h followed by washing in physiological solution. Microorganisms Preparation. Vaccinia virus strain LIVP was obtained by FSRI SRC VB Vector from the D.I. Ivanovsky Institute of Virology (Moscow, RF) in 1986. It was passaged VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5121

10 times on embryonated chicken eggs. Virus-containing material was produced by cultivation in cell culture 4647 (kidney cells of Cercopithecus aethiops monkey) followed by freezing/thawing the infected cell culture three times in supporting modified Eagle’s medium (ICN Biomedicals, Inc., Aurora, Ohio). The standard method for counting the plaques forming on cell culture was used to determine the virus concentration in samples. The initial virus-containing suspension was diluted in supporting medium supplemented with antibiotics. Six 10-fold dilutions were prepared, and 100 µL was put onto a confluent monolayer of cells 4647 in 24-well plates (Costar, Pleasanton, California). The virus adsorption was carried out at 37 °C for 1 h in a CO2 incubator. The plate was shaken every 10-15 min, the fluid was aspirated from the wells in 1 h, and 2 mL of 1% agar (Difco) was added on RPMI 1640 medium (containing 2% fetal calf serum and antibiotics) to each well. Infected cells were incubated for 2 days at 37 °C in a CO2 incubator. Subsequently, the cell monolayer was stained with a dye prepared on the basis of gencyan violet to count the plaques in the wells. The results were calculated as the number of plaque-forming units (PFU) per 1 mL of suspension. The virus concentration was 107-108 PFU/mL. The initial virus-containing suspension was frozen and stored at -70 °C. Influenza virus strain A/Aichi/2/68 (H3N2) obtained from the D.I. Ivanovsky Institute of Virology RAMS, which had undergone 12 passages on mice and 3 passages on embryonated chicken eggs (ECE), was used in the work. The developed and used series of virus-allantoic fluid was stored at -70 °C. Influenza virus concentration in virus-allantoic fluid (VAF) was determined with a standard titration method by infecting 9-11-day ECE. Hank’s medium supplemented with antibiotics was used for virus dilution; 100 µL of each serial 10-fold dilution of virus-containing fluid was inoculated to 6 ECE. After 2-day incubation of infected ECE at 35.5 °C, the presence of the virus in them was determined by hemagglutination reaction (HAR) of chicken erythrocytes. The influenza virus concentration in VAF was expressed in log10 EID50/mL (logarithm 50% of embryonic infecting doses in 1 mL). Six serial 10-fold dilutions of initial samples were prepared to determine the virus concentration in samples. Dilutions were prepared in 4.5 mL of Hank’s solution supplemented with antibiotics. The test tubes were placed on ice when preparing the dilutions. At the next stage, 300 µL of each dilution (from 0 to 6) of initial sample was inoculated to 6 ECE. After 2-day incubation of infected ECE at 35.5 °C, the presence of the virus in them was determined by HAR of chicken erythrocytes. The influenza virus concentration in samples was expressed in log10 EID50/mL. Mycobacterium smegmatis was grown in liquid LB medium at the temperature of 30 °C on a thermostatted shaker for 3 days. 10-fold dilutions were prepared from the produced culture fluid, and 20 or 50 µL was seeded onto agarized FPA medium. The seedings were incubated in a thermostat at 30 °C. The grown, isolated colonies were counted after 4-5 days of incubation. The results were calculated as the number of colony-forming units (CFU) per 1 mL of suspension. The biological activity of initial mycobacterial suspensions was 107-108 CFU/mL. The sporiferous bacterium Bacillus thuringiensis obtained from the Microorganism Collection of FSRI SRC VB “Vector” was used in the work. The concentration of the initial bacterial suspension was up to 1010 CFU/mL. The biological concentration of bacteria was determined with micromethod in 96well immunological plates. For this purpose, 90 µL of physiological solution was placed into each well. The initial suspension of microorganism strains in the amount of 10 µL was placed into the first well at stirring with a doser. It was the first dilution. After the tip change, 10 µL was collected from the well with the first dilution and transferred to the 5122

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

second well followed by stirring with a doser, which was the second dilution. The procedure was repeated 6-8 times using new doser tips in each case. Suspension dilutions were inoculated by the surface method to agarized RPA medium -10 µL in 3 repeats. Incubation was performed in a thermostat at 30 °C for 18 h. Then, the grown colonies were counted. The spore percentage in initial bacterial samples usually made up 20-30%. The samples’ biological activity was determined in three parallel experiments. The calculations of the microorganisms’ biological activities were performed according to methods described in ref 21. Physical Label. The uranin physical label was added to sprayed suspensions to evaluate the weight of aerosol deposited on glass slides. This dye is often used as a physical label at the study of microorganisms’ survival under the influence of different factors (22-24). It was used further in all the conducted experiments. Uranin was sterilely added to the initial suspension to the final concentration of 10-5 g/mL. Fluorescence intensities of diluted samples proportional to concentration of initial material were measured with Spekol-11 spectrophotometer (Carl Zeiss, Jena, Germany) at uranin concentration lower than 10-7 g/mL, which provided a linear dependence of the label concentration on fluorescence intensity. Experimental values of fluorescence intensities and biological activity of samples were used to calculate the efficiency of inactivation of selected biological agents. Microorganism Deposition from Aerosol. The deposition was performed in a sealed chamber for sterile application of microorganism-containing aerosols to photocatalyst-coated and uncoated glass slides. The detailed description of the chamber and deposition procedure is given in the Supporting Information. Microorganisms Deposition by a Pipet. A prepared suspension of Bacillus thuringiensis (0.2 mL) was poured by pipet on the surface of slides in the dark, the size of the spot was 2.5 cm × 2.5 cm. The slides were dried in a desiccator in the dark under controlled humidity and temperature for 7.5 h. Then, they were weighed in the dark and placed in microcontainers. The specimens with microorganisms were taken from the container immediately before the photocatalytic experiment. Photoinactivation of Microorganisms. Slides with deposited microorganisms were exposed to ultraviolet radiation for various periods of 1-30 min. The radiation source was PHILIPS PL-S 11W/10/2P lamp (λ ) 350-400 nm) installed at a distance of 22 cm from the sample surface (Figure 2S of the Supporting Information); UV irradiance of 0.65 ( 0.05 mW/cm2 was measured with a radiometer (UVX digital radiometer, UVP Inc., USA). From 3 to 12 slides were irradiated simultaneously. All experiments with photocatalyst samples and different controls were repeated three times. After the treatment, experimental and control samples were put into weighing bottles with 10 mL of the lavage fluid, and wash-out was performed on a shaker for 5 min. The fluid was a phisiological solution for bacteria and Hank’s solution for viruses. The lavage fluids from weighing bottles were poured into labeled bottles and placed in a refrigerator at +4 °C. The standard method for counting the plaques forming on cell culture was used to determine the virus concentration in samples and colony forming units were counted for bacteria. Photocatalytic Mineralization of Microorganisms. Slides with deposited photocatalysts and inactivated microorganisms were irradiated by the same UV lamp in a static reactor shown in Figure 3S in the Supporting Information. The concentration of CO2 inside the reactor was measured by means of a gas chromatograph LChM-8 equipped with a methanizer and flame ionization detector (FID). Nine specimens, which are described in Table 1, were used for

TABLE 1. Properties of Specimens with Titania Photocatalysts and Inactivated Bacillus thuringiensis in the Photocatalytic Mineralization Reaction deposited mass of bacterial type of amount of CO2 mass of carbon average rate calculated C no. catalyst/mass, mg deposit, mg deposition evolved, µmol in evolved CO2, mg of CO2 evolution, ppm/h content in deposit, % 0a 1 2 3 4 5 6 7 8 9

TiO2/4 mg TiO2/4 mg TiO2/4 mg TiO2/20 mg TiO2/20 mg Pt/TiO2/20 mg Pt/TiO2/20 mg Pt/TiO2/20 mg TiO2/20 mg Pt/TiO2/20 mg a

10 7 13 3 2 2 3 1 4 4

pipet pipet pipet aerosol aerosol aerosol aerosol aerosol pipet pipet

6 28 51 65 46 49 66 24 88 88

0.07 0.34 0.61 0.78 0.55 0.59 0.79 0.29 1.10 1.10

30 26 55 590 333 568 806 435 505 808

0.7 4.9 4.7 26.0 27.5 29.5 26.3 29.0 27.5 27.5

Carbon dioxide evolution without UV-irradiation. Experimental error was about 10%.

FIGURE 2. Inactivation of Bacillus thuringiensis aerosol over TiO2 and Pt/TiO2 under UV irradiation and in the dark. FIGURE 1. Inactivation of Mycobacterium smegmatis aerosol over TiO2 and Pt/TiO2 under UV irradiation and in the dark. photocatalytic mineralization. The reaction was carried out until the amount of CO2 finished increasing. A blank dark experiment was carried out as well: the glass with inactivated Bacillus thuringiensis and pure TiO2 was placed in the sealed reactor. The amount of carbon dioxide evolved without illumination was measured.

Results and Discussion Photocatalytic Inactivation of Aerosol Deposited Microorganisms. Figure 1 shows logarithmic kinetic plots of Mycobacterium smegmatis biological activity over undoped TiO2 and Pt/TiO2 under UV irradiation and in dark. The highest rate of inactivation is observed over Pt/TiO2: activity decreases in about 50 times during 30 min of irradiation while over TiO2 the activity decreases by about 1 order of magnitude during this time. In the dark experiments over both TiO2 and Pt/TiO2, the activity decreased only approximately by half. The kinetic plots are well-linearized in logarithmic coordinates with correlation coefficient R ) -0.99 and -0.96, probability P < 0.01 giving the values of the first-order rate coefficient 0.14 ( 0.009 and 0.07 ( 0.01 min-1 for UV irradiated Pt/TiO2 and TiO2, respectively. In the dark experiments, the rate coefficient was 0.021 and 0.016 min-1, respectively. These results demonstrate that Mycobacterium smegmatis undergoes inactivation by 98% in 30 min over Pt/TiO2. One can expect with a high probability that similar pathogenic microorganism Mycobacterium tuberculosis will also be inactivated very efficiently and photocatalytic filters with Pt/TiO2 catalyst rather than TiO2 should be used for air treatment.

Figure 2 shows experimental data on photocatalytic and dark inactivation of aerosol deposited Bacillus thuringiensis. This bacterium is morphologically similar to Bacillus anthracis but is not pathogenic. In these experiments, the rate of disinfection over Pt/ TiO2 and TiO2 was the same. Biological activity decreased about by 1 order of magnitude during 30 min of irradiation. Inactivation in the dark was not confirmed because the difference between the initial and final activity was within the range of experimental error. The kinetic plots are linearized in logarithmic coordinates with R ) -0.97 and -0.98 with P e 0.005 giving the values of first-order rate coefficient 0.06 ( 0.01 and 0.05 ( 0.01 min-1 for Pt/TiO2 and TiO2, respectively. The spore-forming bacteria of genus Bacillus in the dry form were previously tested for inactivation by photocatalytic oxidation over TiO2 P25 (14, 17). The study by Vohra et al. (14) utilized impregnation of Bacillus cereus spores on metal plate and polyester fabric from spore aqueous suspension with subsequent drying. The rate coefficient of inactivation can be estimated from kinetic plots as 0.005 and 0.007 min-1 for TiO2 and Ag/TiO2 coated substrates, respectively, at UV irradiance of 5 mW/cm2. The lower rate coefficient compared to our result above can be ascribed to higher resistance of bacterial spores to inactivation as compared to the mixed vegetative form and spores (17, 25, 26), higher activity of TiO2 Hombifine compared to TiO2 Degussa at the gas-solid interface, and different methods of bacteria deposition. In the study by Pal et al. (17), bacteria were coated on TiO2-impregnated cellulose acetate membrane filters using filtration. The first-order rate coefficient of Bacillus subtilis inactivation was 0.006 min-1 at irradiance 0.013 mW/cm2 and 0.2 min-1 at irradiance 4.3 mW/cm2. The higher rate VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5123

FIGURE 3. Inactivation of vaccinia virus aerosol over TiO2 and Pt/TiO2 under UV irradiation and in the dark.

FIGURE 4. Inactivation of influenza virus aerosol over TiO2 and Pt/TiO2. coefficient could be associated with the method of inactivation measurements: the treated filters were placed face down on agar plates. Thus, only viable bacteria located on the surface of the filter have chances to be detected while those located deeper could not be detected. Comparison of rate coefficients of aerosol and impregnation deposited bacteria shows that aerosol deposited bacteria are generally inactivated faster. Figure 3 demonstrates kinetic plots of inactivation of aerosol deposited vaccinia virus. The highest inactivation rate was observed over Pt/TiO2 that provided about 30-fold disinfection during 30 min of UV irradiation. The kinetic plots are linearized in log coordinates with R ) -0.97 and -0.98 and P e 0.005, and the first-order rate coefficients of inactivation for irradiated Pt/TiO2 and TiO2 are equal to 0.1 ( 0.01 and 0.08 ( 0.009 min-1, respectively. In the dark, the rate coefficient of inactivation is 0.009 and 0.006 min-1 for Pt/TiO2 and TiO2, correspondingly. Inactivation over platinized TiO2 is marginally faster than over Pt/TiO2. The last studied microorganism was influenza A (H3N2) virus. Figure 4 demonstrates log plot of kinetics of inactivation over undoped and platinized sulfated TiO2. Only one dark test was performed for this virus, with undoped TiO2, since the reported above dark tests showed very similar inactivation rates over Pt/TiO2 and TiO2 in dark. The large scattering of points obtained over TiO2 and Pt/TiO2 in dark in Figure 4 5124

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

reflects large errors in PFU technique applied. The 30 min irradiation of Pt/TiO2 catalyst with aerosol deposited virus resulted in 99.8% inactivation. Linearization was satisfactory with R ) -0.92 and -0.54, P e 0.27, and first-order rate coefficient of disinfection for Pt/TiO2 and TiO2 0.18 ( 0.06 and 0.06 ( 0.05 min-1, correspondingly. The activity of Pt/ TiO2 was marginally higher. The results demonstrate that bacteria and viruses deposited from aerosol were inactivated over Pt/TiO2 and TiO2 films under UVA irradiation. The highest resistance to inactivation was shown by Bacillus thuringiensis. These observations are in agreement with same radical mechanisms that are operating in photocatalytic oxidation (25, 27). Previous studies revealed that cell death in TiO2 photocatalytic reaction takes place as a result of cell membrane, cell wall damage (28), leakage of internal cell components (28, 29), and damage to intracellular components (30). There are two main mechanisms of photocatalytic oxidation initiationsdirect by photogenerated holes and indirect by hydroxyl radicals (31-33). However, damage to internal cell components can be caused by radical species or stable oxidizers like H2O2 (30) only because such a long-range electron transfer is improbable. Platinized sulfated TiO2 used in the present study possesses increased oxidizing photocatalytic activity (18) that is employed to decompose bacteria and viruses faster than over pristine TiO2. It also has slightly higher inactivating ability in the dark. Photocatalytic Mineralization of Deposited Microorganisms. For a photocatalytic filter, in order to function continuously in air sterilization, its surface has to be cleaned from dead remains of microbes. Jacoby et al. were first who demonstrated that bacteria E. coli underwent partial conversion into inorganic products in contact with UV irradiated TiO2 (15). In the present work, prolonged irradiation time is used and effects of bacteria deposition method, bacteria mass, photocatalyst mass, and type are investigated for Bacillus thuringiensis complete mineralization. Figure 5 shows kinetic curves of CO2 production under UVA light and in the dark from bacteria deposited on TiO2 using the pipet method. The part A represents kinetics of CO2 evolution under UV-irradiation. Curves for the both samples have sigmoid shape that corresponds to a chemical reaction with an induction period. In the first stage of the reaction (0-30 h), the large molecules of organic compounds constituting bacteria are transformed into the products of partial oxidation under UV-irradiation, so the rate of CO2 evolution is not very high. In the second stage, the partial oxidation products are converted into carbon dioxide and the rate of CO2 evolution increases. After 150 h, the amount of CO2 reaches a plateau. One can see that the time of plateau reaching is the same for specimens 1 and 2, although the amount of microorganisms and carbon dioxide evolved is not the same. The amount of carbon dioxide evolved is proportional to the mass of deposited microorganisms. The rate of the photocatalytic carbon dioxide evolution in the middle part of the experiment (50-100 h) for specimen no. 2 is about two times higher than that for specimen no. 1. Thus, in the middle stage of reaction, kinetics obeys the first-order dependence. Figure 5B shows that the amount of CO2 evolved from bacteria on TiO2 in the dark is very low compared to irradiated samples 1 and 2. The evolution of CO2 can be caused by the breathing of live microorganisms or decay of dead cells in the sample. Data on photocatalytic mineralization of microorganisms are summarized in Table 1 and include the average rate of CO2 evolution, the final amount of CO2 generated, and carbon content in bacterial deposits calculated from amount of CO2 evolved. Experiment 0 performed without irradiation results in the production of CO2 corresponding to a carbon content

FIGURE 5. CO2 concentration profiles resulting from Bacillus thuringiensis deposited on TiO2. (A) With UV-irradiation. (B) Without UV-irradiation. TiO2 mass ) 4 mg, bacteria mass ) 0-10, 1-7, and 2-13 mg.

FIGURE 6. Dependence of CO2 evolved in microorganism oxidation vs time under UV-irradiation on the pure and platinized TiO2 (experiments 4 and 5 in Table 1). 0.7%. The carbon content in dry bacteria generally constitutes approximately 40-60% by weight (34) while the dry mass content in bacteria ranges from 31 to 57% (35). Thus, the carbon weight content can be estimated as 12-34%. Therefore, dark conditions lead to only minor mineralization of bacteria. Experiments 1 and 2 performed with TiO2 mass 4 mg and bacterial mass 7 and 13 mg resulted in CO2 corresponding to about 5% carbon from bacteria mass. This is considerably lower than estimated above. Dark catalyst coloration was observed after the experiment completed which is an indication of catalyst deactivation. Indeed, such large loads of organic materials containing various heteroatoms are expected to generate nonvolatile products covering active sites of the TiO2 surface and stopping oxidation at some intermediate stage. Further experiments were performed with larger TiO2 mass 20 mg and smaller bacteria loadings. One can see that for specimens 3-9 the calculated carbon content varies from 26 to 29%. These values are in a good agreement with the estimate of carbon content in bacteria of 12-34%. It should be noted that CO2 evolution stopped at the end of the experiments. Jacoby et al. (15) performed E. coli mineralization, but it did not complete even though it lasted longer (80 h). They obtained a similar carbon content of 27%. Thus photocatalytic bacterium oxidation allows one to reach the full mineralization provided the amount of the photocatalyst should be high enough. In our experiments, a 5-fold excess of TiO2 was found to be enough for complete mineralization. The next step of our investigation was to compare the undoped and platinized TiO2. Figure 6 shows the kinetics of photocatalytic carbon dioxide formation during the cell mass oxidation on TiO2 and Pt/TiO2. One can see that the photocatalytic oxidation is faster on the surface of platinized titania. Platinization increases the rate of the photocatalytic CO2 production by a factor of 1.7 (nos. 4 and 5; Table 1). Earlier we described the effect of the platinum deposition on the rate of organophosphorous compounds photocatalytic mineralization (20). There are several possible reasons of enhancement of photocatalytic activity of platinized TiO2 catalyst. Supporting platinum is

FIGURE 7. Dependence of the CO2 photocatalytic production rate on the microorganisms mass. thought to enhance charge separation in semiconductor and decrease electron-hole pairs recombination. It is often supposed to be the main reason. The other reasons are dark oxidation reactions on the Pt surface, increase of the adsorption constant by introducing Pt particles, and even photocatalytic reaction on the platinum surface. The rate of photocatalytic carbon dioxide production in the case of specimen no. 8 is higher than in the cases of specimen nos. 1 and 2 likely due to the photocatalyst higher content. In the case of specimen nos. 3-7, which are prepared by aerosol deposition, the photocatalytic activity enhancement by the platinum deposition is about 40-70%. Table 1 shows that the rate of the photocatalytic CO2 production grows with the amount of cell mass deposited on the surface of the test glass. We built the dependence of the CO2 photocatalytic production rate vs the microorganism mass. Figure 7 shows that the rate of the photocatalytic carbon dioxide evolution grows linearly with the deposited microorganism mass at the same photocatalyst (Pt/TiO2) mass. Thus, this catalyst amount is enough for the immediate cell mass photocatalytic oxidation. Our experiments have shown the full photocatalytic mineralization of the Bacillus thuringiensis on the undoped and platinized photocatalyst TiO2. The rate of the photocatalytic carbon dioxide production grows with both the cell mass increase and the photocatalyst mass increase. The photocatalyst platinization increases the bacterium mineralization rate likely due to better charge carrier separation in the doped semiconductor photocatalyst. The aerosol microorganism deposition is preferable in comparison to the pipet deposition because it is more representative of the conditions of real gas phase photocatalytic systems.

Acknowledgments We gratefully acknowledge the support of grants of NATO SfP-981461 as well as ISTC 3305, SB RAS Integration Nos. 70 and 36, Russian Federal Innovation Agency via the program “Scientific and Educational cadres”, and RAS Presidium No. 27.56.

Supporting Information Available Experimental details of the aerosol deposition chamber and setups for microorganism inactivation and mineralization. VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

5125

This material is available free of charge via the Internet at http://pubs.acs.org/.

Literature Cited (1) Carp, O.; Huisman, C. L.; Reller, A. Photoinduced reactivity of titanium dioxide. Prog. Solid State Ch. 2004, 32, 33–177. (2) Parmon, V. N. Abiogenic catalysis in Nature. Colloid Surfaces A. 1999, 151 (1-2), 351–365. (3) Malato, S.; Fernandez-Ibanez, P.; Maldonado, M. I.; Blanco, J.; Gernjak, W. Decontamination and disinfection of water by solar photocatalysis: Recent overview and trends. Catal. Today. 2009, 147, 1–59. (4) Ireland, J. C.; Klostermann, P.; Rice, E. W.; Clark, R. M. Inactivation of Escherichia-coli by titanium dioxide photocatalytic oxidation. Appl. Environ. Microbiol. 1993, 59 (5), 1668– 1670. (5) Wei, C.; Lin, W. Y.; Zainal, Z.; Williams, N. E.; Zhu, K.; Kruzic, A. P.; Smith, R. L.; Rajeshwar, K. Bactericidal activity of TiO2 photocatalyst in aqueous-media - towards a solar-assisted water disinfection system. Environ. Sci. Technol. 1994, 28 (5), 934– 938. (6) Kim, D. S.; Kwak, S.-Y. Photocatalytic inactivation of E. coli with a mesoporous TiO2 coated film using the film adhesion method. Environ. Sci. Technol. 2009, 43, 148–151. (7) Li, Q.; Xie, R.; Li, Y. W.; Mintz, E. A.; Shang, J. K. Enhanced visible-light-induced photocatalytic disinfection of E. coli by carbon-sensitized nitrogen-doped titanium dioxide. Environ. Sci. Technol. 2007, 41, 5050–5056. (8) McCullagh, C.; Robertson, J. M. C.; Bahnemann, D. W.; Robertson, P. K. J. The application of TiO2 photocatalysis for disinfection of water contaminated with pathogenic microorganisms: a review. Res. Chem. Intermed. 2007, 33, 359–375. (9) van Grieken, R.; Marugan, J.; Sordo, C.; Martinez, P.; Pablos, C. Photocatalytic inactivation of bacteria in water using suspended and immobilized silver-TiO2. Appl. Catal., B 2009, 93 (1-2), 112–118. (10) Wu, P.; Xie, R.; Imlay, J. A.; Shang, J. K. Visible-light-induced photocatalytic inactivation of bacteria by composite photocatalysts of palladium oxide and nitrogen-doped titanium dioxide. Appl. Catal., B 2009, 88, 576–581. (11) Chen, F.; Yang, X.; Wu, Q. Photocatalytic oxidation of Escherichia coli, Aspergillus niger and formaldehyde under different ultraviolet irradiation conditions. Environ. Sci. Technol. 2009, 43, 4606–4611. (12) Seven, O.; Dindar, D.; Aydemir, S.; Metin, D.; Ozinel, M. A.; Icli, S. Solar photocatalytic disinfection of a group of bacteria and fungi aqueous suspensions with TiO2, ZnO and Sahara desert dust. J. Photochem. Photobiol. A. 2004, 165, 103–107. (13) Zan, L.; Fa, W.; Peng, T.; Gong, Z. Photocatalysis effect of nanometer TiO2 and TiO2-coated ceramic plate on Hepatitis B virus. J. Photochem. Photobiol. B 2007, 86, 165–169. (14) Vohra, A.; Goswami, D. Y.; Deshpande, D. A.; Block, S. S. Enhanced photocatalytic inactivation of bacterial spores on surfaces in air. J. Ind. Microbiol. Biotechnol. 2005, 32, 364–370. (15) Jacoby, W. A.; Maness, P. C.; Wolfrum, E. J.; Blake, D. M.; Fennel, J. A. Mineralization of Bacterial Cell Mass on a Photocatalytic Surface in Air. Environ. Sci. Technol. 1998, 32, 2650–2653. (16) Vohra, A.; Goswami, D. Y.; Deshpande, D. A.; Block, S. S. Enhanced photocatalytic disinfection of indoor air. Appl. Catal., B 2006, 65, 57–65. (17) Pal, A.; Pehkonen, S. O.; Yu, L. E.; Ray, M. B. Photocatalytic inactivation of Gram-positive and Gram-negative bacteria using fluorescent light. J. Photochem. Photobiol. A. 2007, 186, 335– 341.

5126

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010

(18) Kozlov, D. V.; Vorontsov, A. V. Sulphuric acid and Pt treatment of the photocatalytically active titanium dioxide. J. Catal. 2008, 258, 87–94. (19) Chen, F.; Yang, X.; Xu, F.; Wu, Q.; Zhang, Y. Correlation of photocatalytic bactericidal effect and organic matter degradation of TiO2 Part I: observation of phenomena. Environ. Sci. Technol. 2009, 43, 1180–1184. (20) Kozlova, E. A.; Vorontsov, A. V. Noble metal and sulfuric acid modified TiO2 photocatalysts: Mineralization of organophosphorous compounds. Appl. Catal., B 2006, 63, 114–123. (21) Ashmarin, I. P., Vorobyov, A. A., Eds. Statistical Methods in Microbiological Studies; Medgiz: Leningrad, U.S.S.R., 1962; in Russian. (22) Sattar, S. A.; Ijaz, M. K.; Johnson-Lussenburg, C. M.; Springthorpe, V. S. Effect of relative humidity on the airborne survival of rotavirus SA11. Appl. Environ. Microbiol. 1984, 47, 879–881. (23) Ijaz, M. K.; Sattar, S. A.; Johnson-Lussenburg, C. M.; Springthorpe, V. S. Comparison of the airborne survival of calf rotavirus and poliovirus type 1 (Sabin) aerosolized as a mixture. Appl. Environ. Microbiol. 1985, 49, 289–293. (24) Rechmenskii, S. S. Essays of Experimental Aerobiology; Medicine: Moscow, 1973; in Russian. (25) Kuhn, K.; Chaberny, I. F.; Massholder, K.; Stickler, M.; Benz, V. W.; Sonntag, H.-G.; Erdinger, L. Disinfection of surfaces by photocatalytic oxidation with titanium dioxide and UVA light. Chemosphere. 2003, 53, 71–77. (26) Grinshpun, S. A.; Adhikari, A.; Honda, T.; Kim, K. Y.; Toivola, M.; Rao, K. S. R.; Reponen, T. Control of aerosol contaminants in indoor air: combining the particle concentration reduction with microbial inactivation. Environ. Sci. Technol. 2007, 41, 606– 612. (27) Rincon, A. G.; Pulgarin, C.; Adler, N.; Peringer, P. Interaction between E. coli inactivation and DBP-precursors s dihydroxybenzene isomers s in the photocatalytic process of drinkingwater disinfection with TiO2. J. Photochem. Photobiol. A. 2001, 139, 233–241. (28) Lu, Z.-X.; Zhou, L.; Zhang, Z.-L.; Shi, W.-L.; Xie, Z.-X.; Xie, H.-Y.; Pang, D.-W.; Shen, P. Cell Damage Induced by Photocatalysis of TiO2 Thin Films. Langmuir 2003, 19, 8765–8768. (29) Sunada, K.; Kikuchi, Y.; Hashimoto, K.; Fujishima, A. Bactericidal and Detoxification Effects of TiO2 Thin Film Photocatalysts. Environ. Sci. Technol. 1998, 32, 726–728. (30) Huang, Z.; Maness, P.-C.; Blake, D. M.; Wolfrum, E. J.; Smolinski, S. L.; Jacoby, W. A. Bactericidal mode of titanium dioxide photocatalysis. J. Photochem. Photobiol. A. 2000, 130, 163–170. (31) Kozlova, E. A.; Smirniotis, P. G.; Vorontsov, A. V. Comparative study on photocatalytic oxidation of four organophosphorus simulants of chemical warfare agents in aqueous suspension of titanium dioxide. J. Photochem. Photobiol. A. 2004, 162, 503– 511. (32) Nadtochenko, V.; Denisov, N.; Sarkisov, O.; Gumy, D.; Kiwi, J. Laser kinetic spectroscopy of the interfacial charge transfer between membrane cell walls of E coli and TiO2. J. Photochem. Photobiol. A. 2006, 181, 401–407. (33) Nadtochenko, V. A.; Sarkisov, O. M.; Nikandrov, V. V.; Chubukov, P. A.; Denisov, N. N. Inactivation of pathogenic microorganisms in the photocatalytic process on nanosized TiO2 crystals. Russ. J. Phys. Chem. 2008, 2 (1), 105–114. (34) Fagerbakke, K. M.; Heldal, M.; Norland, S. Content of carbon, nitrogen, oxygen, sulfur and phosphorus in native aquatic and cultured bacteria. Aquatic Microbial. Ecology 1996, 10, 15–27. (35) Bratbak, G.; Dundas, I. Bacterial dry matter content and biomass estimation. Appl. Environ. Microbiol. 1984, 48 (4), 755–757.

ES100156P