Disinfection of Bacillus spp. spores in drinking water by TiO 2. photocatalysis as a model for Bacillus anthracis

Size: px

Start display at page:

Download "Disinfection of Bacillus spp. spores in drinking water by TiO 2. photocatalysis as a model for Bacillus anthracis"

Marybeth Murphy
6 years ago
Views:

1 Disinfection of Bacillus spp. spores in drinking water by TiO 2 photocatalysis as a model for Bacillus anthracis R. Armon*, G. Weltch-Cohen* and P. Bettane** *Faculty of Civil Engineering, Environmental & Water Resources Engineering, Technion, Haifa, Israel **IDF- Medical Corp., Tel-Hashomer, Tel-Aviv, Israel Abstract During the last decade the use of titanium dioxide has been the focus of water purification studies for photocatalytic degradation of organic compounds. Various studies have shown that TiO 2 photocatalysis is a very efficient process for removal (by mineralization) of a large variety of hazardous chemicals. However, the potential use of this technology for water disinfection has been essentially unexplored. Only a few papers have described the photocatalytic destruction of microbial cells, such as E. coli bacteria, MS2 bacteriophages and recently B. fragilis phages, D. radiophilus. The mechanism of photocatalysis in the presence of TiO 2 is the enhanced formation of hydroxyl (HO ) radicals, active in oxidation processes. The HO radicals have a significant effect on chemical oxidation of anthropogenic organic compounds in the environment. Complete mineralization of many organic substances is possible in aqueous systems, when sufficient HO radical flux can be generated in situ. Various water treatment technologies inherently produce HO radicals in relatively minuscule quantities (i.e., < M). Examples of such processes include ozonation, direct photolysis of hydrogen peroxide, and radiolysis. In contrast to the above system, steadystate HO concentrations of the order of 10 9 M can be generated in UV-irradiated aqueous suspensions of immobilised particles of titanium dioxide. The use of TiO 2 for microbial inactivation and disinfection of potable water is suggested, since free radicals such as HO might act as a strong biocide, because of its high oxidation potential and nonselective reactivity. In the present study, two bacilli strain spores (B. subtilis and B. cereus) were tested for photocatalytic inactivation in water as simulators of B. anthracis spores. B. subtilis was selected for its high resistance to disinfection and B. cereus for its phylogenetic proximity to B. anthracis. Two UV sources were used: 1) monochromatic UV lamp with irradiation intensity of 7mW/cm 2 at 365nm; and 2) Natural sunlight (irradiation intensity at 365nm of ~4 mw/cm 2 between 12:00 and 14:00 hours). TiO 2 at 0.25g/L was found to be the optimal concentration needed for the reduction of four orders of magnitude in B. subtilis spores viability after irradiation for 300 minutes. B. cereus subjected to similar photocatalysis conditions was reduced by five orders of magnitude revealing lower endurance to this process. Comparison of artificial and natural (sunlight) UV irradiation source on B. subtilis resulted in increased inactivation of 5 orders of magnitude in favour of sunlight. Combined inactivation by photocatalytic process (UV 365nm) and detrimental activity of UV at 265nm can explain this result. There was no difference between the two irradiation sources when B. cereus was tested. Under both irradiation types, B. cereus was reduced by four orders of magnitude during 300 minutes time interval. Additional experiments including TiO 2 concentration, irradiation intensity, water depth, initial spore number, etc. were performed. Taking into account that B. anthracis spores have hydrophobic properties, the photocatalytic process seems to be the method of choice in water disinfection eliminating the possibility of by products formation such as halogens. Keywords Bacillus anthracis modelling; bacillus spp spores; disinfection; photocatalysis; titanium dioxide Introduction During the last decade the use of titanium dioxide has been the focus of water purification studies for photocatalytic degradation of organic compounds (Al Ekabi and Serpone, 1988). Various studies have shown that TiO 2 photocatalysis is a very efficient process for removal (by mineralization) of a large variety of hazardous chemicals (Al Ekabi and Serpone, 1988; Ollis et al., 1991; Kormann et al., 1991). However, the potential use of this technology for water disinfection has been essentially unexplored. Only a few papers described the photocatalytic destruction of microbial cells such as E. coli bacteria (Ireland, et al., 1993), coliforms (Watts et al., 1997), MS2 Water Science and Technology: Water Supply Vol 4 No 2 pp 7 14 IWA Publishing

2 8 bacteriophages (Sjogren and Sierka, 1994), Pseudomonas (Biguzzi and Shama, 1994) and recently B. fragilis phages, D.radiophilus (Armon et al., 1997, 1999). The mechanism of photocatalysis in the presence of TiO 2 is the enhanced formation of HO radicals, active in oxidation processes. The hydroxyl radicals (HO ) have a significant effect on chemical oxidation of anthropogenic organic compounds in the environment (Al-Ekabi and Serpone, 1988; Buxton and Greenstock, 1988; Haag and Hoigne, 1985). Complete mineralization of many organic substances is possible in aqueous systems, when sufficient HO radicals flux can be generated in situ (Ollis et al., 1991; Prairie et al., 1993). Various water treatment technologies inherently produce HO radicals in relatively minuscule quantities (i.e., <10 12 M) (Haag and Hoigne, 1985). Examples of such processes include ozonation, direct photolysis of hydrogen peroxide, and radiolysis. In contrast to the above system, steadystate HO concentrations of the order of 10 9 M can be generated in UV-irradiated aqueous suspensions of immobilised particles of titanium dioxide (Sjogren and Sierka, 1994; Kormann et al., 1988; Teichner and Formenti. 1985). The use of TiO 2 for microbial inactivation and disinfection of potable water, is suggested, since free radicals such as HO might act as a strong biocide, because of its high oxidation potential and nonselective reactivity (Ireland et al., 1993; Prairie et al., 1993). TiO 2 in the anatase crystalline form behaves as a classical semiconductor (Ireland et al., 1993). Illumination of TiO 2 on easy dispersions by light with wavelength of less than 400 nm generates excess electrons in the conduction band (e cb ) and positive holes in the valence band (h+ vb ). The following equation describes this phenomenon: TiO 2 + hv = e cb + h+ vb. H 2 O molecules or OH groups adsorbed on TiO 2 particle surface react to form HO radicals according to the following equation: h + vb + H 2O(ads) = HO + H+ or h + vb + OH (sur) = HO Excess electrons in the conduction band react with molecular oxygen to form superoxide ions, e cb + O 2 = O 2 which further disproportionate to form more HO radicals: 2O H 2 O = 2 HO +2OH + O 2 (Ireland et al., 1993) Studies dealing with bacterial inactivation, clearly showed that in the absence of significant amounts of inorganic radical scavengers, successful microorganisms inactivation by TiO 2 illuminated at the far UV light could be promoted (Ireland et al., 1993). Sporeforming bacteria of Bacillus strains were also investigated for photocatalytic disinfection with good results (Pham et al., 1995). The present study is the first attempt to inactivate photocatalytically spores of B. subtilis and B. cereus as a model for the main biological warfare element: B. anthracis (Inglesby, 1999). The spore-forming Bacillus cereus is genetically very closely related to B. anthracis whereas Bacillus subtilis is highly resistant to a variety of stresses. Materials and methods Experimental set Figure 1 outlines the components of the experimental set. Briefly, an UV lamp 7 mw cm 2 (intensity) (VL-215.BLB, 15 W) equipped with No. 254 nm emission filter was held above a 250 ml plastic container at different distances (cm). The plastic container was placed on

3 UV lamp Experimental container Magnetic stirrer Figure 1 The experimental set for UV photocatalytic inactivation of microorganisms with TiO 2 top of a magnetic stirrer. Heat up of the container content was prevented by an isolation layer in between the stirrer and the container. UV intensity was periodically measured with a LongWave ultraviolet Measuring Meter (Model J-221, UVP Ltd., Upland, CA., USA). Drinking water (our laboratory) was previously dechlorinated and filter sterilized. Bacillus spp. spores Spores were introduced in a final volume of 100 ml water at spores/ml (B. subtilis) and spores/ml (B. cereus) respectively. Ampoules of 1 ml stock containing B. subtilis (ATCC 11778) or B. cereus (ATCC 6633) spores were obtained from Difco Co. (USA). Original concentrations were B. subtilis spores/ml and B. cereus spores/ml. Spore enumeration Survival fraction was enumerated by direct or dilution plating on R2A agar following membrane filtration (0.45 mm porosity size) (APHA, 1992). Agar plates were incubated at 36 C for 24 h. Colonies formed from vegetative cells were enumerated in duplicates. TiO 2 The best anatase preparation of TiO 2 (P-25, Degussa Huels AG Co.) were used in the present study. TiO 2 P-25 was a present from Degussa Huels AG Company (Frankfurt, Germany) and its distributors in Israel. Experimental procedure Water containing different concentrations of TiO 2 was inoculated with various concentrations of spores and after 5 minutes of initial mixing, the content was irradiated with a black UV lamp emitting 3.4 mw/cm 2 light intensity at 360 nm. Lamp distance from the water surface controlled the irradiation intensity as measured by longwave ultraviolet measuring meter. Results Previous results showed that the optimal concentration of titanium dioxide (in powder form) to be used in water photocatalysis is 1 g/l (Robert). However, owing to spores high resistance to disinfection by other means (i.e. chlorination) several TiO 2 concentration were tested for inactivation in suspension of B. subtilis spores (Figure 2). The results obtained revealed a different pattern of inactivation as shown in Figure 2. After 120 minutes from experimental onset no reduction in spore viability was observed. After this period a sigmoid reduction curve was observed, typically to microorganism inactivation (like chlorine, H 2 O 2, etc.). At 0.25 and 1 g/l TiO 2 the reduction of spore viability was >4 orders 9

4 10 7 Viable count of spores/ml 0 g/l 0.25 g/l 1 g/l 3 g/l Irradiation Time (min) Figure 2 Photocatalytic inactivation of B. subtilis spores at different TiO 2 concentrations (0.25, 1 and 3 g/l) of magnitude while at 3 g/l the reduction was >3 orders of magnitude. As a result of these experiments it was decided to continue photocatalysis in water suspensions at 0.25 g/l titanium dioxide concentration. It showed the best results repetitively and also is cost saving. Performing photocatalysis in a country with natural sunlight for the most of the year is a Table 1 Sunlight irradiation intensity at 360 nm as a function of day hour (measured at Technion, Haifa, Israel) Day hour Irradiation intensity at 360 nm (mw/cm 2 ) 10: : : : : : : : mw/cm 2 (control) 1 mw/cm mw/cm 2 7 mw/cm Time (min) Figure 3 Photocatalytic inactivation of B. subtilis spores by various irradiation intensities (1, 3.4 and 7 mw/cm 2 ) at 0.25 g/l TiO 2

5 0.25 g/l 0 g/l (control) Irradiation time (min) Figure 4 Photocatalytic inactivation of B. cereus spores as a function of time (TiO g/l and 3.4 mw/cm 2 UV 360 nm irradiation) major advantage. In our case, Israel has enough sunlight for most of the year with very high irradiation during the summer and auatumn. Table 1 shows the UV irradiation at 360 nm as measured in Israel during a typical June day. As shown, the peak irradiation is at midday (3.9 mw/cm 2 ) while the high irradiation intensity is approximately 6 hours long. To test the influence of irradiation intensity, a standard experimental UV lamp (360 nm wavelength) was placed at different distances from the spore s suspension in order to achieve various intensities. Figure 3 shows three irradiation intensities applied onto B. subtilis spores mixed with TiO 2 : 1, 3.4 and 7 mw/cm 2. The inactivation results clearly show that increased irradiation intensity does not particularly increase the inactivation rate. The results from the 3.4 and 7 mw/cm 2 intensities were almost similar as can be seen in Figure 3. At this point it can be realized that sunlight (at approximately the same irradiation intensity of 3.4 mw/cm 2 ) can be efficiently utilized as a natural source of energy. In the present study, B. cereus spores were selected as a model for B. anthracis due to B. cereus B. subtilis Irradiation time (min) Figure 5 Comparison of photocatalytic inactivation of two Bacillus strains in water: B. subtilis and B. cereus (TiO g/l and 3.4 mw/cm 2 UV 360 nm irradiation) 11

6 10 7 Sunlight UV lamp Irradiation time (min) Figure 6 Photocatalytic inactivation of B. subtilis spores by two irradiation sources: sunlight and UV lamp (TiO g/l and 3.4 mw/cm 2 UV 360 nm irradiation) sunlight UV lamp Irradiation time (min) Figure 7 Photocatalytic inactivation of B. cereus spores by two irradiation sources: sunlight and UV lamp (TiO g/l and 3.4 mw/cm 2 UV 360 nm irradiation) 12 their philogenetic closeness. Figure 4 shows B. cereus spores inactivation under standard conditions (as performed with B. subtilis). The reduction in viable spores was the same order but faster compared with B. subtilis. The lag period of no inactivation was shorter (60 min.) This picture is better emphasised in Figure 5 where B. subtilis was compared with B. cereus. Clearly, B. cereus is inactivated faster and by one order of magnitude higher. These results are encouraging because of the similarity of B. anthracis to B. cereus. As already mentioned above, Israel has sunlight for long periods of time and almost all seasons. Figure 6 represents a comparison of two irradiation sources: a laboratory UV lamp and direct sunlight. Sunlight is more effective by shortening the lag period to less than 80 minutes and the reduction in spore viability reached > 5 logs. The same experiment was performed with B. cereus (Figure 7). In this case the viability reduction was similar, as well the lag period.

7 Conclusions Photocatalytic inactivation of microorganisms by TiO 2 is an efficient process for disinfection of water. It also has an additional advantage that most disinfectants do not have: it can mineralise a large variety of pollutants found in water. From our experience the engineering aspects have to be studied and further developed to prevent TiO 2 waste (though its price is relatively low: 23 DM /kg). As a whole we can state the following: 1. The optimal TiO 2 concentration for efficient inactivation of B. subtilis and B. cereus spores in water is 0.25 g/l. 2. The irradiation intensity of 3-4 mw/cm 2 is sufficient to inactivate spores. This is the average intensity of sunlight in Israel and other Mediterranean countries. 3. B. cereus is genetically closely related to B. anthracis and showed a pattern of very good inactivation. B. subtilis was used as a model of more resistant spores and revealed that photocatalysis with TiO 2 can be a very good means for water disinfection in case of biological warfare, with fewer side effects compared with formaldehyde (Manchee et al., 1994). 4. Sunlight is an efficient means to photocatalyse spores due to its irradiation at 360 nm, but also for its 254 nm germicidal irradiation that combined with the first proved to be lethal to spores. From previous results (data not shown) B. subtilis spores as well B. cereus were not inactivated by direct sunlight. 5. Finally the TiO 2 concentration of 0.25 g/l proved to be as efficient or more so compared with 1 g/l usually used by others for photocatalysis of vegetative bacterial cells. It is known that above certain concentrations a shadowing effect will occur which obstructs the irradiation intensity. 6. B. anthracis spores were reported to be hydrophobic by nature, therefore their inactivation in water on the upper layer with TiO 2 is an easy task. Acknowledgements The authors thank the distributors of Degussa Huels in Israel for supplying TiO 2 as a gift for this study; The Israel Defence Forces, Medical Corp., and The Water Research Institute, Technion, for partial funding of the present project. References Al-Ekabi H., and Serpone, N. (1988). Kinetic studies in heterogeneous photocatalysis. 1. Photocatalytic degradation of chlorinated phenols in aerated aqueous solutions over TiO 2 supported on a glass matrix. J. Phys. Chem. 92, APHA (1992). Standard Methods for the Examination of Water and Wastewater, 18th edition. American Public Health Association, Washington, DC. Armon, R., Laot, N., Narkis, N. and Neeman, I. (1997). Photocatalytic inactivation of different bacteria and bacteriophages in drinking water at fifferent TiO 2 concentration with or without exposure to O 2. J. Adv. Oxid. 3(2), Biguzzi, M. and Shama, G. (1994). Effect of titanium dioxide concentration on the survival of Pseudomonas stutzeri during irradiation with near ultraviolet light. Lett. Appl. Microbiol. 19, Buxton, G.V. and Greenstock, C.L. (1988). Critical review of rate constants for reaction of hydrated electrons, hydrogen atoms and hydroxyl radicals (OH/O) in aqueous solutions. J. Phys. Chem. Ref. Data 17, Haag,W.R., and Hoigne, J. (1985). Photo-sensitised oxidation in natural water via OH radicals. Chem. 14, Inglesby, T.V. (1999). Anthrax as a biological weapon. J. Am. Med. Assoc. 281, Ireland J.C. et al. (1993). Inactivation E. coli by TiO 2 photocatalytic oxidation. Applied and environmental microbiology, 59, Kormann, C., Bahnemann, D.W. and Hoffman, M.R. (1988). Peroxide production on illuminated suspensions of TiO 2, ZnO and desert sands. Environ. Sci. Technol. 22, 798. Kormann, C., Bahnemann, D.W. and Hoffman, M.R. (1991). Photolysis of chloroform and other organic molecules in aqueous TiO 2 suspensions. Environ. Sci. Technol. 25,

8 Laot, N., Narkis, N., Neeman, I., Bilanoviç, D. and Armon, R. (1999). TiO 2 photocatalytic inactivation of selected microorganisms under various conditions: sunlight, intermittent and variable irradiation intensity, CdS augmentation and entrapment of TiO2 into sol-gel. J. Adv. Oxid Manchee, R.J, Broster, M.G., Stagg, A.J., Hibbs, S.E. (1994). Formaldehyde solution effectively inactivates spores of Bacillus anthracis on the Scottish Island of Gruinard. Appl. Environ. Microbiol. 60, Ollis, D.F., Pelizzetti, E. and Serpone, N. (1991). Photocatalyzed destruction of water contaminants. Environ. Sci. Technol. 25, Pham, H.N., McDowell, T., Wilkins, E. (1995). Photocatalytically-mediated disinfection of water using TiO2 as a catalyst and spore-forming Bacillus pumilus as a model. J. Environ. Sci. and Health A30(3), Prairie, M.R., Evans, L.R., Stange, B.M., Martinez, S.L. (1993). An investigation of titanium dioxide photocatalysis for the treatment of water contaminated with metals and organic chemicals. Environ. Sci. and Technol., 27, Sjogren J.C, and Sierka. R.A. (1994). Inactivation of phage MS2 by iron-aided TiO 2 photocatalysis. Applied and Environmental Microbiology, 60, Teichner, S.J. and Formenti, M. (1985). Heterogenous photocatalysis. In: Photoelectrochemistry, Photocatalysis and Photoreactors (Schiavello M. ed.), pp Dordrecht: D. Reidel. Watts, R.J., Kong, S., Orr, M.P., Miller, G.C. and Henry, B.E. (1995). Photocatalytic inactivation of coliform bacteria and viruses in secondary wastewater effluent. Water Res. 29,

Chapter - III THEORETICAL CONCEPTS. AOPs are promising methods for the remediation of wastewaters containing

Chapter - III THEORETICAL CONCEPTS 3.1 Advanced Oxidation Processes AOPs are promising methods for the remediation of wastewaters containing recalcitrant organic compounds such as pesticides, surfactants,