Environ. Sci. Techno/. 1995, 29, 501-505
TiOt-Mediated Photochemical Disinfection of Escherichia coli Using Optical Fibers TADASHI M A T S U N A G A * A N D MINA OKOCHI Department of Biotechnology, Tokyo University of Agriculture and Technolog, Koganei, Tokyo 184, Japan
W e have constructed a system for photochemical disinfection of bacteria using a suspension of titanium dioxide and diffuse-light emitting optical fibers (DLEOFs). Disinfection of bacteria at high Ti02 concentrations was light-limited when conventional optical fibers were used to supply light to the cell suspension. However, when using DLEOFs that emit light laterally from their surface, light distribution throughout a suspension of Escherichia coliwas greatly improved even in the presence of high Ti02 concentrations. A 4-fold increase in photochemical disinfection was obtained at high Ti02 concentrations using DLEOFs rather than conventional optical fibers for light supply. The use of DLEOFs should provide a generally applicable method for photodisinfection of water supplies.
Introduction Irradiation of semiconductor materials with light causes the formation of electron-hole pairs (e-, h+), and the reaction of these carriers with species in solution leads to the products of redox reactions (1). n-Type semiconductor powders such as TiOz have been used for photochemical oxidation of acetic acid (2),cyanide (3, 4 , hydrocarbons (a,sulfite (31,and water (6).Research into the application of Ti02 to oxidize chemical compounds in water has also expanded (7-9). We have reported previously that Saccharomyces cerevisiae, Escherichia coli, Lactobacillus acidophilus, and Chlorella vulgaris were adversely affected using photoexcited semiconductor particles (10). In this case, photoelectrochemical oxidation of bacterial cells caused a decrease in respiratoryactivityand cell death (10). We have also described the construction of a continuous sterilization system employing semiconductor particles immobilized on an acetylcellulose membrane (11). Disinfection using TiOzhas also been applied to the inactivation of phage MS2 (12) and to the disinfection of bacteria in pond water (13). Several reports have also been published on the biological applications of semiconductor particles (14-16). Chlorine is generally used to disinfect tap water, but the generation of carcinogenic substances such as trihalomethanes has become a problem. Ti02-mediated disinfection can also be applied to the decomposition of such carcinogenic substances. In the present study, diffuse-lightemitting optical fibers (DLEOFs)that emit light laterally from their surface were used to improve light supply in the reactor. DLEOFs may be used to supply light more efficiently than other methods since loss of light energy is reduced. We have investigated operating conditions for efficient disinfection using semiconductor particles and optical fibers. The optimum illumination time, light intensity, and Ti02 concentration were determined. In addition, disinfection of E. coli by Ti02 particles using either conventional optical fibers or DLEOFs was compared. This work forms the basis for developing an environmentally clean water purification process that can use solar energy.
Materials and Methods Preparationof Bacteria and SemiconductorSuspensions. A total of 25 mg of titanium dioxide particles (anatase type, Aerosil P-25, Nippon Aerosil Ltd, Tokyo, Japan) was suspended in 100 mL of 0.1 M phosphate buffer (pH 7.01, sterilized by autoclaving, and homogeneously suspended by sonication. TiOzsuspension was diluted to the required concentration. E. coli strain LE 392 (ATCC39713) was used as a model microorganism for disinfection studies. E. coli cells were cultured aerobically at 37 "C for 12 h in 10 mL of Luria-Bertani(LB) medium (pH 7.0) containing 1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl after preculture for 12 h under the same conditions. Cells were centrifuged at 1700g for 10 min, washed three times, and resuspended in 0.1 M phosphate buffer (pH 7.0). Cell concentration was determined using a hemocytometer. Cell density was adjusted to the required final concentration * Corresponding author; direct fax: +81-423-85-7713; telephone: +81-423-88-7020; e-mail address:
[email protected].
0013-936x/5/092900501$09.00/0 0 1995 American Chemical Society
VOL. 29, NO. 2, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
501
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(B) FIGURE 1. Schematic diagram of experiment ~pparatusforTiOrmndi*sdphotochemical disinfection of optical fibers (A) and conventional optical fibers (B).
(103-107 ceUs/mL), and cell suspensions were added to the Ti02 suspension. After being mixed for 30 s, samples (1mL) were injected into a glass micro tube (diameter; 6 mm x 50 mm). Preparaton of DLEOFs. DLEOFs consisting of a poly(methyl methacrylate) (PMMA) core and a sheath of fluororesin (1mm in diameter, 500 mm length; Mitsubishi Rayon Co. Ltd., Tokyo, Japan) were prepared by removing thefluororesinwithethylacetateandrougheningthe surface of a 20-mm length of one end of the optical fiber. Light intensity at the DLEOF surface (quantum flux density, mEinstein m-z s-’) was measured using a photometer (MinoltaModel T-1M) connected to an integrating sphere (Optel Ltd., Tokyo, Japan). The integrating sphere has a conversion coefficient K, determined using a calibration methatconvertsmeasuredlightintensityOx) toluminous flux(cdsr). The conversioncoefficient Kwas 0.003 686 925 where measured light intensity x K = luminous flux (14). To measure the diffise-light emitting efficiency of a DLEOF, an integrating sphere was used to determine the quantity of light emitted from the roughened surface of the fiber (emitted diffuse light) and the total amount of light emitted from the fiber (output light) for a single DLEOF. Total output light was measured by insertingthe roughened part ofthe DLEOFintotheintegratingsphere.Light emitted from the end of the DLEOF was also measured by just inserting the end of the fiber into the integrating sphere. The calculated emitted diffuse light is the theoretical amount of light emitted from the roughened surface of the fiber. Calculated emitted diffuse light = (total output light - light emitted from the end of DLEOF). A photometer was used to measure light emitted from the surface of the DLEOFs. DLEOFs were placed on a black surface, and the photometer was placed in contact with the DLEOFs for light intensity measurement. Msinfeaion heedwe. The disinfection experiment was started by irradiating liquid in a glass microtube from 602 m ENVIRONMENTAL SCIENCE &TECHNOLOGY I VOL. 29, NO. 2,1995
Foliusing dtfusn-light emitting
above with light from a 300-Wxenon lamp (wavelength; 400-700 nm) using a conventional optical fiber bundle. When using DLEOFs, the fiber bundle (each DLEOF was arranged on the 2-mm center) was immersed in the suspension. Figure 1 shows a schematic diagram of the experimental apparatus. The reaction vessel (glass microtube) was covered with aluminum foil to prevent loss oflight energy. The temperature ofthe reactionvessel was kept at 25 “C using a water-jacket throughout the experiment. Thesampleswerestirredat650rpmwithamagnetic stirrer to reduce settling of Ti02 panicles. Measurement of Viable Cells. The number of viable cells in the sample was determined by plating appropriate dilutions of the sample onto LB agar plates and counting colonies that appeared after 24-h incubation at 37 “C.The numbers of required replicate plates were as follows. At an initial cell concentration of 2 x lo3 and 10‘ cellslml, one replicate plate was used. At lo5and lo6 cells/mL, two replicate plates were used. At lo7cellslml, three replicate plates were used.
Results and Discussion TiOr-Mediated Disinfection Using Conventional Optical Fibers. The decrease in E. coli viable cell concentration was investigated when cell suspensionswere incubated in the presence of TiOz panicles (0.4 PglrnL). E. coli viable cell concentration decreased gradually with illumination time when using conventional optical fibers for illumination,anddisinfectionwasalmostcompleteafter2 h (Figure 2). A decrease in cell concentration was not observed (suMvalratio was greater than 90%)under light irradiation when semiconductor particles were absent or when cells were incubated with Ti02 suspension in the dark. Disinfection experimentswere performed in duplicate and were reproducible with an average relative error of 10%. These results confirm that the presence of photoexcited Ti02 particles disinfect E. coli cells and that nonirradiated TiOz
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Time (min) FIGURE2. Time course of E colicell concentrationon Ti02-mediated disinfection using conventional opticel fibers for light supply. Samples were incubated with (M) and without (0) Ti02 particles (0.4 pg/mL) under light irradiation (Sa mEinstein m-z s-').
particles are not toxic to the cells. After disinfection, the suspension containing disinfected cellswas filteredthrough a 0.45 pm membrane filter. The filtrate was added to a fresh suspension of E. coli cells (withoutTiOz)and incubated for 15 min (data not shown). No decrease in viable cell number was observed. This suggests that residual toxic substances were not formed during disinfection. The irradiation time required for disinfection of E. coli cells of 2 x lo3 cellslmL by photoexcited Ti02 particles using conventional optical fiber is 2 h. The effect of light intensity on disinfection of E. coli was also investigated. The initial cell concentration was adjusted to 2 x lo3 cellsImL, and the TiOzconcentration was 0.4pglmL. When the light intensitywas above40 mEinstein m-2 s-l ,survivalratio decreased as light intensityincreased. The survival ratio of E. coli was 1%at 60 mEinstein m-2 s-l (data not shown). Therefore subsequent experimentswere performed at intensities above 60 mEinstein m-2 s-l. Effect of Ti02 Concentration on E. coli Disinfection Using Conventional Optical Fibers. The effect of TiOz concentration on survival ratio of E. coli cells is shown in Figure 3. When the concentration of Ti02 was above pglmL, the survivalratio decreased sharplywith increasing concentration. The minimum survival ratio was obtained at 2.5 pglmL Ti02 concentration. However, when the concentration of TiOz was above 2.5 pgImL, the survival ratio increased rapidly with increasingTi02 concentration. This may be due to a decrease in light transmission caused by the increase in TiOz concentration. If the light transmission decreased, photoexcited Ti02 particles might be present only in the light-irradiated surface of the liquid. Light Intensity as a Function of Depth from the Surface at Various Ti02 Concentrations. Differentconcentrations of TiOz (2.5, 25, and 250 pglmL) were used (Figure 4). A submersible photosensor was used to measure the light intensity at various depths. The light intensity decreased sharply with increasing depth. At a Ti02 concentration of 250pglmL, the light intensitywas reduced due to a shading effect. Therefore, reduced E. coli disinfection at high TiOz density appears to be due to reduced light transmission. Light Efllciencyand Intensity When Using DLEOFs for Light Supply. To improve light distribution, we supplied
Ti02 concentration ( pg / ml ) FIGURE 3. Effect of Ti02 concentration on E. colisurvival ratio using conventional optical fibers. A cell suspension containing 2 x 19 cells/ml was incubated for 2 h under light irradiation(100mEinstein m-2 s-0 (M) and dark condition (0).
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Distance (cm) FIGURE 4. Light intensity as a function of depth from the surface at Ti02 concentrations of 2.5 (O), 25 (A), end 250 pg/mL (0).
light using DLEOFs that emit light laterallyfrom their surface (17, IS). When using DLEOFs, the amount of input light was the same as when using conventional optical fibers. The diffuse-lightemittingefficiencyof a DLEOF was defined as (directly measured emitted diffuse light)I (calculated emitted diffuse light). Calculated emitted diffuse light = total output light - light emittedfromthe end of the DLEOF (Table 1). The average total output light was 13.4cd sr, and the average output light from the end of a DLEOF was 0.78 cd sr. Calculated emitted diffuse light was 12.6 cd sr. Therefore, 94% of total diffuse light energy was emitted evenly from the DLEOF surface. The experimentally measured quantity of light emitted from the DLEOF surface was 12.5 cd sr. The diffuse-light emitting efficiency was calculated as 99%, and all the light was emitted from the DLEOF surface and was not converted into heat. The light intensity at the fiber surface was almost constant when DLEOFs were used. The light intensity at the surface of the fiber was 1.6 mEinstein mW2s-l. Total output light was 112 cd sr. These data were obtained when VOL. 29, NO. 2, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY 1 SO3
TABLE 1
100
Diffuse4ight Emitting Efficiency of DLEOF" diff use-light luminous emitting flux (cd sr) efficiency (YO)
light
total output light output light from DLEOF end calculated emitted diffuse light experimentally measured emitted diffuse light a
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Cell concentration (cells / ml) FIGURE 6. Effect of cell concentration on E. colisuwival ratio. light was supplied for 2 h at Ti02 concentlstion of 250 &mL The same amount of light (110 cd sr) was supplied from a xenon lamp when using DLEOFs and conventional optical fibers. (0)light irradiation by DLEOFs. light intensity at the surface of the DLEOFs (surface araa; 382 I&) was 4.9 mEinstein n r 2 s-l. (0)light irradiation by conventional optical fibers. LigM intensity at the surface of the suspension was 67 mEinstein s-l. 1
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Ti02 concentration ( pg / ml ) FIGURE 5. Effect of Ti02 concentration on €. colisuwival ratio using diffuse-light emitting optical fiber. A cell suspension containing 1 x 103 calls/ml was incubated for 2 h under ligM irradiation (5.4 mEinstein rn-z s-l, surface area was 509 mm2)( 0 )and in the dark condition (0).
the photometer was placed directly on the surface of 16 fibers in air. To measure light intensity in suspensions of E. coli (2 x lo3cellslml) and Ti02 (250pglmL),the detection part of the photometer was wrapped with a transparent vinyl chloride sheet and immersed in the suspension. The suspension containing E. coli and Ti02 was stirred at 650 rpm. Wrapping the detection part of the photometer reduced the light intensityreceivedby lo%, so the measured light intensity in air was 1.4 d i n s t e i n mT2sT1. Measured light intensity at the fiber surface was 1.3 mEinstein m-2 s-l in the presence of a suspension of E. coli and Ti02. In the disinfection reactor using DLEOF, optical fibers were arranged at 2-mm centers, and minimum light intensity was assumed to occur 0.5 mm from the surfaceof a DLEOF. The light intensity 0.5 mm from the DLEOF in a suspension of TiOn and E. coli was 1.1 mEinstein m-2 s-l. Thus, the minimum light intensity experienced by E. coli cells remained high. Using DLEOFs, decreased light transmission due to high density Ti02 in the solution could be prevented. Therefore,the decrease in disinfectionefficiency caused by localizationof photoexcited Ti02 particles could be overcome. Effect of Ti02 Concentration on E. coli Disinfection Using DLEOFs. The effect of Ti02 concentration on the survival ratio of E. coli after light irradiation using DLEOFs is shown in Figure 5. The same amount of light was introduced when the light intensity of 100 mEinstein m-2 s-l was obtained by using conventional optical fibers. 504
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 2.1995
Survival ratio decreased as TiOz concentration increased. However, above a TiOz concentration of 2.5 pglmL, an increase in survival ratio was not observed. The survival ratio was 18%at a Ti02 concentration of 250 pglmL when light was supplied using DLEOF as compared to 81%when conventionalopticalfiberwas used. Consequently,a 4-fold increase in photochemical disinfectionwas obtained using DLEOF at high Ti02 concentration. Light supply appears to be the rate-limiting step for disinfection at high Ti02 concentrations. Therefore,it is possible to kiU bacteria more efficiently at high Ti02 concentrations using DLEOF. Effect of Initial Cell Concentration on Survival Ratio. The effect of cell concentration on the E. coli survival ratio using conventional optical fibers and DLEOFs is shown in Figure 6. Using conventional optical fibers, very little disinfectionwas observed at TiOzconcentration of 250pgl mL containing E. coli suspension of lo6cellslml. Survival ratio was above 80% at the initial E. coli concentration of 2 x lo3cellslmL to lo7cellslmL when conventional optical fibers were used for light supply. On the other hand, 40% of E. coli cells were disinfected even at a cell concentration of lo6cellslmLwhen DLEOFs were used. Disinfectionusing W - A lamp (300-400 nm) might be more efficient since Ti02 is activated by wavelengths less than or equal to 380 nm. However, we chose a xenon lamp with a spectrum similar to sunlight to simulate the use of solar energy in the future. It was shown that disinfection of a high cell concentration of E. coli was more effective using DLEOFs for light supply. In conclusion, optimum conditions for disinfection of E. coli byTiO2 particles using optical fibers were as follows: 2 h of light illumination, 60 mEinstein mT2s-l of light intensity, and over 0.4pglmL Ti02 concentration. Diffuselight emitting optical fiber illumination can be used effectivelyfor disinfection of high cell densities of bacteria using high Ti02 concentrations. The present study describes an improved light supply system for disinfection of E. coli using Ti02 particles. The
main advantage of using diffuse-lightemittingoptical fibers is the high surface area to volume ratio, enabling more efficient photoexcitation of Ti02 and, as a result, more efficient disinfection. Disinfectionusing Ti02 can also be used to disinfect other microorganisms (10, 12) and has also been applied to the disinfection of bacteria in pond water (13). Therefore, this method may be generally applicable to the disinfection of a wide range of microorganisms in water. Future work is also necessary to determine whether other water-contaminating organisms could be disinfected.
Literature Cied (1) Fujishima, A.; Honda, K. Nature (London) 1972,238,37-38. (2) Kraeutler, B.; Bard, A. J. I. Am. Chem. SOC.1978,100,2239-2240. (3)Frank, S. N.; Bard, A. J. J. Phys. Chem. 1977,81,1484-1488. (4) Frank, S. N.; Bard, A. J. 1.Am. Chem. SOC. 1977,99,303-304. (5)Kawai, T.; Sakata, T. Nature (London) 1980,286,474-476. (6) Schrauzer, G. N.; Guth,T. D.J. Am. Chem. SOC. 1977,99,71897193. (7)Borgarello, E.;Kiwi, J.; Pelmetti, E.; Visca, M.; Gratzel, M.J. Am. Chem. SOC. 1981,103,6324-6329. (8) Matthew, R. W. J. Phys. Chem. 1987,91,3328-3333. (9)Ollis, D. F.; Pelizzetti, E.; Serpone, N. Environ. Sci. Technol. 1991, 25,1523-1529.
(10)Matsunaga, T.; Tomoda, R.; Nakajima, T.; Wake, H. FEMS MicrobioI. Lett. 1985,29, 211-214. (11)Matsunaga, T.; Tomoda, R.;Nakajima, T.; Nakarnura, N.; Komine, T. Appl. Environ. Microbiol. 1988,54, 1330-1333. (12) Sjogren, J. C.; Sierka, R. A. Appl. Environ. Microbiol. 1994,60, 344-347. (13) Ireland, J. C.; Klostermann, P.; Rice, E. W.; Clark, R. M. AppZ. Environ. Microbwl. 1993,59, 1668-1670. (14)Wei, C.; Lin,W.; Zainal, Z.; Williams, N. E.; Zhu,K.; h i c , A.; Smith, R. L.; Rajeshwar, K. Environ. Sci. Technol. 1994,28,934938. (15)Fujishima, A.; Cai, R.; Hashimoto, K.; Sakai, H.; Kubota,Y. Dace Met. Environ. 1993,3, 193-205. (16)Shumilin, I. A.; Nikandrov,V.V.; Popov,V. 0.;Krasnovsky,A.A. FEBS Lett. 1992,306,125-128. (17)Takano, H.; Furu-une, H.; Burgess, J. G.; Manabe, E.; Hirano, M.; Okazaki, M.; Matsunaga, T. Appl. Biochem. Biotechnol. 1993, 39/40,159-167. (18)Matsunaga, T.; Takeyama, H.; Sudo, H.; Oyarna, N.; Ariura, S.; Takano, H.; Hirano, M.; Burgess, J. G.; Sode, K.; Nakarnura, N. Appl. Biochem. BiotechnoZ. 1991,28/29,157-167.
Received for review July 5,1994. Revised manuscript received October 28, 1994. Accepted November 2, 1994.*
ES9404126 @
Abstract published in AdvanceACSAbstracts, December 15,1994.
VOL. 29, NO. 2, 1995 I ENVIRONMENTAL SCIENCE 81TECHNOLOGY
BOB
