Environ. Sci. Technol. 2003, 37, 4901-4904
Soluble Sunscreens Fully Protect E. coli from Disinfection by Electrohydraulic Discharges W.-K. CHING, A. J. COLUSSI,* AND M. R. HOFFMANN W. M. Keck Laboratories, California Institute of Technology, Pasadena, California 91125
We show that the ultraviolet radiation emitted, rather than the thermal/pressure shocks or the chemical species that are generated in these events, is the lethal agent that inactivates Escherichia coli colonies exposed to electrohydraulic discharges, EHD. Disinfection is completely suppressed in the presence of 300 nm is deemed to be inactive toward DNA damage. The fraction of actinic radiation factinic that could be used for disinfection can be calculated by integration of Planck’s spectral distribution function for a blackbody at 50 kK (34)
factinic )
∫
dλ 8πhc 180 nm 5 λ [exp(hc/λkT) - 1] 300 nm
σSBT4
) 0.033
(3)
i.e., Jactinic ) factinic × J ) 3.3 MW cm-2 ) 3.8 × 1024 photons (λ ) 230 nm) cm-2 s-1 or about 0.26 mEinsteins per pulse. If λ (CFU-1 cm2) and βλ (L mg-1 cm-1) are the spectral extinction coefficients of E. coli suspensions and sunscreen solutions, respectively, and γλ is the spectral photochemical efficiency for the generation of thymine dimers (normalized to γ180 nm ) 1) (35), then the actinic radiation absorbed at λ by the E. colonies per unit volume per s within a spherical shell of thickness dR at a distance R from the discharge center is given by
Iλ,R ) Jλ,R0
()
R0 2 -{λ[E. coli]+βλ[SS]}R e λ[E. coli]γλ4πR2dR R
(4)
which upon integration over R and summation over λ yields
1 - e-{λ[E. coli]+βλ[SS]}Rmax
300 nm
I)A
∑
180 nm
Jλ,R0λ[E.coli]γλ
λ[E. coli] + βλ[SS]
(5)
Discussion A conservative estimate of the lethal fluences can be obtained from the actinic photon flux: Jactinic ) 3.8 × 1024 photons cm-2 s-1 (λ ) 230 nm) computed above, multiplied by the average absorption cross-section of the cells. The average absorption cross-section of the cells from 180 to 300 nm can be computed as the blackbody weighted average
〈cell
∫ 〉) ∫
300 φ dλ 180 λ λ 300 φ dλ 180 λ
) 1.30 × 10-9 cm2 CFU-1
(6)
where φλ is the integrand in eq 3. Therefore, each cell absorbs Jactinic × cell ) 5.0 × 1015 photons CFU-1 s-1, i.e., a photon is absorbed every 0.2 fs. The lethal radiation dose may be computed as the fluence times the effective lethal exposure time: 2.5 EHD × 40 µs ) 100 µs. Hence, about 5.0 × 1015 photons CFU-1 s-1 × 10-4 s ) 5.0 × 1011 photons CFU-1 are actually required to inactivate 50% of the bacterial population by EHD! A clue to the low efficiency of high-intensity radiation is provided by the fact that at photon fluences higher than 3 × 1017 photons cm-2 the first excited triplet and singlet excimer states of purine and pyrimidine DNA bases, such as thymine, are promoted to higher states by absorption of a second photon with unitary probability (14, 37). The actinic fluence of EHD, 3.8 × 1020 photons cm-2 per pulse, exceeds the threshold fluence suggesting that a disinfection mechanism involving higher excited states is expected to operate in this case (17, 37, 38). In contrast, continuous exposure of cells with cell ) 1.3 × 10-9 cm2 CFU-1 to 15 W germicidal lamps (λ ) 254 nm) having typical photon fluxes of about 4.5 mW cm-2 ) 5.8 × 1015 photons cm-2 s-1 leads to the absorption of 6.5 × 106 photons CFU-1 s-1 or a photon every 150 ns. Using the previously determined lethal LD50 ) 102 s (or 1.7 min at 7 × 107 CFU mL-1) for such lamps (4), we obtain a lethal dose of about 6.6 × 108 photons CFU-1 or about 750 times smaller than for EHD disinfection. VOL. 37, NO. 21, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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The lifetimes of the lowest singlet S1 and triplet T1 excited states of nucleic acids are 1-10 ps and ∼1 µs, respectively (16, 37). Our calculations show that, on average, the intervals between photon absorption by cells are ∼0.2 fs under EHD and ∼150 ns under germicidal lamps. Therefore, low-intensity photochemistry is monophotonic. Consecutive photon absorption in the high-intensity regime can promote S1, T1 to quasi-Rydberg states, leading to ionization (11). In this case, the bleaching of the S1 and T1 states by the absorption of a second photon (5, 37) competes with the formation of thymidine dimers. The ionized species produced by highintensity UV radiation may be scavenged by cell membrane proteins. The destruction of proteins in E. coli is, however, biologically less important than DNA damage (5). At intensities higher than 106 W cm-2, the uvr and rec cell repair systems are inhibited (39), and single- and double-strand breaks occur (17-19). Actually, the nonexponential decay of E. coli colonies viability observed in Figure 1a may be explained by a multihit mechanism of deactivation (40). In summary, high-intensity UV emission is the dominant, if not the exclusive, mechanism of disinfection in the EHD process. At sufficiently high sunscreen concentrations (above ∼50 mg BP9 L-1) the viability of E. coli colonies is unaffected after many discharges, suggesting that mechanical rupture or oxidative attack by the chemical species generated in the discharges are insignificant relative to the damaged induced by radiation. A detailed model of the disinfection process that incorporates the spectral distribution of plasma emissions and photon absorption by water, E. coli, and the soluble sunscreen as well as of the disinfection efficiency provides a satisfactory description of the dependence of LD50 with sunscreen concentration. The model reveals that the mechanism of disinfection by electrohydraulic discharges is qualitatively different and substantially less efficient than that induced by low-intensity UV radiation sources.
Literature Cited (1) Lang, P. S.; Ching, W.-K.; Willberg, D. M.; Hoffmann, M. R. Environ. Sci. Technol. 1998, 32, 3142-3148. (2) Willberg, D. M.; Lang, P. S.; Hochemer, R. H.; Kratel, A.; Hoffmann, M. R. Environ. Sci. Technol. 1996, 30, 2526-2534. (3) Anpilov, A. M.; Barkhudarov, E. M.; Christofi, N.; Kop’ev, V. A.; Kossyi, I. A.; Taktakishvili, M. I.; Zadiraka, Y. Lett. Appl. Microbiol. 2002, 35, 90. (4) Ching, W.-K.; Colussi, A. J.; Sun, H. J.; Nealson, K. H.; Hoffmann, M. R. Environ. Sci. Technol. 2001, 35, 4139. (5) Gorner, H. J. Photochem. Photobiol. B: Biol. 1994, 26, 117. (6) Ravanat, J. L.; Douki, T.; Cadet, J. J. Photochem. Photobiol. B: Biol. 2001, 63, 88. (7) Angelov, D.; Spassky, A.; Berger, M.; Cadet, J. J. Am. Chem. Soc. 1997, 119, 11373. (8) Nikogosyan, D. N.; Letokhov, V. S. Riv. Nuovo Cimento 1983, 6, 1. (9) Nikogosyan, D. N.; Angelov, D. A.; Oraevsky, A. A. Photochem. Photobiol. 1982, 35, 627. (10) Ma, J. H.; Lin, W. Z.; Wang, W. F.; Han, Z. H.; Yao, S. D.; Lin, N. Y. J. Photochem. Photobiol. B: Biol. 2000, 57, 76.
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(11) Reuther, A.; Nikogosyan, D. N.; Laubereau, A. J. Phys. Chem. 1996, 100, 5570. (12) Crespo-Hernandez, C. E.; Arce, R. Photochem. Photobiol. 2002, 76, 259. (13) Goez, M.; Zubarev, V. Chem. Phys. 2000, 256, 107. (14) Oraevsky, A. A.; Nikogosyan, D. N. Chem. Phys. 1985, 100, 429. (15) Schulte-Frohlinde, D.; Bothe, E. Pulse radiolysis of nucleic acids in aqueous solutions; CRC Press: Boca Raton, FL, 1991. (16) Angelov, D.; Berger, M.; Cadet, J.; Getoff, N.; Keskinova, E.; Solar, S. Radiat. Phys. Chem. 1991, 37, 717. (17) Masnyk, T. W.; Minton, K. W. Photochem. Photobiol. 1991, 54, 99. (18) Schulte-Frohlinde, D.; Simic, M. G.; Gorner, H. Photochem. Photobiol. 1990, 52, 1137. (19) Bothe, E.; Gorner, H.; Opitz, J.; Schulte-Frohlinde, D.; Siddiqi, A.; Malgorzata, W. Photochem. Photobiol. 1990, 52, 949. (20) Kochevar, I. E.; Walsh, A. A.; Green, H. A.; Sherwood, M.; Shih, A. G.; Sutherland, B. M. Cancer Res. 1991, 51, 288. (21) Arrage, A. A.; Phelps, T. J.; Benoit, R. E.; White, D. C. Appl. Environ. Microbiol. 1993, 59, 3545. (22) Cockell, C. S.; Knowland, J. Biol. Rev. 1999, 74, 311. (23) Groniger, A.; Sinha, R. P.; Klisch, M.; Hade, D. P. J. Photochem. Photobiol. B: Biol. 2000, 58, 115. (24) Schick, J. M.; Dunlap, W. C. Annu. Rev. Physiol. 2002, 64, 223. (25) Krinsky, N. I. Pure Appl. Chem 1979, 51, 649. (26) Garcia-Pichel, F.; Castenholz, R. W. J. Phycol. 1991, 27, 395. (27) Proteau, P. J.; Gerwick, W. H.; Garcia-Pichel, F.; Castenholz, R. Experientia 1993, 49, 825. (28) Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed.; Kroschwitz, J. I., Ed.; Wiley: New York, 1991; Vol. 17. (29) Bhasikuttan, A. C.; Singh, A. K.; Palit, D. K.; Sapre, A. V.; Mittal, J. P. J. Phys. Chem. A 1998, 102, 3470-3480. (30) Willberg, D. M.; Lang, P. S.; Hochemer, R. H.; Kratel, A.; Hoffmann, M. R. Chemtech 1996, 26(4), 53. (31) Standard Methods for the Examination of Water and Wastewater, 17th ed.; Clesceri, L. S., Greenberg, A. E., Trussell, R. R., Eds.; APHA, AWWA, and WPCF: Washington, DC, 1989. (32) Kratel, A. W. H. Pulsed Discharges in Water. Doctoral Dissertation, Division of Engineering & Applied Science, California Institute of Technology, Pasadena, 1996. (33) Robinson, J. W.; Ham, M.; Balaster, A. N. J. Appl. Phys. 1973, 44, 72-75. (34) Berry, R. S.; Rice, S. A.; Ross, J. Physical Chemistry; Wiley: New York, 1980. (35) Matsunaga, T.; Hieda, K.; Nikaido, O. Photochem. Photobiol. 1991, 54, 403. (36) In Non-Thermal Plasma Techniques for Pollution Control Part B; Penetrante, B. M., Schultheis, S. E., Eds.; Springer-Verlag: New York, 1993. (37) Masnyk, T. W.; Nguyen, H. T.; Minton, K. W. J. Biol. Chem. 1989, 264, 2482. (38) Lang, H.; Riesenberg, D.; Zimmer, C.; Bergter, F. Photochem. Photobiol. 1986, 44, 565-570. (39) Nikogosyan, D. N.; Oraevsky, A. A.; Zavilgelsky, G. B. Photobiochem. Photobiophys. 1986, 10, 189. (40) Harm, W. Biological effects of ultraviolet radiation; University Press: Cambridge, 1980.
Received for review February 27, 2003. Revised manuscript received August 7, 2003. Accepted August 11, 2003. ES034182J
