A pure culture of Microcystis aeruginosa (NIES-98) was exposed to low-pressure (LP) or medium-pressure (MP) UV lamps and subsequently incubated under white light fluorescent lamps allowing both photoreactivation and photosynthesis. During incubation, profiles of the number of existing cells and UV-induced DNA damage were determined for each sample. The growth of Microcystis aeruginosa was inhibited by the exposure to LPUV or MPUV. Only a minor difference was observed between LPUV and MPUV both in the cell number and the DNA damage. UV-induced DNA damage just after UV irradiation was almost the same regardless of the UV fluence or the UV lamp. Meanwhile, the UV-induced DNA damage was repaired during 1day incubation after UV exposure, and the number of DNA damage appeared somehow proportional to the UV fluence after 1day incubation. A comparison between the cell number and the number of DNA damage implied that the UV-induced DNA damage mainly contributed to the cell number reduction of Microcystis aeruginosa
Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 55 - Low- or Medium-pressure UV Lamp Inactivation of Microcystis aeruginosa SAKAI Hiroshi *1 , OGUMA Kumiko *2 , KATAYAMA Hiroyuki *3 , OHGAKI Shinichiro *4 Department of Urban Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, JAPAN (E-mail: *1:h_sakai@env.t.u-tokyo.ac.jp, *2:oguma@env.t.u-tokyo.ac.jp, *3:katayama@env.t.u-tokyo.ac.jp, *4:ohgaki@env.t.u-tokyo.ac.jp) ABSTRACT A pure culture of Microcystis aeruginosa (NIES-98) was exposed to low-pressure (LP) or medium-pressure (MP) UV lamps and subsequently incubated under white light fluorescent lamps allowing both photoreactivation and photosynthesis. During incubation, profiles of the number of existing cells and UV-induced DNA damage were determined for each sample. The growth of Microcystis aeruginosa was inhibited by the exposure to LPUV or MPUV. Only a minor difference was observed between LPUV and MPUV both in the cell number and the DNA damage. UV-induced DNA damage just after UV irradiation was almost the same regardless of the UV fluence or the UV lamp. Meanwhile, the UV-induced DNA damage was repaired during 1day incubation after UV exposure, and the number of DNA damage appeared somehow proportional to the UV fluence after 1day incubation. A comparison between the cell number and the number of DNA damage implied that the UV-induced DNA damage mainly contributed to the cell number reduction of Microcystis aeruginosa. KEY WORDS Microcystis aeruginosa, Low-pressure (LP) UV Lamp, Medium-pressure (MP) UV Lamp, Endonuclease Sensitive Site (ESS) Assay, Photoreactivation INTRODUCTION The presence of algae in drinking water source can have a significant impact on the subsequent water treatment. Algae produce undesirable odorous compounds such as 2-methylisoborneol (2-MIB) or Geosmin (1, 2, 3) and also produce toxic compounds such as microcystin (4, 5, 6). Therefore, how to control algae in the source of drinking water has received considerable attention. The most effective and drastic way to control algal growth is to reduce nutrient load into lakes or reservoirs (7). However, because of the significant internal loading in most reservoirs and lakes, especially from bottom sediment, controlling the external nutrient-load alone is not sufficient to prevent seasonal algal blooms (8). In order to inhibit the excessive algal growth and to reduce its impact on water treatment, many water treatment plants apply copper sulfate to their target lakes or reservoirs. Currently, however, there is a growing concern against the use of copper sulfate, mainly because it also has an impact on non-target creatures other than algae. Meanwhile, some water treatment utilities apply chlorine in order to inhibit the growth of algae, but chlorine reacts with the precursors of by-products in water Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 56 - to produce cancer-causing by-products such as trihalomethane (9). Compared with the use of chemical compounds, UV treatment has a certain advantages to improve the defects of such treatments. UV exposure has a feature of low residuality in its germicidal effects and therefore has a less impact on the ecosystem in the target watersheds. Another advantage is that UV treatment gives relatively lower disinfection byproducts (10), because UV reacts with the target DNA much more efficiently than with chemical agents in water. With these advantageous characteristics UV treatment is expected to become an alternative to conventional treatment against excessive algal growth. As for the system of UV irradiation, most conventional UV lamps are low pressure UV lamps (LPUV) while medium pressure UV lamps (MPUV) have also been used. Previously, the application of LPUV was investigated for the purpose of growth inhibition of Microcystis aeruginosa (11) while the use of MPUV has not yet been investigated so far. In order to apply UV to inhibit the excessive algal growth, much attention should be paid to photoreactivation. In the photoreactivation process, UV-induced DNA damage can be repaired by the activity of photolyase with the energy of visible light. Once microorganisms are exposed to UV light and the DNA is damaged, those organisms can not reproduce themselves and therefore the growth is inhibited. However, when UV-damaged DNA is repaired by photoreactivation process, the microorganisms can reproduce themselves normally. Hence, it can be easily presumed that photoreactivation process may impair the efficacy of UV treatment. But, still, there have been no investigation about photoreactivation of M. aeruginosa and its effect on UV treatment with the engineering point of view. In this study, the scope of using UV-radiation to control algal growth was assessed using M. aeruginosa as test species. M. aeruginosa was selected for the experiment because of its frequent association with seasonal algal blooms. The specific objectives were (i) to study the effect of UV-radiation on the inactivation of algae; (ii) to study the level of photoreactivation by directly investigating the number of DNA damage, and (iii) to study the contribution of DNA damage to the UV inactivation of M. aeruginosa. MATERIALS AND METHODS Microorganism Axenic culture of planktonic blue-green algae M. aeruginosa (NIES-98) was obtained from National Institute for Environmental Studies (NIES, Tsukuba, Japan) and then grown in M-12 media, whose composition is listed in Table1. Cultures were maintained at 25 °C in an incubation chamber (BITEC-400L, Shimadzu) with controlled lighting. Fluorescent lamps (FL20SW-B, GE/Hitachi) were used as the light source with an automated light/dark cycle of 12 h/12 h. The light intensity during the lighting phase was set at 1500 lux. Table1 Composition of M-12 Media Ingredients Concentration NaNO 3 100mg/l K 2 HPO 4 10mg/l MgSO 4 ・7H 2 O75mg/l CaCl 2 ・2H 2 O40mg/l Na 2 CO 3 20mg/l Fe-Citrate 1mg/l pH 8 Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 57 - UV Irradiation and following Incubation Axenic culture of M. aeruginosa was grown for 10 days in the M-12 media to reach a concentration of about 10 6 cells/ml. To assess the effect of UV-irradiation, 40ml of cultured samples were irradiated in glass petri dishes 90 mm in diameter, which were pre-treated to be chlorine demand free glasswares described previously (12). Two types of UV lamps were used in the experiment, which are monochromatic low-pressure UV lamp (15W×2, GE/Hitachi) and polychromatic medium-pressure UV lamp (330W, B410MW, Ebara). The germicidal intensity of the light emitted from each UV lamp was standardized by determining the irradiance of light at 254 nm with a biodosimeter using F-specific RNA coliphage Qβ (13). Briefly, a pure-culture suspension of phage Qβ at an initial concentration of 2.0 × 10 5 PFU/ml was exposed to the LP or MPUV lamps to determine the inactivation curves by a double-agar layer method with LB agar (Merck) by using Escherichia. coli K-12 strain F + (A/λ) as the host organism. The rate of inactivation of phage Qβ for each lamp was compared with the inactivation rate constant for phage Qβ at 254 nm to determine the irradiance values for the LP and MPUV lamps (0.4 mW/cm 2 and 1.5 mW/cm 2 , respectively). The irradiance values were fixed throughout the experiment, and the UV fluence were controlled by changing the exposure time. After irradiation, the samples were incubated for 7days in an incubation chamber (1500 lux fluorescent light, 25°C temperature, 12 h/12 h-light/dark cycle) in 100 ml Erlenmeyer flask. Cell number The number of cells in the samples was enumerated by a fluoresence microscope (BH2, Olympus) using plankton counting chamber (MPC-200, Matsunami Glass, Japan). Cells showing chlorophyll fluorescence were enumerated as alive cells and others were considered as dead cells. Alive cells of M. aeruginosa were counted just before and after UV irradiation, as well as 1, 3, 5, and 7 days after UV irradiation. All experiments were carried out in triplicates. ESS assay Endonuclease Sensitive Site (ESS) assay An ESS assay allows the recognition of pyrimidine dimers in DNA at ESS by the treatment of DNA with a UV endonuclease, which incises a phosphodiester bond Fig.1 Low Pressure UV System 580 mm 500 mm 240 mm 90 mm Glass Petri Dish Low Pressure UV Lamp Shutter Magnetic Stirrer Fig.2 Medium Pressure UV System 220 mm Medium Pressure UV Lamp 380mm 160 mm Glass Petri Dish Mangetic Stirrer Stirrer Bar Sample 4.5 mm 60mm 90 mm Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 58 - specifically at the site of pyrimidine dimers. The molecular lengths of fragmented DNA are determined by alkaline agarose gel electrophoresis, followed by a theoretical calculation to obtain the number of ESS (14). This assay was applied in the survey of UV-irradiated health-related microbes such as Escherichia coli, Cryptosporidium parvum and Legionella pneumophila (15, 16, 17, 18). The conditions for the ESS assay used in this study were basically the same as those described previously (15). After the irradiation procedures, the M. aeruginosa suspensions were centrifuged (1,800×g, 20 min), and the pellets were subjected to DNA extraction step (Genomic-tip; Qiagen). DNA extraction was performed according to the protocols provided by Qiagen, with a minor modification. The extracted DNA was concentrated by using centrifugal filter devices (Centricon; Millipore) and resuspended in a UV endonuclease buffer containing 30 mM Tris (pH 8.0), 40 mM NaCl, and 1 mM EDTA. The concentrated DNA was treated with a UV endonuclease from Micrococcus luteus, prepared by the method of Carrier and Setlow (19), at 37°C for 45 min. The reaction was stopped by the addition of an alkaline loading dye preparation containing following ingredients as a final concentration (100 mM NaOH, 1 mM EDTA, 2.5% Ficoll, and 0.05% bromocresol green). The DNA samples were electrophoresed at 0.6 V/cm for 16 h on 0.35 or 0.5 % alkaline agarose gels in an alkaline buffer containing 30 mM NaOH and 1 mM EDTA along with a molecular length standard, T4dC+T4dC/BglI digest mixture (7GT; Wako). After electrophoresis, the gels were stained in a 0.5µg/ml solution of ethidium bromide, photographed, and analyzed (Gel Doc 1000 Molecular Analyst; Bio-Rad). The midpoint of the mass of DNA was photographically determined by determining the median migration distance of each sample, which was converted into the median molecular length (L med ) of the DNA relative to the migration patterns of the molecular length standards. The average molecular length (L n ) of the DNA was obtained by using the equation of Veatch and Okada (20): L n = 0.6 × L med . The number of ESS per base was calculated as follows (21): ESS/base = [1/L n (+UV)] - [1/L n (-UV)], where L n (+UV) and L n (-UV) are the average molecular lengths of UV-irradiated and nonirradiated samples, respectively. RESULTS Cell number Figures 3 and 4 show the inactivation profiles of after LPUV or MPUV exposure, respectively. The horizontal axis shows the time passed after UV exposure, while the vertical axis shows the cell density in the unit of 10 3 cells/ml . The cell density of control samples without any UV exposure is also shown as references. In samples exposed to 1800 [mWs/cm 2 ] of UV, the cell density became below the detection limit only 2days after the exposure to LPUV or MPUV. In samples exposed to 600 [mWs/cm 2 ] of UV, the reduction of cell density was observed 3days after the LPUV exposure or 5days after the MPUV exposure, and the final reduction compared with the control samples was 1.5 log for LPUV and 1.2 log for MPUV after 7days incubation. In samples exposed to 180 [mWs/cm 2 ] of LPUV, a clear reduction was observed after 5days to show a 0.9 log difference from the control samples after Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 59 - 7days incubation. In samples exposed to 180 [mWs/cm 2 ] of MPUV, only a slight reduction of cell density was observed and difference from control samples was only 0.4 log after 7days incubation. Regardless of UV lamps and UV fluence, no significant reduction of cells was observed just after UV irradiation. ESS assay ESS assay was applied to the UV irradiated and non-irradiated M. aeruginosa cells just after UV irradiation and after 1day incubation. DNA was extracted from the concentrated cells, to determine the UV-induced DNA damage mainly composed of pyrimidine dimers. Figure 5 shows the number of UV-induced DNA damage in each sample. Just after UV irradiation, the number of DNA damage was almost the same regardless of the UV fluence and only a minor difference was observed between LPUV and MPUV. The number of ESS ranged between 1.96~3.42 [10 -4 ESS/base]. It could be speculated that the UV exposure produced too many pyrimidine dimers to determine quantitatively, which caused no observable difference among different samples and fluences. In contrast, in the samples incubated for 1day after UV exposure, the number of UV-induced DNA damage appeared somehow proportional to the UV fluence. This could be because DNA damage was repaired during 1day incubation after UV exposure. When the UV fluence was 180 or 600 [mWs/cm 2 ], some of the DNA damage was repaired and photoreactivation could play a major role in this repair. The incubating condition used in this experiment includes 12 h of light phase in the daily light-dark cycle, which could be long enough to cause photoreactivation. 1 10 100 1000 10000 -101234567 Time after UV irradiation [day] Cell Density [10 3 cells/ml]l Control LP- 180 LP- 600 LP- 1800 UV Irradiation 1 1 10 100 1000 10000 -101234567 Time after UV irradiation [day] Cell Density [10 3 cells/ml]l Control MP-180 MP-600 MP-1800 UV Irradiation 1 Fig.3 Cell density profiles of M. aeruginosa under white light after LPUV irradiation Fig.4 Cell density profiles of M. aeruginosa under white light after MPUV irradiation Fig.5 Number of UV-Induced DNA Damage in M. aeruginosa -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 LP-180 LP-600 LP-1800 MP-180 MP-600 MP-1800 Number of UV-Induced DNA Damage [10 -4 /base] Just after UV Irradiation (n=6) After 1day Incubation (n=5) Journal of Water and Environment Technology, Vol.3, No.1, 2005 - 60 - DISCUSSION Figures 6 and 7 show the results of each measurement item as a function of UV fluence. In Fig.6, the UV fluence was compared with the net reduction of cells after 7days incubation. As shown in Fig.6, the net reduction of cells after 7days incubation suggested a proportional relationship with the UV fluence. In Fig.7, the UV fluence was compared with the number of UV-induced DNA damage after 1day incubation. As shown in this figure, DNA damage showed the tendency to increase along with the increase of the UV fluence. Comparing Fig.6 with Fig.7, it might be implied that the number of UV-induced DNA damage after 1 day incubation may have contributed to the net cell reduction of M. aeruginosa after 7 days incubation. CONCLUSIONS (i) The growth of M. aeruginosa was inhibited by either LPUV or MPUV irradiation and its effect was proportional to the UV fluence. 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Blackett, D. C. Monteleone, R. B. Setlow, B. M. Sutherland, and J. C. Sutherland. (1986) Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application of pyrimidine dimers. Anal. Biochem. 158:119-129 . relevant to the occurrence of musty odor in Lake Kasumigaura. Wat. Res. 32 : 35 49 -35 54 3) Chang, J. C. H., Ossoff, S. F., Lobe, D. C., Dofrman, M. H., Dumais,. University of Tokyo, 7 -3- 1 Hongo, Bunkyo-ku, Tokyo, 1 13- 8656, JAPAN (E-mail: *1:h_sakai@env.t.u-tokyo.ac.jp, *2:oguma@env.t.u-tokyo.ac.jp, *3: katayama@env.t.u-tokyo.ac.jp,