Low- or Medium-pressure UV Lamp Inactivation of Microcystis aeruginosa

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

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

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

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 106 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 × 105 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/cm2 and 1.5 mW/cm2, 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

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 103 cells/ml The cell density of control samples without any UV exposure is also shown as references

In samples exposed to 1800 [mWs/cm2] 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/cm2] 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/cm2] of LPUV, a clear reduction was observed after 5days to show a 0.9 log difference from the control samples after

7days incubation In samples exposed to 180 [mWs/cm2] 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/cm2], 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

Time after UV irradiation [day]

3 cells

Control LP-180 LP-600 LP-1800

UV Irradiation

10 100 1000 10000

Time after UV irradiation [day]

3 cells

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

-4 /b

Just after UV Irradiation (n=6) After 1day Incubation (n=5)

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 The net reduction of cells after 7days incubation was over 4 log after 1800 [mWs/cm2] of either LPUV or MPUV, 1.2 log after 600 [mWs/cm2] of MPUV, 1.5 log after 600 [mWs/cm2] of LPUV, 0.9 log after 180 [mWs/cm2] of LPUV, and 0.4 log after 180 [mWs/cm2] of MPUV

(ii) In samples after the exposure to LPUV or MPUV at the fluence of 180 and 600 [mWs/cm2], most of the UV-induced DNA damage was repaired

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