Effects of solar radiation on viability of two strains of antarctic
Effects of solar radiation on viability of two strains of antarctic bacteria EMILI0 R. MARGUET, Cdtedra de Biologia Celulary Molecular, Facultad de Ciencias Naturales, Universidad Nacional de la
Patagonia, Chubut, Argentina E. WALTER HELBLING, VIRGINIA E. VILLAFAIE, and OSMUND HOLM-HANSEN, Polar Research Program, Scripps Institution of
Oceanography, University of California at San Diego, La Jolla, California 92093-0202
ince El-Sayed (1988) called attention to the potential danger S to the southern ocean ecosystem from the enhanced ultraviolet-B (UV-B) radiation which results from the seasonal ozone hole over Antarctica, numerous studies have assessed the impact of solar ultraviolet radiation (IJVR) on marine organisms (see Weiler and Penhale 1994). With just a few exceptions (e.g., Karentz 1994, pp. 93-110), little effort has been devoted to determining the impact of UVR on marine bacteria. This is a notable omission in efforts to assess the impact of enhanced UV-B radiation on the dynamics of the food web in antarctic waters, because bacterioplankton are believed to have a dominant role in the cycling of organic carbon and nutrients in marine waters (Azam, Smith, and Hoffibaugh 1991). In this article, we present preliminary data obtained at Palmer Station (64.70S 64.10W) during the months of November and December 1993 on the effects of solar UVR on viability of isolated bacterial strains. Using a sterile polycarbonate (1-liter) bottle from the bow of a slow-moving Zodiak, we took samples of surface waters. Incident solar radiation was monitored continuously (recorded every minute) using a spectroradiometer (PUV-510, Biospheri cal Instrument, Inc.) that measures four channels of UVR [305, 320,340, and 380 nanometers (nm)] as well as photosynthetically available radiation (PAR). Different bacterial strains were isolated from natural populations and identified according to Austin (1988). Once identified, single strains were maintained on a solid medium at 4°C until used in various experiments. Two strains, Acinetobacter sp. and Bacillus sp. (gram negative and gram positive, respectively), were used for the experiments described below. Before use in any experiment, the bacteria were transferred to a marine broth and incubated at 4°C until the culture was well into the exponential growth phase (about 18 hours). At that time, 0.5 milliliters of the culture was dispensed into replicate quartz tubes containing 50 milliliters of sterile seawater; the final concentration was between 105 to 106 cells per milliliter. The experimental samples were exposed to natural solar radiation in an outdoor incubator with flowing surface sea water for temperature control. Two types of experiments were performed: • incubations where the solar radiation was attenuated by means of neutral density screen down to 3 percent of the incident radiation, and • incubations to assess the importance of the SOS-repair system. Duplicate or triplicate samples were used in all treatments, in which the samples were exposed to three different spectral irradiance regimes:
• samples in quartz tubes (received all radiation), • samples in Pyrex tubes covered with Mylar film (received UV-A and PAR), and • samples in Pyrex tubes covered with Plexiglas UF-3 (received only PAR). The induction of the SOS system was done by incubating the samples with nalidixic acid (Piddock and Walters 1992) at a concentration of 50 percent of minimal inhibitory concentration (MIC) for 1 hour immediately preceding the experimental exposure period; the concentrations used were 10 micrograms per milliliter for Bacillus and 20 micrograms per milliliter for Acinetobacter.
After the incubations, the viability was determined by the method of counting of colonies on agar. Plates of agar Schaedler were inoculated with 50 microliters of each of the dilutions of the original samples (final dilutions of 10 ) 10, and 10- 5 ) and incubated for 48 hours at 20°C. Colonies were counted on dilution plates that had between 50 to 250 colonyforming units. Data in figure 1 show bacterial viability (as survival fraction) as a function of the mean irradiance to which the cells were exposed during the incubation period. Both strains of bacteria showed a decrease in the survival fraction with increased irradiance. The total inhibition, however, was more dramatic in Bacillus (figure 1B) than in Acinetobacter (figure 1A). Both strains showed some inhibition due to PAR at high irradiances, but the greatest inhibition was due to UVR. At the highest irradiance, more than 70 percent of the total inhibition due to UVR was due to UV-A whereas the rest was due to UV-B. Data from the experiments designed to study the importance of the SOS system in repairing IJVR damage are shown in figure 2. When the strains were transferred directly from the laboratory conditions (see above) and exposed to solar radiation, they showed a decrease in the survival fraction, especially in the treatments receiving UV-B (280-320 nm) and UV-A (320-400 nm). As mentioned above, Bacillus (figure 2B) also showed a decrease in viability due to high PAR. When the SOS system was activated before the exposure to solar radiation, however, the survival fraction was higher in all treatments but particularly so for the samples exposed to UVR. Figure 2A illustrates that when the SOS system was induced in Acinetobacter, no significant difference in viability between the samples exposed to UV-A+UV-B (quartz treatment) as compared to the cells exposed to just UV-A (Mylar treatment) was detected. This finding suggests that the SOS response was very effective in repairing damage caused by UV-B radiation. The viability in both these treatments, however, was still lower than in the
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iO°
I1 I .---
A
10.1 C
10 Cl)
ioio4 L
Quartz Mylar
P-400
100
101 C 0 0 Co Co >
io-
C')
ioioFigure 1. Survival fraction of antarctic bacteria as a function of incident solar irradiance (mean value during the incubation period). Inset in each graph indicates symbols for the treatments. QU indicates quartz (received UV-B+UV-A+PAR); Myl indicates Mylar filter (received UV-A+PAR); P-400 indicates Plexiglas filter (received only PAR). A. Acinetobacter strain. B. Bacillus strain.
I r
Quartz Mylar
P.400
Figure 2. Survival fraction of two strains of antarctic bacteria before and after the SOS-repairing system has been induced. Bars indicate the mean and 1 standard deviation of data collected during 4 different days. The dark bars indicate samples before induction of the SOS system and the light bars samples after induction of the SOS system. The overall mean PAR irradiance during the incubation was 0.0926 microeinsteins per square centimeter per second, the mean UVR at 320 nm was 15.23 microwatts per square centimeter, whereas the mean ozone column concentration was 290 Dobson units. The treatments were as follows: quartz (received UV-B+UV-A+PAR); Mylar (received UV-A+PAR); and P-400 (received only PAR). A. Acinetobacter strain. B. Bacillus strain.
samples not exposed to any UVR (UF-3 treatment). Although Bacillus showed higher viability in all treatments when the SOS system was induced, the treatment receiving UV-B was still significantly lower than the others. Our data indicate that bacterioplankton in antarctic waters are strongly inhibited by solar UVR and to a much lesser extent by high fluences of PAR. Although differential sensitivity of different strains to solar radiation is evident, both UV-A and LTV-B apparently can seriously affect the viability and functioning of these marine bacteria. As shown by our experiments, however, this deleterious effect of UVR on bacterioplankton in the Antarctic may be mitigated to varying extent by induction of the SOS system. Whether or not this SOS response can be induced in natural assemblages of bacterioplankton in situ must be addressed in future studies. We thank all the personnel at Palmer Station for their generous help during the field season. This research was supported by National Science Foundation grant OPP 92-20150. E.M. Marguet was at Palmer Station from 18 November 1993 to 3 January 1994; E.W. Helbling and V.E. Villafañe were at Palmer Station from 3 October 1993 to 3 January 1994; and 0. Holm-Hansen was at the Station from 3 October to 15 November 1993.
References Austin, B. 1988. Marine microbiology. Cambridge: Cambridge University Press. Azam, F., D.C. Smith, and J.T. Hollibaugh. 1991. The role of microbial loop in antarctic pelagic ecosystems. Polar Research, 10(1), 239-243. El-Sayed, S.Z. 1988. Fragile life under the ozone hole. Natural History, 97(10), 72-80. Karentz, D. 1994. Ultraviolet tolerance mechanisms in antarctic marine organisms. In S. Weiler and P. Penhale (Eds.), Ultraviolet
radiation in Antarctica: Measurements and biological effects
(Antarctic Research Series, Vol. 62). Washington, D.C.: American Geophysical Union. Piddock, L.J.V., and R.N. Walters. 1992. Bactericidal activities of five quinolona for E. coli strains with mutations in gene encoding the SOS response or cell division. Antimicrobial Agents and Chemotherapy, 36,814-825. Weiler, S., and P. Penhale (Eds.). 1994. Ultraviolet radiation inAntarctica: Measurements and biological effects (Antarctic Research Series, Vol. 62). Washington, D.C.: American Geophysical Union.
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Patagonia, Chubut, Argentina E. WALTER HELBLING, VIRGINIA E. VILLAFAIE, and OSMUND HOLM-HANSEN, Polar Research Program, Scripps Institution of
Oceanography, University of California at San Diego, La Jolla, California 92093-0202
ince El-Sayed (1988) called attention to the potential danger S to the southern ocean ecosystem from the enhanced ultraviolet-B (UV-B) radiation which results from the seasonal ozone hole over Antarctica, numerous studies have assessed the impact of solar ultraviolet radiation (IJVR) on marine organisms (see Weiler and Penhale 1994). With just a few exceptions (e.g., Karentz 1994, pp. 93-110), little effort has been devoted to determining the impact of UVR on marine bacteria. This is a notable omission in efforts to assess the impact of enhanced UV-B radiation on the dynamics of the food web in antarctic waters, because bacterioplankton are believed to have a dominant role in the cycling of organic carbon and nutrients in marine waters (Azam, Smith, and Hoffibaugh 1991). In this article, we present preliminary data obtained at Palmer Station (64.70S 64.10W) during the months of November and December 1993 on the effects of solar UVR on viability of isolated bacterial strains. Using a sterile polycarbonate (1-liter) bottle from the bow of a slow-moving Zodiak, we took samples of surface waters. Incident solar radiation was monitored continuously (recorded every minute) using a spectroradiometer (PUV-510, Biospheri cal Instrument, Inc.) that measures four channels of UVR [305, 320,340, and 380 nanometers (nm)] as well as photosynthetically available radiation (PAR). Different bacterial strains were isolated from natural populations and identified according to Austin (1988). Once identified, single strains were maintained on a solid medium at 4°C until used in various experiments. Two strains, Acinetobacter sp. and Bacillus sp. (gram negative and gram positive, respectively), were used for the experiments described below. Before use in any experiment, the bacteria were transferred to a marine broth and incubated at 4°C until the culture was well into the exponential growth phase (about 18 hours). At that time, 0.5 milliliters of the culture was dispensed into replicate quartz tubes containing 50 milliliters of sterile seawater; the final concentration was between 105 to 106 cells per milliliter. The experimental samples were exposed to natural solar radiation in an outdoor incubator with flowing surface sea water for temperature control. Two types of experiments were performed: • incubations where the solar radiation was attenuated by means of neutral density screen down to 3 percent of the incident radiation, and • incubations to assess the importance of the SOS-repair system. Duplicate or triplicate samples were used in all treatments, in which the samples were exposed to three different spectral irradiance regimes:
• samples in quartz tubes (received all radiation), • samples in Pyrex tubes covered with Mylar film (received UV-A and PAR), and • samples in Pyrex tubes covered with Plexiglas UF-3 (received only PAR). The induction of the SOS system was done by incubating the samples with nalidixic acid (Piddock and Walters 1992) at a concentration of 50 percent of minimal inhibitory concentration (MIC) for 1 hour immediately preceding the experimental exposure period; the concentrations used were 10 micrograms per milliliter for Bacillus and 20 micrograms per milliliter for Acinetobacter.
After the incubations, the viability was determined by the method of counting of colonies on agar. Plates of agar Schaedler were inoculated with 50 microliters of each of the dilutions of the original samples (final dilutions of 10 ) 10, and 10- 5 ) and incubated for 48 hours at 20°C. Colonies were counted on dilution plates that had between 50 to 250 colonyforming units. Data in figure 1 show bacterial viability (as survival fraction) as a function of the mean irradiance to which the cells were exposed during the incubation period. Both strains of bacteria showed a decrease in the survival fraction with increased irradiance. The total inhibition, however, was more dramatic in Bacillus (figure 1B) than in Acinetobacter (figure 1A). Both strains showed some inhibition due to PAR at high irradiances, but the greatest inhibition was due to UVR. At the highest irradiance, more than 70 percent of the total inhibition due to UVR was due to UV-A whereas the rest was due to UV-B. Data from the experiments designed to study the importance of the SOS system in repairing IJVR damage are shown in figure 2. When the strains were transferred directly from the laboratory conditions (see above) and exposed to solar radiation, they showed a decrease in the survival fraction, especially in the treatments receiving UV-B (280-320 nm) and UV-A (320-400 nm). As mentioned above, Bacillus (figure 2B) also showed a decrease in viability due to high PAR. When the SOS system was activated before the exposure to solar radiation, however, the survival fraction was higher in all treatments but particularly so for the samples exposed to UVR. Figure 2A illustrates that when the SOS system was induced in Acinetobacter, no significant difference in viability between the samples exposed to UV-A+UV-B (quartz treatment) as compared to the cells exposed to just UV-A (Mylar treatment) was detected. This finding suggests that the SOS response was very effective in repairing damage caused by UV-B radiation. The viability in both these treatments, however, was still lower than in the
ANTARCTIC JOURNAL - REVIEW 1994
264
iO°
I1 I .---
A
10.1 C
10 Cl)
ioio4 L
Quartz Mylar
P-400
100
101 C 0 0 Co Co >
io-
C')
ioioFigure 1. Survival fraction of antarctic bacteria as a function of incident solar irradiance (mean value during the incubation period). Inset in each graph indicates symbols for the treatments. QU indicates quartz (received UV-B+UV-A+PAR); Myl indicates Mylar filter (received UV-A+PAR); P-400 indicates Plexiglas filter (received only PAR). A. Acinetobacter strain. B. Bacillus strain.
I r
Quartz Mylar
P.400
Figure 2. Survival fraction of two strains of antarctic bacteria before and after the SOS-repairing system has been induced. Bars indicate the mean and 1 standard deviation of data collected during 4 different days. The dark bars indicate samples before induction of the SOS system and the light bars samples after induction of the SOS system. The overall mean PAR irradiance during the incubation was 0.0926 microeinsteins per square centimeter per second, the mean UVR at 320 nm was 15.23 microwatts per square centimeter, whereas the mean ozone column concentration was 290 Dobson units. The treatments were as follows: quartz (received UV-B+UV-A+PAR); Mylar (received UV-A+PAR); and P-400 (received only PAR). A. Acinetobacter strain. B. Bacillus strain.
samples not exposed to any UVR (UF-3 treatment). Although Bacillus showed higher viability in all treatments when the SOS system was induced, the treatment receiving UV-B was still significantly lower than the others. Our data indicate that bacterioplankton in antarctic waters are strongly inhibited by solar UVR and to a much lesser extent by high fluences of PAR. Although differential sensitivity of different strains to solar radiation is evident, both UV-A and LTV-B apparently can seriously affect the viability and functioning of these marine bacteria. As shown by our experiments, however, this deleterious effect of UVR on bacterioplankton in the Antarctic may be mitigated to varying extent by induction of the SOS system. Whether or not this SOS response can be induced in natural assemblages of bacterioplankton in situ must be addressed in future studies. We thank all the personnel at Palmer Station for their generous help during the field season. This research was supported by National Science Foundation grant OPP 92-20150. E.M. Marguet was at Palmer Station from 18 November 1993 to 3 January 1994; E.W. Helbling and V.E. Villafañe were at Palmer Station from 3 October 1993 to 3 January 1994; and 0. Holm-Hansen was at the Station from 3 October to 15 November 1993.
References Austin, B. 1988. Marine microbiology. Cambridge: Cambridge University Press. Azam, F., D.C. Smith, and J.T. Hollibaugh. 1991. The role of microbial loop in antarctic pelagic ecosystems. Polar Research, 10(1), 239-243. El-Sayed, S.Z. 1988. Fragile life under the ozone hole. Natural History, 97(10), 72-80. Karentz, D. 1994. Ultraviolet tolerance mechanisms in antarctic marine organisms. In S. Weiler and P. Penhale (Eds.), Ultraviolet
radiation in Antarctica: Measurements and biological effects
(Antarctic Research Series, Vol. 62). Washington, D.C.: American Geophysical Union. Piddock, L.J.V., and R.N. Walters. 1992. Bactericidal activities of five quinolona for E. coli strains with mutations in gene encoding the SOS response or cell division. Antimicrobial Agents and Chemotherapy, 36,814-825. Weiler, S., and P. Penhale (Eds.). 1994. Ultraviolet radiation inAntarctica: Measurements and biological effects (Antarctic Research Series, Vol. 62). Washington, D.C.: American Geophysical Union.
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