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. Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
PAR Photosynthetic Active Radiation, 400–700 nm is sufficient to sustain growth and the UV-B is attenuated. In eutrophic waters with high concentrations of phytoplankton,
the UV-B radiation is attenuated within the first meter of the water column Kirk, 1994; ¨
Hader, 1997, while UV-B in more oligotrophic regions can penetrate several meters into the water column Smith et al., 1992. The phytoplankton cells respond differently to
varying PAR intensities, e.g. under light-saturated conditions, the growth rates are high, and the cell concentration of light-capturing pigments e.g. chlorophyll a and fuco-
xanthin are low, while the opposite is found under low-light conditions Falkowski, 1980; Goericke and Welschmeyer, 1992. Excessive irradiance can inhibit the growth
rates and induce production of photoprotective pigments Demers et al., 1991.
UV-B radiation can also have a major impact on algal cells, and some of these effects can be hard to distinguish from the response to excessive light, e.g. the production of
photoprotective pigments Goericke and Welschmeyer, 1992, while other look alike responses to low-light conditions, e.g. lower growth rates Buma et al., 1996. Even
though some of the responses to either low-light or excessive light conditions appear to be similar to UV-B effects, the physiological changes in the algae cells may be
completely different. For example can reduced growth in UV-B exposed algal cells originate from DNA damages Mitchell and Karentz, 1993, damage to the photo-
synthesis apparatus Smith et al., 1992; Cullen and Neale, 1994, or changed nutrient metabolism Behrenfeld et al., 1995; Garde and Gustavson, 1999. The aim of the
present study was to assess the responses of algal cells to UV-B radiation and different PAR intensities. The impact of the different light regimes and light intensities on growth,
14
uptake of C, excretion of dissolved organic carbon DOC, cell morphology, and
pigmentation was conducted on a culture of the marine prymnesiophyte, Emiliania huxleyi. We choose E
. huxleyi as test organism because this algae is one of the most dominant algal species in oceanic waters Brown and Yoder, 1994, also at higher
latitudes where the impact of increased UV-B radiation due to ozone depletion is ¨
believed to be most pronounced Vernet and Smith, 1997; Bjorn et al., 1998.
2. Materials and methods
2.1. Culture conditions and irradiation A non-calcifying flagellated strain of Emiliania huxleyi Lohman Hay and Mohler,
isolated from Oslo Fjord in 1990, was grown in batch cultures at 158C in K30 medium Keller et al., 1987. No coccolith production took place during the experiment. Prior to
the experiment, four cultures of E . huxleyi were acclimated to a light intensity of 106
22 21
22
mmol photons m s
light source: Pope 36 W m with a light dark cycle of 16:8 h,
in 3-l Erlenmeyer bottles. Three bottles were made of Pyrex and one of quartz. Several cell doublings were observed to allow for adequate light acclimation. The experiment
started at the beginning of a light period, i.e. t 5 0, when the algae were in an exponential growth phase. One bottle was kept at original light intensity, 106 mmol
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photons m s
ML, one exposed to lower light intensity, 53 mmol photons m s
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LL, and one exposed to higher light intensity, 176 mmol photons m s
HL. The
K . Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
101
light intensity was regulated by adding or removing black tulle from the outside of the glass bottles. The black tulle reduced the light intensity equally at wavelengths from 200
to 700 nm. The algae in the quartz bottle were exposed t 5 0 to artificial UV-B
22
radiation Phillips TL 20 W 12 RS with an intensity of 0.52 W m UV, in addition
22 21
to the original light intensity, 106 mmol photons m s
. The UV-B tubes were covered by a film of cellulose acetate, which absorbs wavelength , 280 nm, i.e. UV-C radiation.
In order to minimize the change of the filter properties of the film, the cellulose acetate was preburnt for 48 h at a distance of 1 m from four UV-B lamps. The UV-B spectra
22 22
21
provided a biologically effective radiation UV-B of 0.185 W m
666 J m h
,
BE
when weighted with Setlow’s DNA spectrum normalised to 300 nm Setlow, 1974.
22
Daily UV-B up to 1700 J m
may be considered realistic UV-B intensities for
BE
temperate regions during summer months, according to Behrenfeld et al. 1993. The spectras and intensities of the PAR and UV-B lamps Fig. 1 were measured with a
spectroradiometer, OL-754 Optronics Laboratories, INC. The spectrophotometer was calibrated against a Deuterium lamp standard for the spectral range between 200 and 400
nm.
2.2. Experimental procedure
6
At the start of the experiment, each culture had a density of 0.75 3 10 60.061 3
6 14
10 cells per ml and was inoculated with 1200 mCi NaH CO solution The
3 14
International Agency for C Determination, VKI, Hørsholm, Denmark, giving a final
21
concentration of 400 mCi l . Subsamples for cell number, cell morphology, uptake of
14 14
C, excretion of DO C, and pigment concentration were taken regularly during the 28 h of the experiment.
2.2.1. Enumeration and cell volume Samples for microscopic enumeration of algal cells were fixed in a Lugol solution and
stored at 58C. At least 100 cells were counted, and the growth rates were calculated. The cell diameter for at least 50 cells was measured in the microscope, and cell volume was
estimated by assigning the cells to the geometric shape of a sphere. The dimension was multiplied by a factor 1.1 to compensate for shrinkage due to fixation Choi and
Stoecker, 1989. For bacterial enumeration, samples were fixed in glutaraldehyde and stored at 58C. Acridine orange-stained samples were filtered onto 0.2-mm black
polycarbonate filters and counted in an epifluorescence microscope Hobbie et al., 1977.
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2.2.2. Determination of PO C and DO C
14
The activity of particular organic carbon PO C was determined from triplicate 5-ml subsamples filtered onto 0.45-mm cellulose nitrate filters 25 mm. To remove labeled,
dissolved inorganic carbon, the filters were fumed in acid for 5 min and subsequently placed in a scintillation vial with 10 ml Ecoscint A scintillation cocktail. The filtrate
14
from the PO C filtrations was collected for determination of dissolved organic carbon
14
excreted from the algal cells DO C. The aquatic solution was acidified to eliminate
102 K
. Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
Fig. 1. The spectras and light intensities to which cultures of Emiliania huxleyi were exposed. A Intensities of the PAR lamps, and B intensity of the artificial UV-B tubes. HL denotes high light 176 mmol photons
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m s
, ML denotes Medium Light 106 mmol photons m s
, LL denotes Low Light 53 mmol photons
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22
m s
, and UV denotes exposure to UV-B 0.52 W m . The UV was, in addition to the UV-B light,
exposed to PAR with the same intensity as ML. The spectras and light intensities were measured outside the Erlenmeyer bottles. Note the difference in units of the Y-axes.
inorganic carbon and degassed for 24 h, after which Instagel scintillation cocktail was added. All radioactivity counting was carried out using a liquid scintillation counter
Beckman Model 1802. The release of extracellular organic material was expressed as
14
percentage of total assimilated C Zlotnik and Dubinsky, 1989:
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14
DOC excretion 5 DO C 3 100 PO C 1 DO C Dissolved inorganic carbon DIC was measured by acidification of 1 ml sample with a
subsequent quantification of CO in an IR gas analyser.
2
K . Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
103
2.2.3. Pigments For pigment analyses i.e. chlorophylls and carotenoids, 50–200 ml samples were
collected on glass fibre filters Whatman GF C, 47 mm and immediately frozen in liquid nitrogen, after which they were stored until analysis. The filters were extracted in
100 acetone, sonicated on ice for 10 min and stored at 58C for 24 h Bidigare, 1991. A mixture of 1.0 ml pigment extract and 0.3 ml H O were injected into a Shimadzu-
2
LC10 HPLC system, and the pigments were analysed according to Wright et al. 1991, ¨
with modifications as described in Schluter and Havskum 1997. The HPLC system
14
was calibrated with pigment standards from The International Agency for C De-
termination, VKI, Hørsholm, Denmark. 2.3. Statistical test of data
For all data collected an ANOVA: Two-factor analysis was used. P values ,0.05 were regarded as significant.
3. Results
