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
The number of E . huxleyi cells in all light regimes was of the same order of
magnitude during the first 15 h of exposure Fig. 2A. Cell divisions took place during the dark period, and the cell number increased significantly in all but the UV-B-exposed
light regimes. The cell volume in the cultures only exposed to PAR decreased parallel to
3 3
the increase in cell number from 58 mm at 15 h to 24–31 mm at 24 h in LL, ML and
3
HL, while the cell volume in the UV-B-exposed cells increased significantly to 80 mm
3
during the first hours of the dark period but declined to 55 mm at the end, i.e. t 524 Fig. 2B. Microscopic observations revealed that the enlarged UV-B-exposed cells did
not look normal, since the vacuole area within the cells increased very markedly. The change in cell volume and appearance was notable already after 4 h of exposure, i.e. 2.7
22
kJ m UV-B
, in the UV-B-exposed culture. No difference in bacterial abundance was
BE
observed between the four light treatments data not shown.
14
Uptake of C took place only during light periods in all light regimes Fig. 3A.
14
PO C was significantly highest in the HL and ML cultures after 24 h of incubation. The
14
PO C in UV and LL cultures was only half the activity in HL and ML. Despite a lack
14
of cell division, the UV-B-exposed culture had the same incorporation rate of C as the
14
culture exposed to LL. The PO C was actually higher during the first 10 h of incubation in the UV-B-exposed culture compared to LL, but thereafter and for the rest of the
14
experimental period, PO C was slightly higher in LL than in UV. The activity per cell was highest in the UV-B-exposed cells after 24 h of incubation data not shown,
although growth based on cell division was not observed in the cells. The DOC
14
excretion followed the levels of the PO C and increased during the experiment in HL and ML from 0.3 to 1.6, while a smaller increase from 0.3 to 0.6 was found in LL
and UV-B Fig. 3C. The algal cells from the UV and the ML cultures received the same intensity of PAR
light, differing only in UV-B load. The variance in algal response between these two
104 K
. Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
Fig. 2. A Cell number, and B cell volume for cultures of Emiliania huxleyi grown at four different light conditions. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as in Fig. 1.
14 14
cultures is thus regarded to be due to the added UV-B exposure. The PO C and DO C
22
were significantly inhibited at UV-B doses higher than 2.7 kJ m
Fig. 4. The
BE
inhibition increased as the UV-B dose increased. No recovery from the UV-B induced inhibition was observed after 8 h in the dark.
In general, the cultures exposed to only PAR light exhibited an identical increase in the concentration of chlorophyll a chl a during the light period, and the chl a
concentration remained constant during the dark period Fig. 5A. No changes in chl a concentration, however, were observed in the UV-B-exposed culture. The carotenoids,
i.e. the sum of fucoxanthin fuco, 199-hexanoyloxyfucoxanthin 199-hex, diadinox- anthin DD, diatoxanthin DT, and b-carotene, increased in the cultures exposed to
PAR intensities alone, while the carotenoids in the UV-B-exposed cells were more or less constant during the experiment Fig. 5B. The ratio between the carotenoids and chl
a was significantly lower in the LL, while no major difference was observed between ML, HL and UV Fig. 5C.
The chl a concentration per cell was more or less constant in all light regimes during
21
the first 7 h of incubation, around 190 fg chl a cell , whereupon it increased at different
rates in all light regimes until the dark period Fig. 6A. The highest increase in chl a per
K . Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
105
14 14
Fig. 3. A PO C, B DO C, and C estimated DOC excretion for cultures of Emiliania huxleyi grown at four different light conditions. The horizontal bar indicates the dark period from 16 to 24 h. Abbreviations as
in Fig. 1. Error bars in A and B represent the standard deviation n 53.
21
cell was found in ML, with a concentration up to 357 fg chl a cell . The cell
21
concentration of fuco was significantly higher in LL, 266 fg fuco cell , than in any of
the other light regimes while there was no change in cell concentration of fuco in UV Fig. 6B. In contrast, the cell concentration of 199-hex was highest in ML, 318 fg
21
199-hex cell , and lowest in LL Fig. 6C. HL and UV also displayed an increase in the
199-hex content per cell during the light periods Fig. 6C. The cellular concentration of
21
DD was significantly highest in UV, 109 fg DD cell Fig. 6D, and lowest in LL
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. Garde, C. Cailliau J. Exp. Mar. Biol. Ecol. 247 2000 99 –112
Fig. 4. The effect of the biologically effective UV-B dose, UV-B weighted with Setlows DNA spectrum
BE 14
14
Setlow, 1974 on the PO C and DO C in Emiliania huxleyi cultures exposed to UV-B UV as percentage of the controlML. The vertical bar indicates 8 h in the dark.
during the entire incubation period. The highest DT concentrations per cell were found
21
in ML and HL during the light period, 11 and 12 fg DT cell , respectively, but the DT
concentration declined in the dark period, also in UV Fig. 6E.
4. Discussion
