AQUATIC MICROBIAL ECOLOGY
Aquat Microb Ecol
Vol. 25: 75–86, 2001
Published August 10
Impact of UV-B radiation on microalgae and
bacteria: a mesocosm study with computer
modulated UV-B radiation addition
Sten-Åke Wängberg*, Angela Wulff, Claes Nilsson, Ulrica Stagell
Botanical Institute, Göteborg University, PO Box 461, 405 30 Göteborg, Sweden
ABSTRACT: Effects of ambient and enhanced UV-B radiation (UVBR) on marine microbial plankton
communities were assessed in a model ecosystem at Kristineberg Marine Research Station (KMRS)
on the Swedish west coast. The system consisted of 16 aquaria (40 l) filled with surface seawater and
semicontinuously run by replacing 10 l of their contents with filtered seawater twice a day. The
aquaria were placed outdoors and the ambient solar radiation was reduced by 70% using neutral
screens. Four different levels of UVBR were applied, each in 4 replicates: nothing, ambient, ambient
+10% and ambient + 20%. The enhanced UVBR was supplied by fluorescent tubes whose intensity
was modulated by the ambient radiation to give a constant percentage increase. Variables measured
were nutrients (N, P, Si), composition of phytoplankton species and pigments, bacterial and primary
productivity, and bacterial cell numbers. Statistically significant UVBR effects were found for carbon
allocation, size distribution of primary productivity and phytoplankton species composition. It was
also found that UVBR exposure during the development of the phytoplankton communities increased
their sensitivity to UVBR in short-term carbon dioxide fixation measurements. We propose that this
was due to an adaptation of the community to UVBR, including an increased production of components within the photosynthetic apparatus damaged by UVBR. The UVBR had no significant effect on
the total biomass of phytoplankton and bacteria.
KEY WORDS: UV-B radiation · Primary production · Marine microbial plankton · Plankton · Pigment
Resale or republication not permitted without written consent of the publisher
INTRODUCTION
The effects that increased UV-B radiation (UVBR;
280 to 320 nm) have on marine microbial ecosystems
are still unclear. Several observations have shown
that UVBR negatively affects major ecological processes such as carbon dioxide fixation, bacterial growth
and nutrient turnover (e.g., Vincent & Roy 1993, Wängberg et al. 1996a, Davidson 1998). Within phytoplankton communities, a large species-specific UVBR sensitivity has been shown, and enhanced UVBR has been
found to change the species composition (Worrest et al.
1981, Wängberg et al. 1996b). UVBR can also photolyse refractory DOM thereby releasing carbon and
nutrients (Wetzel et al. 1995, Bushaw et al. 1996) leading to increased biological activity (Karentz et al. 1994,
*E-mail: swa@fysbot.gu.se
© Inter-Research 2001
Wängberg et al. 1999, Gustavson et al. 2000). Algae
and bacteria are also capable of avoiding the negative
effects of UVBR by producing screening pigments,
quenching toxic photoproducts or increasing their repair capacity (Mitchell & Karentz 1993, Karentz 1994,
Davidson 1998).
To understand the effects UVBR has on plankton
communities demands long-term experiments which
integrating negative, positive and adaptive processes.
Such experiments are unfortunately rare (Wängberg
& Selmer 1997 and references therein). UVBR effects
on community level are complex and UVBR has been
shown to stimulate the microalgae through either reduction of grazers (Bothwell et al. 1994) or the photolysis effect on DOM (Wängberg et al. 1999). Furthermore, time-scales are important when designing ecologically relevant experiments. Effects on the structure
of algal communities are predominantly found early in
76
Aquat Microb Ecol 25: 75–86, 2001
the experiments (Cabrera et al. 1997, Keller et al. 1997,
Wulff et al. 2000) when the succession is high. Smaller
effects observed later in the experiments can be due to,
for example, competition for resources (nutrient or
space) or UVBR adaptation of the community (Laurion
et al. 1998, Odmark et al. 1998).
Not only the consequences on biomass and productivity are important when analysing UVBR effects
in microbial communities; other important factors include UVBR-mediated changes in size distribution
within the plankton community and carbon allocation
to different biomolecules. Both of these factors are indicative of the status of the community and are
important for the value of phytoplankton as prey. It has
previously been assumed that larger cells are generally
less sensitive to UVBR than smaller cells (Karentz et al.
1991, Bothwell et al. 1993). Wängberg et al. (1996b)
found that the smaller algae were more sensitive if the
analysis was reduced to a single class of diatoms (Centrales), but not when the whole community was analysed. In the study of Laurion et al. (1998) the fraction of
total chlorophyll a (chl a) in picoplankton increased significantly when the community was shielded from
UVBR. Recently Mostajir et al. (1999) found that UVBR
reduced the number of large (5 to 20 µm) but not small
(< 5 µm) phytoplankton, which they assumed was due
to UVBR-induced changes in grazing pressure (reduction in ciliates that graze on small phytoplankton).
Even if UVBR does not reduce the primary productivity, it has been shown to affect the allocation of fixed
carbon. Arts & Rai (1997) showed that the UVBR effects
on carbon allocation are species-specific. Furthermore,
Wängberg et al. (1998) found that the fraction allocated
to polysaccharides increased when the fraction allocated
to low molecular weight (LMW) compounds decreased
during the development of plankton communities under
UVBR exposure. In a diatom-dominated microbenthic
community the fractions allocated to proteins and lipids
increased while the fractions allocated to polysaccharides and LMW compounds decreased after UVBR exposure (Sundbäck et al. 1997).
A complication when comparing different UVBR
experiments is the discrepancy in UVBR levels used.
In most UVBR experiments the only treatment is that
the ambient UVBR is excluded (Bothwell et al. 1993,
Cabrera et al. 1997, Wulff et al. 1999), thus showing
effects of ambient UVBR rather than consequences of
increased UVBR following a reduction of the stratospheric ozone layer. When the UVBR is enhanced artificially, a fixed light intensity is generally added for a
few hours per day (Worrest et al. 1981, Keller et al.
1997, Laurion et al. 1998, Odmark et al. 1998, Mostajir
et al. 1999, Wängberg et al. 1999). This can mimic an
increase of the UV-B dose d–1 following a specific
reduction of the stratospheric ozone layer. There are,
however, substantial natural variations in ambient
radiation due to, for instance, clouds and time of day.
As a consequence, a fixed addition of UVBR can result
in strong differences in the relative increase depending on the time of day and the actual weather situation.
This can produce biased results, as UVBR effects are
dependent on the ratios between UV-B, UV-A and PAR
(Karentz 1994, Prezelin et al. 1994, Franklin & Forster
1997, Laurion et al. 1998). Moroz et al. (1999) recently
showed that the motility of Nitzschia linearis was unaffected on sunny but not on overcast days when
exposed to the same UVBR intensities.
Therefore, the best experimental approach is to
modulate the UVBR enhancement so that a constant
fraction of the incident surface irradiance is achieved.
Such systems have been used by terrestrial ecologists
to achieve realistic simulations of ozone depletion
(e.g., McLoed 1997) and were used in the present
study to investigate UVBR effects on a marine plankton model ecosystem. The exposure system was also
used for an experiment with benthic microbial communities that was run simultaneously with the experiment
presented here (Wulff et al. 2000).
The experiment was specifically designed to study
the effects of ecologically relevant UVBR levels on
growing microbial communities and to determine
whether this affects important functional and structural variables: primary and bacterial productivity,
composition of the phytoplankton community, and
concentration of nutrients and DOC. Special attention
was paid to the carbon allocation and carbon fixation,
which was fractionated in different size classes and
measured at different times of the day.
MATERIAL AND METHODS
Location and weather conditions. The experiment
was performed at Kristineberg Marine Research
Station (KMRS) on the Swedish west coast (58.2° N,
13.4° E) for 10 d, from 3 to 13 June 1997. The weather
during the whole experiment was sunny. The incoming
PAR had typical values of ca 1800 µmol photons m–2 s–1
(midday). Downwelling UVBR was measured between
07:00 and 17:00 h using a cosine-corrected sensor (SUL
240, International Light Inc., Newburyport, MA, USA)
calibrated against a spectroradiometer (OL754, Optronic
Laboratories, Orlando, FL, USA). The maximal UV-B
irradiation on each day was, on average, 0.84 ± 0.11 W
m–2 and the mean UVBR dose d–1 was 3.96 ± 0.52 kJ m–2
(mean ± SD). The stratospheric ozone layer during the
experiment was on average 343 Dobson Unit (DU) ± 4
(Total Ozone Mapping Instruments [TOMS] data).
Experimental set up. The outdoor model ecosystem
consisted of 16 aquaria (40 l: 28.5 × 28.5 × 49 cm [length
Wängberg et al.: Impact of UV-B radiation on microalgae and bacteria
77
× width × height]) made of UVB-transparent Plexiglas
(Fig. 1). The aquaria were placed in opaque plastic
cases covered with UVB-transparent Plexiglas surrounded by a continuous water flow for temperature
control (10 to 14°C). To reduce the light intensity in the
aquaria, strips of black tape were attached to the Plexiglas on top of each box, covering half the area. The
tape reduced the irradiance by 70% (measured at
noon, central in the aquarium). After the experiment
the walls of the aquaria were inspected and no algal
growth was found on them.
The aquaria were initially filled with 40 l of natural
surface seawater from the Gullmar fjord, screened
through a 160 µm net to exclude large grazers. Twice
daily (07:00 and 19:00 h) 10 l of water from each aquarium was replaced with seawater filtered through a tangential-flow filter (Minikros, Microgen Inc., Laguna
Hills, CA, USA; pore size 0.2 µm). The added water
was a mixture of surface (90%) and deep water (10%)
from the KMRS water system. The water in the aquaria
was kept in motion by gentle bubbling.
Each aquarium was randomly given 1 of 4 different
treatments (4 replicates for each treatment): (1) NoUVB,
shielded from UVBR by a Mylar foil (Mylar-D, DuPont,
Wilmington, DE, USA; Fig. 1); (2) AMB, exposed to
ambient UVBR; (3) UVB+, exposed for a 10% increased UVBR; and (4) UVB++, exposed for a 20%
increased UVBR.
Enhanced UVBR was provided by 2 UV-B fluorescent
tubes (TL4W/12, Philips, Eindhoven, The Netherlands)
placed on the Plexiglas screen covering each UVB+ and
UVB++ aquarium. Wavelengths shorter than 290 nm
(UV-C radiation) were screened out by cellulose diacetate film (Erik S. Ekman AB, Stockholm, Sweden;
Fig. 1), which was replaced daily. To ensure equal shading effects, dummies for the tube holders were placed on
the NoUVB and AMB aquaria. The UVBR intensity was
controlled by a computer system linked to a UVBR sensor (International Light SUL 240). The signal from the
sensor was continuously transferred to a computer,
which, through a digital controlled power supply
(Thurlby Thandar Instruments, Huntington, UK), modulated the lamp voltage to desired levels. The applied
voltage varied between 2.3 and 6 V. The composition of
the lamp spectra was almost independent of the voltage
within this range (Fig. 2). The desired levels were determined from the voltage-radiation relation of the lamps
determined before the beginning of the experiment. The
function of the radiation system was checked continuously by a sensor placed under an additional lamp. The
modulation was not totally continuous but had 5 trigger
values (0.1, 0.4, 0.7, 1.0 and 1.3 W m–2, ambient UVBR)
and was run between 07:00 and 17:00 h. Thus, the enhanced levels given by the UV-B fluorescent tubes
(Philips TL4W/12) varied throughout the daily exposure,
mirroring the natural UVBR curve. An example of how
the enhancement worked is shown in Fig. 3. The 10 and
20% enhancement levels refer to unweighted UVBR.
When weighted with a biological weighting function determined on the marine alga Phaeodactylum tricornutum
(Cullen et al. 1992), the enhancement levels were +15
and + 30%, respectively. Daily doses of PAR and UVBR
in the aquaria are given in Table 1. There was some variation among the individual lamps and before the experiment started the efficiency of all lamps was checked and
a selection was made, excluding the most deviant ones.
After the selection, the coefficient of variation between
aquaria was calculated to 15%.
Fig. 1. Transmittance of Mylar foil, Plexiglas, polyethylene
bag, scintillation vial and cellulose diacetate film between 250
and 700 nm. Transmittance was measured on a Shimadzu UV2401PC spectrophotometer
Fig. 2. Lamp spectra depending on applied voltage. The
spectra, recorded at noticed voltages, were normalised to 1
at 300 nm
Aquat Microb Ecol 25: 75–86, 2001
78
Fig. 3. Example of the function of the UV-B radiation (UVBR)
exposure system on 13 June 1997 (Day 10). Shown are the
radiation levels in the aquaria exposed to ambient UVBR
(AMB) and 20% increased UVBR (UVB++) and the additions
from the UV-B lamps
Primary productivity measurements. Primary productivity was measured in separate temperaturecontrolled incubators covered with UV-B-transparent
Plexiglas and neutral screens (nylon nets), thus reducing the ambient radiation by 70%. Enhanced UVBR
was not used during the productivity measurements.
One set of incubators was covered with Mylar foil to
exclude UVBR. Primary productivity was measured by
a 14C-technique to assess 4 different aspects of the primary productivity: total primary productivity, carbon
allocation; size-fractionated primary productivity and
variation during the day.
The sensitivity to UVBR during the incubation was
quantified as the ratio between primary productivity
measurements with Mylar screen (NoUVBR) and incubations without a screen (ambient spectra). If the
UVBR had any negative effect the ratio was >1.
Samples were incubated in polyethylene bags (Whirlpak, Aldrich, Stockholm, Sweden) for size fractionation,
while the other samples were incubated in scintillation
vials (high quality, Packard Instrument Company Inc.,
Meriden, CT, USA); transmittances are shown in Fig. 1.
When the samples were incubated, 10 ml water was
added to each vial and 50 ml to each polyethylene bag.
For total primary productivity 2 µCi 14C-bicarbonate
was added to each vial, and 3 parallels were placed in
each incubator and incubated for 4 h (approx. 09:00 to
13:00 h). After the incubation, 300 µl formaldehyde
(35%) was added and the samples were acidified with
HCl to pH < 2 and bubbled to remove infixed inorganic
carbon. Scintillation cocktail (Beckman, Ready Gel) was
added and the radioactivity (in disintegrations min–1)
was measured on a Beckman scintillation counter
(Model 1802, Beckman Coulter Inc., Fullerton, CA,
USA). For allocation experiments, 5 µCi 14C-bicarbonate
was added to 3 parallels from each aquarium and incubated at the same time as for total primary productivity
but only under ambient radiation. Immediately after the
incubation the water was filtered through a nylon filter
(Gelman Science, Ann Arbor, MI, USA; 0.2 µm). The filter was placed in a scintillation vial, 0.4 ml distilled water was added, and the filter was refrigerated at –20°C
until fractionation. The carbon was fractionated into proteins, LMW compounds, polysaccharides and lipids following the method used by Li et al. (1980). With this fractionation method the LMW fraction includes hydrophilic
compounds that are not collected on a glass fibre filter
(Whatman GF/F). It contains primarily mono- and
oligosaccharides, amino acids and oligopeptides. With
the incubation times used in this study more than 80% of
the 14C-labelled LMW were carbohydrates (S.Å.W. unpubl. data). The lipids were further separated into polar
and neutral lipids on silica columns as described by
Sundbäck et al. (1997). Total primary productivity and
carbon allocation were measured on 4 occasions (Days 0,
3, 6 and 10). All primary productivity measurements included extra samples to which formaldehyde was added
before incubation. The values in these were used to correct for abiotic uptake of 14C-carbonate.
Table 1. Daily ambient and added radiation doses in the aquaria. nd: not determined; UVB+: 10% increase in UV-B exposure;
UVB++: 20% increase in UV-B exposure. *Start time 10:00 h
Date (1997)
June 3
June 4
June 5
June 6
June 7
June 8
June 9
June 10
June 11
June 12
June 13
Day
PAR
(mol photons m–2)
UV-B
(KJ m–2)
UV-B addition UVB++
(kJ m–2)
UV-B addition UVB+
(kJ m–2)
0
1
2
3
4
5
6
7
8
9
10
12.07
14.46
13.28
15.92
14.83
13.24
9.08
11.30
14.04
14.64
12.54
nd
*2.98*
4.05
4.15
4.29
3.61
3.42
3.67
4.35
4.67
4.37
*0.37*
0.58
0.57
0.64
0.47
0.47
0.48
0.66
0.70
0.62
*0.19*
0.29
0.28
0.32
0.24
0.23
0.24
0.33
0.35
0.31
Wängberg et al.: Impact of UV-B radiation on microalgae and bacteria
For postincubation size fractionation of the primary
productivity, 10 µCi 14C-bicarbonate was added to each
bag and incubated for 4 h (09:00 to 13:00 h). After incubation 9 ml of the sample was filtered through polycarbonate filters (Poretics Corp., Livermore, CA, USA) with
3 different pore sizes (0.2, 2 and 10 µm), and 9 ml was
used for measuring total primary productivity. Each filter
was washed with 2 × 1 ml unlabelled seawater and
placed in a scintillation vial, scintillation cocktail was
added and the radioactivity was measured as above. The
radioactivity in the filtrate was measured as for total primary productivity. The recovery of each filtration was
calculated as the sum of the radioactivity on filters and
filtrate divided by the total. Samples with extreme recoveries (>1.5 or < 0.8 of the total) were discarded and
not used in the calculations (22% of all). The mean recovery of the remaining 84 samples was 1.00 ± 0.17. Size
fractionation was done on Days 2 and 9.
The daily variation in photosynthetic activity and its
sensitivity to ambient UVBR were determined in samples taken from the NoUVB and AMB aquaria on
Day 8. Incubations were done as for total primary productivity but for 2 h only, starting at 08:00 h, and again
at 10:00, 12:00, 14:00 and 16:00 h. The UVBR doses received during the incubations were 0.77, 1.53, 1.56 and
0.96 kJ m–2, respectively.
Species composition and photosynthetic pigments.
Species were counted in water preserved with acid
Lugol’s solution in sedimentation chambers. For a species to be included in the data set at least 40 cells had
to be counted. Cell numbers were transformed to cell
volume as in Wängberg et al. (1996b).
For pigment analysis 2 subsamples of 240 ml from
each aquarium were gently filtered onto GF/F and immediately frozen in liquid nitrogen. After 3 wk the filters were transferred to a low-temperature freezer
(–85°C) and stored for up to 3 mo until analysed. Two
ml of 100% methanol was added to the filters and the
extraction and HPLC-analysis continued according to
Wright & Jeffrey (1997) using a Linear 206 detector
(Spectraphysics, Mountain View, CA, USA). As we were
only looking for possible treatment effects, pigments
are expressed as ratios to chl a (area/area). Chl a was
quantified in µg l–1 as described by Wright & Jeffrey
(1997).
Bacterial productivity. Bacterial productivity was
measured using the microcentrifuge 3H-leucine incorporation technique (Smith & Azam 1992). The results
were transformed to µg protein h–1 assuming that 7.3%
(w/w) of the protein is leucine. From each aquarium,
4 samples (1.2 ml) were filled into Eppendorf tubes,
3.33 µCi 3H-leucine was added to each sample, and they
were incubated for approximately 1 h in the incubator
without Mylar foil (Eppendorf tubes, however, absorb
most of the UVBR). The incubations were terminated by
79
adding TCA (5% final concentration) and the samples
were processed according to Smith & Azam (1992).
Bacterial biomass. Bacterial biomass was estimated as
bacterial numbers in formaldehyde-preserved samples
using a flow cytometer (FACS Calibur, Becton-Dickinson,
Franklin Lakes, NJ, USA) after addition of the DNA stain
SYTO-13 (Molecular Probes Inc., Eugene, OR, USA). A
standard solution containing fluorescent beads (Fluoresbrite carboxyl YG, Polyscience Inc., Warrington, PN,
USA) was used (del Giorgio et al. 1996). The accuracy of
the flow cytometer counting was checked by microscopic counting of sample for each measurement session.
Nutrients and DOC. For nutrient analysis of both the
added water and the water in the aquaria (sampled
before each dilution), the samples were filtered through
cellulose acetate filters (Sartorius MiniSart N, 0.45 µm)
and stored at –80°C until analysed on an autoanalyser
system (Traacs, Bran & Luebbe Inc., Buffalogrove, IL,
USA). Samples for DOC were treated the same way
and stored in Pyrex tubes at –20°C until analysis on a
TOC-5000 (Shimadzu, Kyoto, Japan) as in Granéli et
al. (1996). Both nutrients and DOC were measured in
triplicate from each aquarium. The start values (Day 0)
were given from 5 replicates taken before the water
was distributed to the aquaria.
Statistical analyses. Statistical evaluations were done
with ANOVA. Post-hoc analyses were made by the Student-Newman-Keuls test, if necessary, after appropriate transformation. Cochran’s test (Winer et al. 1991)
was used to check for heterogeneous variances. p <
0.05 was accepted for significant differences.
RESULTS
General development independent of treatment
The inorganic nutrient concentrations in the water
added to the aquaria showed some irregularity in
Fig. 4. Nutrient concentration of the water that was used to fill
the aquaria. Each value is the mean of 3 replicates
Aquat Microb Ecol 25: 75–86, 2001
80
ammonium concentration but were otherwise low and
stable during the experiment (Fig. 4). DOC content
was on average 6.5 ± 0.61 mg l–1 during the first 5 d,
increased to 12.6 ± 1.39 mg l–1 between Days 6 and 8,
and thereafter decreased to initial values.
The ammonium concentrations in the water taken out
of the aquaria were significantly higher on Day 3 (407 ±
26 nM) than initial values but decreased to very low values on Days 6 and 10 (80 ± 12 nM). Nitrate + nitrite concentrations were low on Days 3 and 6 (0.44 ± 0.04 µM)
but increased on Day 10 (1.78 ± 0.39 µM), while the
phosphate concentration was more stable with a mean
concentration of 106 ± 5.1 nM during the whole experiment. The silicate concentration decreased from 2.4 ±
0.01 µM on Day 3 to 0.45 ± 0.01 µM on Day 10. The DOC
values increased from Day 0 to Day 3 (11.1 ± 1.25 mg l–1)
but decreased to 4.8 ± 0.38 mg l–1 on Days 6 and 10.
Total algal cell volume (Table 2) and carbon dioxide
fixation (Fig. 5) decreased between Day 0 and Day 3 in
all treatments but recovered during the experiment to
values as high as or higher than initial values. Chl a
values dropped significantly from 3.2 to 1.0 µg l–1
Table 2. Cell volumes of microalgae and heterotrophic flagellates (µm3 106 l–1). The total cell volume is based on more species
than shown. Ciliates are shown as cell numbers. AMB: exposure to ambient UV-B radiation. See Table 1 for other abbreviations
Seed
NoUVB
Dinophytes
Ceratium tripos
Dinophysis acuminata
Dinophysis norvegica
Gonyaulax grindleyi
Heterocapsa triquetra
Pentapharsodinium/
Scrippsiella sp.
Prorocentrum cf. aporum
Prorocentrum micans
Protoperidinium sp.
Protoperidinium bipes
Total ± SE
Cryptophytes
Plagioselmis prolongata
Teleaulax sp.
Total ± SE
Prasinophytes
Micromonas sp.
Nephroselmis sp.
Total ± SE
Total algal cell volume
Ciliates
UVB++
NoUVB
Day 10
AMB
UVB+
UVB++
19.87
1.10
0
0
13.21
0
10.90
0.24
37.48
4.94
0.78
10.08
16.26
0.24
33.13
5.64
0.63
8.80
11.75
0.71
37.57
4.61
0.72
7.17
22.63
0.55
37.84
5.23
0.66
5.31
0
0
1.63
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.28
0
0
0
0
0
0
0.58
0
0
182.83
0
0.30
4.01
0
131.42 ±
10.4
0.33
0
0
0
101.66 ±
30.9
0.52
0
1.46
0
88.25 ±
27.9
0.19
0.15
0
0
120.37 ±
23.2
0
0
0
1.49
3.13 ±
2.5
0
0
0
1.27
1.27 ±
0.7
0
0
0
0.38
0.38 ±
0.3
0
0
0
1.33
3.61 ±
3.6
68.45
60.17
0.26
0.24
0
0
0
0.05
0
71.66
200.83 ±
16.7
63.72
49.44
0.35
0.75
0
0
0.15
0.07
0
39.78
154.40 ±
10.3
53.13
49.64
0
0.50
0
0
0
0
0
54.02
157.28 ±
15.9
72.01
50.67
0.39
0.21
0
0
0
0.05
0
57.44
182.04 ±
22.8
250.91
133.97
0
2.27
136.39
6.52
0.50
3.72
202.74
695.19
1432.21 ±
112.3
343.45
130.02
0.61
3.70
122.23
7.73
0
5.69
244.41
628.10
1485.94 ±
125.6
74.66
74.66
68.47
68.47 ±
23.8
60.88
60.88 ±
21.1
35.90
35.90 ±
21.4
52.24
52.24 ±
18.1
0
0
0
0
0
0
0
0
126.3
85.6
211.9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
597
401 ±
31.7
2947 ±
943
377 ±
54
3501 ±
164
335 ±
23.7
3082 ±
24
348 ±
15.3
3274 ±
240
10.74
10.08
20.83 ±
6.3
1456 ±
112.4
472 ±
112
7.85
6.84
14.69 ±
5.8
1502 ±
124.3
515 ±
124
8.20
5.24
13.44 ±
5.5
1567 ±
161.7
519 ±
162
13.21
7.16
20.37 ±
5.9
1694 ±
156
748 ±
156
Diatoms
Chaetoceros curvisetus
46.62
Chaetoceros danicus
50.98
Chaetoceros decipiens
0
Chaetoceros sp. (solitary)
1.64
Chaetoceros tenuissimus
0
Entomoneis sp.
0
Licmophora sp.
0
Nitzschia reversa
0.63
Pseudonitzschia sp.(solitary) 0
Skeletonema costatum
27.53
Total ± SE
127.40
Prymnesiophytes
Chrysochromulina sp.
Total ± SE
Day 3
AMB
UVB+
34601
287.45
239.70
98.94
92.92
0
0.57
2.17
2.47
119.58
166.70
5.90
9.31
0.42
0.52
3.61
5.77
163.51
184.66
871.20
967.63
1552.78 ± 1670.25 ±
427.8
161.4
81
Wängberg et al.: Impact of UV-B radiation on microalgae and bacteria
Fig. 6. Allocation of carbon to different biochemical classes
(percentage of total carbon fixation). Four hour incubations in
ambient radiation reduced by 70%. Values are mean for all
treatments. Error bars represent SE (n = 16). Significant differences between treatments are indicated by labels over the
bar. Different labels indicate a significant difference. LMW:
low molecular weight compounds; Neu. lip.: neutral lipids;
Pol. lip.: polar lipids; Polys.: polysaccharides
Fig. 5. Primary productivity (incorporation of 14C-bicarbonate
over 4 h). All experiments were done in incubators where the
ambient radiation was reduced by 70%. Error bars indicate
SE (n = 4). Significant differences between treatments are
indicated by labels over the bar. Different labels indicate a
significant difference. (A) Productivity in ambient radiation,
including UVBR; (B) ratio of productivity without UVB to productivity with UVBR
between Day 0 and 3 and stabilised around 1.0 µg l–1
throughout the experiment (Table 3).
The pattern of carbon allocation changed slightly
during the experiment with a significant decrease in
the fraction allocated to proteins and polar lipids and
an increased allocation to LMW compounds (Fig. 6).
According to total cell volume the initial proportions
of different phytoplankton groups were 16, 52 and 30%
for dinoflagellates, diatoms and prymnesiophytes, respectively (Table 2). In general, the cell counts were
confirmed by the pigment concentrations where peridinin and fucoxanthin initially showed high ratios to
chl a (Table 3). From Day 3 onwards the pigment ratios
decreased significantly for the pigments peridinin, 19’-
Table 3. Chlorophylls (Chl) and carotenoids (ratios to chl a) in the different treatments. Values are based on 2 samples from the
4 replicate mesocosms, respectively. ALLO: alloxanthin; βCAR: β-carotene; DDX: diadinoxanthin; DIAT: diatoxanthin; FUCO:
fucoxanthin; HEX: 19’-heaxanoyloxyfucoxanthin; PERI: peridinin; ZEA: zeaxanthin
Day 0
NoUVB AMB UVB+ UVB++
Chl a (µg l–1)
Chl b
Chl c1+c2
Chl c3
HEX
ALLO
βCAR
DDX+ DIAT
FUCO
PERI
ZEA
3.048
0.022
0.451
0.115
0.149
0.412
0.037
0.315
0.385
0.075
0.003
3.114
0.016
0.445
0.107
0.146
0.408
0.026
0.318
0.370
0.074
0.010
2.948
0.019
0.451
0.106
0.146
0.431
0.029
0.333
0.383
0.075
0
2.972
0.021
0.455
0.107
0.148
0.404
0.035
0.337
0.385
0.076
0
Day 3
NoUVB AMB UVB+ UVB++
0.920
0
0.487
0.063
0.114
0.260
0.110
0.408
0.499
0.008
0
0.924
0.478
0.097
0.113
0.252
0.075
0.441
0.519
0.017
0
0.756
0
0.494
0.100
0.114
0.262
0.037
0.415
0.544
0.011
0
0.994
0
0.460
0.096
0.113
0.277
0.051
0.419
0.488
0.016
0
Day 7
NoUVB AMB UVB+ UVB++
1.1594
0
0.509
0.065
0.014
0
0.088
0.359
0.728
0
0.006
1.020
0
0.476
0.070
0.008
0
0.076
0.332
0.658
0
0.008
0.944
0
0.575
0.073
0.008
0
0.066
0.343
0.728
0
0.007
1.000
0
0.595
0.079
0.009
0
0.064
0.378
0.746
0
0
Day 10
NoUVB AMB UVB+ UVB++
0.987
0
0.678
0.141
0.012
0
0.082
0.641
0.724
0
0
0.918
0
0.745
0.093
0
0
0.043
0.630
0.745
0
0.014
0.835
0
0.784
0.116
0
0
0.072
0.579
0.737
0
0
0.880
0
0.681
0.120
0
0
0.080
0.581
0.679
0
0
82
Aquat Microb Ecol 25: 75–86, 2001
Table 4. Summary of effects of UV-B radiation (UVBR) on all variables studied. +:
significant effects (p < 0.05, n = 4); –: non-significant effects; nd: not determined
Microalgal biomass
Microalgal composition
Photosynthetic pigments
Primary productivity
Total productivity
Ratio in productivity ± UVBR
Size fractionation of productivity
Carbon allocation
Bacterial biomass
Bacterial productivity
Nutrients and DOC
Day 3
Day 7
Day 10
–
–
–
nd
nd
–
–
+
–
–
–
+a
+
–
nd
–
–
–
nd
+
–
nd
–
–
+
+b
–
–
–
–
a
Day 2; bDay 9
was larger in the AMB than in the
NoUVB communities. This shows that
the AMB communities were more sensitive to UVBR during the incubation
than the NoUVB communities (Fig. 7B).
At the beginning of the experiment
(Day 2), a smaller fraction of the fixed
carbon was retained on the 10 µm filter in the UVB++ treatment than in the
other treatments. Later in the experiment (Day 9), algae >10 µm fixed relatively more carbon in the AMB treatment than in either the UVB+ or the
UVB++ (Fig. 8).
The fraction allocated to polysaccharides was significantly affected by the
hexanoyloxyfucoxanthin, alloxanthin and chl b, indicating a shift in the microalgal community composition. Furthermore, chl c1+c2, fucoxanthin and diadinoxanthin increased significantly, indicating a dominance of diatoms, which was confirmed by the algal
counts. The cell counts also showed that the small
prasinophytes Micromonas sp. and Nephroselmis sp.
appeared in the latter part of the experiment; this was
not, however, confirmed by the pigment ratios.
The bacterial cell numbers decreased from 2.0 × 106
to 1.0 × 106 cells l–1 between Day 0 and Day 3 and continued to decrease to 0.5 × 106 cells l–1 on Day 10. In
contrast to carbon dioxide fixation, bacterial protein
production increased from 17.2 ± 1.1 ng l–1 h–1 at the
start to 114.3 ± 14.8 on Day 10 despite decreased bacterial cell numbers.
Effects of UVBR
Parameters significantly affected by the UVBR treatment are shown in Table 4.
With incubation under ambient radiation, the primary productivity, as the amount of chl a, was higher
in the NoUVB treatment than the other treatments at
the end of the experiment (not significant, p = 0.073 on
Day 10) (Fig. 5A). The ratio of primary productivity
without to primary productivity with UVBR on Day 10
was significantly larger in the UVB+ than in the
NoUVB treatment (Fig. 5B). This indicates that the primary productivity in UVB+ was more sensitive than
NoUVB to ambient UVBR during the incubation.
In the diurnal variation study on Day 8, significantly
more carbon was fixed by the NoUVB communities than
the AMB when incubated under ambient radiation starting at 10:00, 14:00 and 16:00 h (Fig. 7A). In the incubations started at 10:00 h, the ratio, in primary productivity,
of incubations done with to those done without UVBR
Fig. 7. Primary productivity (incorporation of 14C-bicarbonate
over 2 h) at different times of the day. Ambient radiation
reduced by 70% (with or without UV-B). Error bars represent
SE (n = 4). Significant differences between treatments are
indicated by labels over the bar. Different labels indicate a
significant difference. (A) Productivity in ambient radiation,
including UVBR; (B) ratio of productivity without UVBR to
productivity with UVBR
Wängberg et al.: Impact of UV-B radiation on microalgae and bacteria
83
Fig 9. Percentage of total fixed carbon allocated to polysaccharides. Four hour incubations in ambient radiation reduced
by 70%. Error bars represent SE (n = 4). Significant differences between treatments are indicated by labels over the
bar. Different labels indicate a significant difference
No significant treatment effects were found for algal
and bacterial biomass, composition of photosynthetic
pigments and bacterial leucine incorporation. No significant UVBR effects were found for concentrations of
inorganic nutrients or DOC. There was, however, a
strong tendency to higher phosphate concentrations in
the NoUVB treatment on Day 10 (p = 0.053).
DISCUSSION
Relevance of exposure levels
Fig. 8. Fraction of total carbon fixation retained on filters with
different pore sizes. Four hour incubations in ambient radiation reduced by 70%. Error bars represent SE (n = 4). Significant differences between treatments are indicated by labels
over the bar. Different labels indicate a significant difference.
(A) Day 2; (B) Day 9
UVBR treatment on Days 3 and 7, but in different
ways. On Day 3, a smaller fraction was allocated to
polysaccharides in the NoUVB treatment than in either
the UVB+ or the UVB++ treatments, while on Day 6
more carbon was allocated to polysaccharides in the
NoUVB treatment than in the AMB and UVB++ treatments (Fig. 9).
A significant treatment effect was found in the species composition on Day 10 (Table 2). Two species,
Pseudonitzschia sp. and Nitzschia reversa, were significantly affected by the UVBR treatments. For Pseudonitzschia sp. a significantly (p < 0.007) higher cell volume was found in AMB than in any of the other
treatments. The same pattern was found for N. reversa
but because of non-homogeneous variance it was not
possible to include UVB++ in the statistical analysis.
This study was designed to determine the effects
that a reduction in the stratospheric ozone layer would
have on natural microbial communities in temperate
latitudes. By allowing the ambient radiation to modulate the addition of UVBR, the ratio of UVBR to other
wavelengths was retained. When the weighted intensities and a radiation amplification factor of 1.6 (calculated for a generalised plant spectrum [Caldwell et al.
1986] in Madronich 1993) were used, the 2 enhancement levels corresponded to a 19% and 9% reduction
of the ozone layer. The TOMS data for May to August
1997 and 1998 at the location of the experiment
showed that episodes with more than 9% reduction
from the mean DU levels (353 DU) were found during
15% of the days, but during 1 d only, the reduction
increased by 19%. The UVB+ level is thus relevant for
short-term episodes of a reduced ozone layer during
summertime. If the 1997–98 data are compared with
the mean 1979–80 value (362 DU), there was a > 9%
reduction during > 20% of the days and a 19% reduction during > 4% of the days. The UVB++ level is thus
representative of the highest levels of reduction due to
the long-term decline, to date, in stratospheric ozone,
84
Aquat Microb Ecol 25: 75–86, 2001
thus becoming more relevant as the thinning of the
ozone layer continues. As the UVBR effects at the
water surface, despite being the strongest, are not representative of the whole water column, and primary
productivity is inhibited by PAR and UVAR at the surface on sunny days, we reduced the ambient radiation
by 70% using a neutral screen. As the attenuation in
the water is not uniform within the PAR-UV range an
unavoidable consequence is that the depth to which
this reduction corresponds is wavelength dependent.
To obtain a realistic enhancement of UVBR, we developed a computer-modulated UVBR exposure system, where the intensity of the enhanced radiation was
directly related to the ambient UVBR, important for
keeping realistic ratios of UVBR to other wavelengths.
Similar techniques have earlier been used in terrestrial
experiments (McLeod 1997 and references therein).
Set-up of the model ecosystems
In their natural environment phytoplankton can
have a high net production rate but grazing keeps the
standing stock more or less constant (though under
a high turnover rate). In small-scale experimental
systems the grazing pressure usually becomes too low,
the phytoplankton standing stock increases, and the
turnover rate declines. To avoid this problem, we diluted the community semicontinuously to keep a high
net growth and succession rate without increasing the
standing stock. Through this dilution, less than 1% of
the organisms in the seed community would remain at
the end of the experiment, which gives species favoured by conditions in the aquaria (including different
degrees of UVBR) good opportunities to increase. The
selection pressure also induced a succession independent of the differences in UVBR exposure including
relative increase in diatoms, increased leucine uptake
per bacterial cell and reduced chl a values.
Treatment effects
The results show that ambient UVBR affects marine
plankton communities, not only in surface water but
also down to substantial parts of the euphotic zone,
and that it is affected by the present levels of ozone
reduction. The effects were not dramatic and included
no changes in biomass or total productivity of either
phytoplankton or bacteria. The effects were instead
found on phytoplankton species composition, sizefractionated primary productivity, carbon allocation
and the sensitivity to ambient UVBR in primary productivity. Also Laurion et al. (1998) and Mostajir et al.
(1999) found that the total phytoplankton biomass was
not affected by enhanced UVBR, but changed structurally, although the UVBR exposure level in these
experiments was much higher than ours. To evaluate
fully the ecological significance of the results from the
present study, not only should the experiment be replicated elsewhere, but also it is essential to include more
trophic levels to gain knowledge about possible negative consequences on the whole ecosystem.
That carbon dioxide fixation of communities developed under UVBR exposure were more sensitive to
UVBR than communities shielded from UVBR was also
found in similar experiments performed in the Southern Ocean (Wängberg & Wulff unpubl. data) where the
increased sensitivity was even more pronounced. We
hypothesise that this was an adaptive process that
functions as follows: communities developed under
UVBR that partially inhibit some process or component
build up an over-capacity to compensate for the loss.
When the community is instead shielded from UVBR,
the over-capacity results in increased carbon dioxide
fixation. Potential photosynthetic processes are, for
example, the carbon dioxide fixation protein RUBISCO
or components in the chloroplastic electron transport,
both of which are known to be sensitive to UVBR (Vincent & Roy 1993, Strid et al. 1994).
This hypothesis presumes that the over-capacity is
built up each night, through either repair or new synthesis, but successively gets reduced each day on
UVBR exposure. That the UVBR-induced damages are
compensated during the night is supported by the significant difference in sensitivity between NoUVB and
AMB treatments at 10:00 h, before the communities
had been exposed to high UVBR, but not when the
incubation was started at 12:00 h (Fig. 7B). Differences
in sensitivity to UVBR depending on the time of day
was earlier shown by Prezelin et al. (1994), with the
lowest inhibition found in the middle of the day when
the UVBR to (UVR + PAR) ratio was highest. They suggested that the reduced sensitivity in the middle of the
day is due to a protective mechanism induced during
the morning. This cannot be the case in our experiment, as the tolerance was not enhanced when incubations were started at 12:00 h. An increased sensitivity
to UVBR in the primary productivity of communities
when exposed to UVBR questions the use of short-term
primary productivity measurements to predict the ecological consequences of UVBR.
We found that communities developed under enhanced UVBR had a smaller part of the carbon dioxide
fixation in the largest fraction (>10 µm). Our data support the data published by Mostajir et al. (1999) and
Wängberg et al. (1996b), which show that smaller
phytoplankton are favoured by UVBR, in contrast to
the data of Laurion et al. (1998). The agreement
between the data presented here and in those of
Wängberg et al.: Impact of UV-B radiation on microalgae and bacteria
Wängberg et al. (1996b) might depend on the fact that
both experiments were marine and went through the
same type of succession from dinoflagellates to diatoms. In contrast, the succession described by Laurion
et al. (1998) was limnic and towards an increase in the
proportion of picocyanobacteria.
As the incubations before filtration in our experiment
lasted 4 h, there is a risk that carbon fixed by small
algae that are grazed on during the incubation were
retrieved in the wrong size class. We could not estimate this risk. The lack of effects on ciliate cell numbers indicates, however, that a possible effect was
treatment independent. Thus it should not have
affected the differences found between treatments.
As in Wängberg et al. (1998) a UVBR-mediated
increase in the fraction allocated to polysaccharides
was balanced with a decreased allocation to LMW
compounds at the beginning of the experiment. On
Day 7, the pigment composition indicated that diatoms
dominated the communities, and the fraction allocated
to polysaccharides was decreased by the UVBR treatment as in the diatom-dominated microbenthic communities reported by Sundbäck et al. (1997). It is not
possible to say whether these changes between the
early and later parts of the experiment were due to different species or whether it was a physiological
response to the UVBR exposure. Our incubations for
measuring carbon allocation were all done under
ambient radiation with the aim to study the long-term
changes in carbon metabolism. Goes et al. (1996) have
shown that UV radiation (UV-A + UV-B) has an acute
effect on carbon allocation in diatom-dominated phytoplankton communities. They found that UV radiation
inhibited the synthesis of storage carbohydrates that
were easily hydrolysed but not of the more stable
structural carbohydrates. Their fractionation procedure differs from ours but agrees with our results in the
early part of the experiments.
In the present study, the only species affected by UVBR
were the pennate diatoms Pseudonitzschia sp. and
Nitzschia reversa, both belonging to Biraphidae. Our
findings agree with those of Karentz et al. (1991), who
found that the pennate diatom Nitzschia kerguelensis
was more sensitive than the centric diatoms to UVBR, a
difference that may be explained by differences in morphology (cell shape, chloroplasts) as well as habitat.
That the effects of UVBR on development of microbial communities depend on the nutrient condition was
clearly shown in microbenthic communities by Wulff et
al. (2000), where addition of nutrients (P, N and Si)
reduced the UVBR effects. We found no effects on concentration of dissolved nutrients at the p < 0.05 level,
but a tendency was found for phosphate on Day 10,
with lower concentrations in all UVBR-exposed communities than in AMB. If the NoUVB is compared with
85
all the UVBR-exposed communities, the effect is significant (p = 0.005). This was probably due to a larger
uptake of phosphate, as we do not know any way in
which UVBR directly can reduce the phosphate concentration. Interestingly, several other studies have
indicated that the demand for phosphate is increased
by UV-B exposure (Hessen et al.1995, Wängberg et al.
1998, Wängberg et al. 1999).
CONCLUSIONS
From this 10 day experiment to assess the effects of
ambient and enhanced UVBR on marine microbial
communities on the Swedish west coast we reached
the following conclusions:
• Ambient UVBR does not directly threaten the phytoplankton or bacterial biomass in pelagic water, nor does
the enhancement in UVBR that has occurred following
short- and long-term reductions in stratospheric ozone.
• Ambient and enhanced UVBR, however, induce several changes within the phytoplankton community,
including changes in carbon allocation, size distribution and species composition, that might change the
ecological interactions between phytoplankton and
other parts of the marine food web.
• UVBR exposure during the development of phytoplankton communities changes their sensitivity to UVBR
in short-term carbon dioxide fixation measurements.
Acknowledgements. We thank Kristineberg Marine Research
Station for providing excellent working facilities. We also
thank Wilhelm Granéli and colleagues at the Limnological
Institute in Lund University for help with the DOC measurements. Financial support was provided by research grants
from the Swedish Environmental Protection Agency, the
Swedish Natural Science Research Council, and the Swedish
Council for Planning and Coordination of Research. Additional support was given by funds from Hierta-Retzius, Wilhelm and Martina Lundgren, Captain Carl Stenholm and
Helge Ax:son Johnson.
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Editorial responsibility: Karin Lochte,
Rostock, Germany
Submitted: July 9, 2000; Accepted: April 25, 2001
Proofs received from author(s): July 19, 2001
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