Rol2008e

Rol2008e
RESEARCH ARTICLE
Photosynthetic response of Nodularia spumigena to UVand
photosynthetically active radiation depends on nutrient
(N and P) availability
Michael Y. Roleda, Malin Mohlin, Bagmi Pattanaik & Angela Wulff
Department of Marine Ecology, Marine Botany, Gothenburg University, Gothenburg, Sweden
Correspondence: Present address:
Michael Y. Roleda, Institute for Polar Ecology,
University of Kiel, Wischhofstrasse 1-3, Bldg.
12, D-24148 Kiel, Germany. Tel.: 149 431
600 1235; fax: 149 431 600 1210; e-mail:
[email protected]
Present addresses: Malin Mohlin,
Department of Marine Ecology, Marine
Botany, Gothenburg University, PO Box 461,
SE 405 30 Gothenburg, Sweden.
Bagmi Pattanaik, MSU-DOE Plant Research
Laboratory, Michigan State University, 322
Plant Biology, East Lansing, MI 48824, USA.
Angela Wulff, Department of Marine Ecology,
Marine Botany, Gothenburg University, PO
Box 461, SE 405 30 Gothenburg, Sweden.
Received 18 March 2008; revised 7 July 2008;
accepted 10 July 2008.
First published online 27 August 2008.
DOI:10.1111/j.1574-6941.2008.00572.x
Editor: Riks Laanbroek
Keywords
cyanobacteria; effective quantum yield;
nitrogen fixation; P–E curve; phosphorus.
Abstract
Biomass of N. spumigena is distributed within the dynamic photic zone that
changes in both light quantity and quality. This study was designed to determine
whether nutrient status can mitigate the negative impacts of experimental
radiation treatments on the photosynthetic performance of N. spumigena.
Cyanobacterial suspensions were exposed to radiation consisting of photosynthetically active radiation (PAR = 400–700 nm), PAR1UV-A ( = PA, 320–700 nm),
and PAR1UV-A1UV-B ( = PAB, 280–700 nm) under different nutrient media
either replete with external dissolved nitrate (N) and orthophosphate (P; designated as 1N/1P), replete with P only ( N/1P), or replete with N only
(1N/ P). Under low PAR (75 mmol photons m2 s1), nutrient status had no
significant effect on the photosynthetic performance of N. spumigena in terms of
rETRmax, a, and Ek. Nodularia spumigena was able to acclimate to high PAR
(300 mmol photons m2 s1), with a corresponding increase in rETRmax and Ek.
The photosynthetic performance of N. spumigena cultured with supplemental
nitrogen was more susceptible to experimental PAR irradiance. Under UVR,
P-enrichment in the absence of additional external N ( N/1P) induced lower
photoinhibition of photosynthesis compared with 1N/ P cultures. However, the
induction of NPQ may have provided PSII protection under P-deplete and
PAR1UVR conditions. Because N. spumigena are able to fix nitrogen, access to
available P can render them less susceptible to photoinhibition, effectively
promoting blooms. Under a P-deficient condition, N. spumigena were more
susceptible to radiation but were capable of photosynthetic recovery immediately
after removal of radiation stress. In the presence of an internal P pool in the Baltic
Sea, which may be seasonally available to the diazotrophic cyanobacteria, summer
blooms of the resilient N. spumigena will persist.
Introduction
Phototrophs require essential macronutrients for photosynthesis and growth. Phytoplankton primary production
in freshwater systems is usually controlled by phosphorus
(P) availability, whereas in marine environments elemental
nitrogen (N) is more commonly the limiting nutrient
(McCarthy & Carpenter, 1983). Diazotrophic cyanobacteria,
that have the ability to fix atmospheric dinitrogen (N2) may,
ecologically, be at an advantage to other phytoplankton in
nitrogen-limited environments.
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Freshwater inflow containing high concentrations of
inorganic and organic nutrients from land drainage and
river discharge contributes to eutrophication of coastal
waters. Based on research at various spatial and temporal
scales, a scientific consensus has emerged that nitrogen
represents the largest pollution problem in coastal waters
(NRC, 2000). Excess phosphorus in estuaries and brackish
water can, however, interact with the available nitrogen to
affect ecological structure and function adversely (Howarth
& Marino, 2006). Management efforts in the Baltic Sea have
seen the decrease of external phosphorus and nitrogen load
FEMS Microbiol Ecol 66 (2008) 230–242
231
Light and nutrient effects on photosynthesis of Nodularia
by c. 30% in the 1990s (Pitkänen et al., 2001). However, due
to the poor oxygen condition at the sediment–water interface, there is an increased benthic release of dissolved
inorganic phosphorus (DIP) concentration from the internal phosphorus pool (Pitkänen et al., 2001).
Cellular synthesis of nucleic acids and membrane
phospholipids, and energy transfer through tri- and biphosphorylated nucleotides requires phosphorus. In diazotrophic cyanobacteria, lack of available phosphorus can
result in decreased photosynthesis, growth, and cellular
phosphorus content, and loss of polyphosphate storage
granules. Low phosphorus availability, coupled with low
light, can further suppress heterocyst formation with a
subsequent reduction in N2 fixation and cellular nitrogen
content, and loss of their prominent gas vacuoles (Thompson et al., 1994).
In the Baltic Sea, the annual recurrence of massive
cyanobacterial blooms occurs during late spring and summer (Finni et al., 2001; Stolte et al., 2006) dominated
by Nodularia spumigena, Aphanizomenon flos-aquae, and
Anabaena spp. These taxa are able to adjust their vertical
position in the water column facilitated by their gas vesicles,
which can potentially expose them to high solar radiation
during certain periods of the day.
Summer bloom-forming cyanobacteria may possess
adaptive mechanisms to limit damage by excessive amounts
of photosynthetically active radiation (PAR) and UV radiation (UVR). Photoinhibition occurs not only as a photoprotective energy dissipation mechanism but also upon
photoinactivation or photodamage to the D1 protein of
photosystem II (PSII) reaction centers under conditions of
excess PAR. Exposure to UVR could further reduce maximum photosynthetic rates and efficiency. The targets of
UVR are numerous, which include: decrease in electron flow
from reaction centers to plastoquinone, damage to the
oxidizing site and reaction center of PSII, and degradation
of parts of the D1/D2 heterodimer, among others (Grzymski
et al., 2001; Turcsányi & Vass, 2002). On the other hand,
UVR protection mechanisms include the production of
UV-absorbing compounds such as mycosporine-like amino
acids (MAAs). Synthesis of MAAs was reported in several
cyanobacteria when exposed under UVR (e.g. Wulff et al.,
2007; Pattanaik et al., 2008).
Initiation of a cyanobacterial bloom is related to a
number of abiotic factors including nutrients, temperature,
and hydrographic conditions. Abiotically driven mass occurrence of N. spumigena is reportedly dependent on
phosphorus reserves and an optimum temperature, whereas
A. flos-aquae occurs in cooler water and is more dependent
on wind-induced mixing and upwelling of nutrients
(Kononen, 1992; Karjalainen et al., 2007). Despite the fact
that the diazotrophic hepatotoxic species N. spumigena is
exposed to high solar radiation during the mid- to late
FEMS Microbiol Ecol 66 (2008) 230–242
summer bloom in the Baltic Sea, this is, to our knowledge,
the first study that has looked into the multifactorial
interactive effects of radiation and nutrient condition on
the photosynthetic rate and efficiency of N. spumigena.
Recently, Wulff et al. (2007) reported an isolate-specific
response of N. spumigena under UVR and nutrient-replete
conditions.
Most studies of Baltic N. spumigena focused on monitoring bloom dynamics and the regulating factors (e.g. nutrient
and temperature), hepatoxin production, and other physiological and biochemical parameters, except that very little is
known of its photosynthetic performance under different
light and nutrient regimes. The seasonal occurrence and
morphological characteristics of N. spumigena confine the
bloom within the dynamic photic zone that changes in both
light quantity and quality. In the presence of both, or the
absence of one of the essential nutrients, nitrogen and
phosphorus, this study aims to better understand the role
played by nutrients in affecting the photosynthetic process.
Whether the adverse effects of radiation (PAR and PAR1
UVR) are eased by supplemental nutrient fertilization is
discussed.
Materials and methods
Culture material
Nodularia spumigena Mertens (KAC 71) isolated from the
Baltic Sea was obtained from the Kalmar Algal Collection
(KAC), Kalmar University, Sweden. The culture, hereafter
called Nodularia (in reference to this study), was inoculated
into seven salinity f/2 medium in sterile 500-mL Nunc
bottles and left to grow for 2 weeks at 17 1 1C under a
16 : 8 h light : dark cycle of 75 mmol photons m2 s1.
Nutrient treatments
Natural deep seawater (salinity 37) collected at Kosterfjorden, Sweden, was filtered (GF/F) and diluted with MilliQwater to reduce salinity to seven. The growth medium
consisted of three treatments: (1) a medium with a replete
external dissolved nitrate (NO3, 955 mM) and orthophosphate (PO3
4 , 45 mM) designated as 1N/1P, corresponding
to the f/2 medium; (2) a medium with a replete external
dissolved orthophosphate only designated as N/1P; and
(3) a medium with a replete external dissolved nitrate only
designated as 1N/ P. The nutrients initially present in the
seven salinity seawater were 2.8 mM nitrogen and 0.7 mM
phosphorus.
Irradiation treatments
PAR (400–700 nm) of 300 mmol photons m2 s1 was obtained from 10 white fluorescent tubes (GE Polylux XL,
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232
F36W/860, Great Britain). In another setup, UVR
(280–400 nm) was supplemented with the addition of four
UVA-340 fluorescent tubes (Q-Panel, Cleveland, OH). To
cut off different wavelength ranges from the spectrum
emitted by the fluorescent tubes, quartz bottles (25 mL)
were covered with one of the following filters: Ultraphan
transparent (Digefra GmbH, Germany), Folanorm (Folex
GmbH, Germany), or Ultraphan URUV Farblos corresponding to the PAR1UV-A1UV-B (PAB), PAR1UV-A
(PA), and PAR treatments, respectively. UVR was measured
using a Solar Light PMA 2100 radiometer equipped with the
UV-A sensor PMA 2110 and the UV-B Sensor PMA 2106
(Solar Light, Philadelphia). Adjusted UVR below the cut-off
filters was 8.90 Wm2 UV-A and 0.65 Wm2 UV-B. The
available PAR was measured using a cosine quantum sensor
attached to a LI-COR data logger (LI-1000, LI-COR Biosciences, Lincoln, Nebraska). After high PAR (300 mmol
photons m2 s1) treatment, samples were exposed to low
PAR for recovery (1 and 4 h) and during the circadian
rhythm experiment (16 : 8 h light : dark photoperiod) under
10 and 60 mmol photons m2 s1, respectively.
Experimental design
To determine the photosynthetic capacity of Nodularia
under different nutrient conditions, 100 mL of stock culture
was diluted to 200 mL with corresponding media into 12
Nunc bottles corresponding to four replicates per nutrient
treatment. The cultures were maintained in semi-continuous growth for 1 week by replacing 50% and 25% of the
volume on day 4 and day 7 of the corresponding media,
respectively. On the second week, the cultures were grown as
batch culture until the end of the experiment. The Nunc
bottles were laid prostrate on the exposure table with the lid
closed. The bottles were gently shaken many times every day
and randomly moved around the exposure table to ensure
equal light treatment. The experiment was conducted inside
a temperature-controlled room at 17 1 1C. The average
temperature under the lamps was 20 2 1C.
To determine the circadian pattern in photosynthetic
efficiency under different radiation (PAR, PA, PAB) and
nutrient conditions (1N/ P, N/1P), 24 quartz bottles
were prepared. From the above experimental culture,
a 120 mL suspension was obtained from the high-lightacclimated 1N/ P- and N/1P-grown cyanobacteria,
respectively. Filled into each 25-mL quartz bottle was
10-mL suspension. The 12 quartz bottles corresponding
to a specific nutrient treatment were divided into three
groups and assigned the radiation treatment to obtain four
replicates for each radiation and nutrient treatment
combination. Corresponding 10 mL of new media were
added to each bottle. The quartz bottles were randomly
arranged in the exposure table and covered with corre2008 Federation of European Microbiological Societies
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M.Y. Roleda et al.
sponding filter foils. The bottles were gently shaken many
times every day.
Chlorophyll fluorescence measurements
The photosynthetic efficiency of Nodularia was measured
as variable fluorescence of PSII using a Water Pulse Amplitude Modulation fluorometer (Water-PAM) consisting of
the Emitter-Detector Unit Water-ED and PAM-Control
Universal Control Unit connected to a PC operated
with WINCONTROL software (Heinz Walz GmbH, Effeltrich,
Germany) (Roleda et al., 2006). The Water-PAM is specialized to study a highly diluted cell suspension and applicable
to study cyanobacterial photosynthesis using measuring
light-exciting chlorophyll fluorescence peaking at 650 nm.
Although the photosynthetic physiology and fluorescence
pattern of cyanobacteria differ in important respects from
those of plants, cyanobacterial DF/Fm 0 presents a useful
integrated measure of PSII activity (Campbell et al., 1998b).
Cell suspension was filled into 5-mL quartz cuvettes to
determine effective quantum yield, DF/Fm 0 , calculated as
(Fm 0 –F)/Fm 0 . F is the fluorescence yield of the irradiationadapted sample and Fm 0 is the maximum fluorescence yield
when a saturating pulse of 600-ms duration (c. 2750 mmol
photons m2 s1) was applied inside the Emitter-Detector
Unit. F0 was first measured with a red measuring light pulse
(c. 0.3 mmol photons m2 s1, 650 nm).
Rapid photosynthesis (in terms of relative electron transport rate, rETR = PFR DF/Fm 0 ; PFR = photon fluence rate
of PAR) vs. irradiance (E) curves (P–E curve) of Nodularia
under different nutrient conditions was measured in lowlight-acclimated samples before the start of the experiment
and in high-light-treated samples at day 7 (n = 3, chosen at
random from the four replicates). Cyanobacteria suspension
was exposed to increasing actinic red light intensity, making
up to eight points at 21, 30, 47, 69, 98, 137, 227, and
341 mmol photons m2 s1. Each actinic light treatment was
performed for 30 s before application of a saturating pulse to
determine rETR. Data points were plotted and curve fits
were calculated with the Solver Module of MS-Excel using
the least squares method comparing differences between
measured and calculated data. The hyperbolic tangent
model of Jassby & Platt (1976) was used to estimate P–E
curve parameters as:
rETR ¼ rETRmax tanh a EPAR rETRmax 1
where rETRmax is the maximum relative electron transport
rate, tanh is the hyperbolic tangent function, a is the
electron transport efficiency, and E is the PFR of PAR. The
saturation irradiance for electron transport (Ek) was calculated as the light intensity at which the initial slope
of the curve (a) intercepts the horizontal asymptote
(rETRmax).
FEMS Microbiol Ecol 66 (2008) 230–242
233
Light and nutrient effects on photosynthesis of Nodularia
To evaluate the photosynthetic performance of the
cyanobacteria grown in different media, 2 mL of the suspension was drawn from the Nunc bottles and DF/Fm 0 of
300 mL of the high-light-acclimated suspension was measured immediately. The rest of the suspension was exposed
to low light and DF/Fm 0 was measured after 1 and 4 h.
Photosynthesis was measured on the 1st, 4th, 7th, 9th and
14th day.
The interactive effect of radiation and nutrient conditions
on the circadian pattern of photosynthesis of Nodularia was
measured six times within the day and every 2 days for 1
week. DF/Fm 0 was measured under low PAR at 1 h after
daylight (07 : 00 h), at 1, 4, and 7 h after high PAR and UVR
(10 : 00, 13 : 00, and 16 : 00 h), and under low PAR at 1 and
4 h after the end of UV exposure (18 : 00, 21 : 00 h). The nonphotochemical-quenching (NPQ) parameter after a 7-h
exposure to light stress (high PAR1UVR) was derived
according to the equation: NPQ = (Fm–Fm 0 )/Fm 0 . For every
measurement, the 300 mL suspension used was discarded.
Nutrient analysis
Samples were regularly taken during the course of the timeseries experiment on the day photosynthesis was measured.
Nitrate in medium was quantitatively reduced to nitrite by
running through a cadmium column coated with metallic
copper. The nitrite produced was determined by diazotizing
with sulfanilamide and coupling with N-(1-naphthyl)-ethylenediamine to form a colored azo dye measured spectrophotometrically at 550 nm. Phosphate in the medium was
allowed to react with a composite reagent containing
molybdic acid, ascorbic acid and trivalent antimony. The
resulting complex was reduced to give a blue solution
measured at 885 nm. The standard nutrient analyses were
described by Parson et al. (1984).
Statistical analysis
Data were tested for homogeneity of variance (Levene
Statistics). Corresponding transformations (square root)
were performed to heteroskedastic data. Data on time-series
measurements were subjected to repeated measure ANOVA
(RMANOVA, P o 0.001) to determine the main effects of
nutrient enrichment and radiation treatment, and their
interactive effect on photosynthetic yield. The remaining
available nutrient in the medium under different radiation
treatments was also tested using appropriate ANOVA
(P o 0.001). This was followed by Duncan’s multiple range
test (DMRT, P = 0.05). When a significant interaction
was observed, significant subgroups were determined by
plotting the means of each dependent factor against the
level of each independent (main) factor. Groupings were
based on a post hoc multiple comparison test. Statistical
FEMS Microbiol Ecol 66 (2008) 230–242
analyses were performed using the
Chicago, IL).
SPSS
program (SPSS,
Results
The rapid P–E curve parameters showed a variable response
between low-light- and high-light-acclimated Nodularia
(Fig. 1). The estimated slope alpha (a), a parameter for the
performance of both light-harvesting and photosynthetic
conversion efficiency, inclined steeply (a = 0.19–0.26; mean0.23 0.08) in low-light-acclimated samples compared with
high-light-acclimated samples with significantly lowinclined slopes (a = 0.05–0.06; mean = 0.05 0.01). Under
low PAR, comparison between different nutrient conditions
showed a relatively steeper a in Nodularia grown in 1N/ P
growth media compared with 1N/1P- and N/1P-grown
Nodularia. One-way ANOVA (P o 0.001) showed an insignificant difference in rETRmax (P = 0.060), a (P = 0.174), and
Ek (P = 0.077) under low PAR. After a 1-week exposure to
high PAR, a was also comparable among all nutrient
treatments (ANOVA, P = 0.254), while a significant difference
was observed in rETRmax and Ek (ANOVA, P = 0.001 and
0.028, respectively). DMRT (P = 0.05) showed rETRmax of
N/1P 4 1N/1P 4 1N/ P while Ek was 1N/1PZ
N/1P 4 1N/ P.
In low PAR-grown Nodularia, the initial effective quantum yields of the PSII (DF/Fm 0 ) were comparable when
stock culture was diluted with corresponding experimental
growth media at 0.523 0.01, 0.526 0.02 and 0.533 0.01
in 1N/1P, N/1P and 1N/ P, respectively. This was
reduced by 44–45% after a 1-day exposure to high PAR (Fig.
2a). The DF/Fm 0 of the semi-continuously grown culture
further decreased by 22% on day 4 in 1N/1P- and
1N/ P-grown cyanobacteria but not in the N/1P treatment where Nodularia was able to maintain their photosynthetic efficiency at a certain ‘optimum’ level. Photosynthetic
performance on day 7 was not significantly different from
the day 4 measurement. When the cultures were grown
without further dilution and nutrient replenishment (batch
culture), DF/Fm 0 on day 9 consequently decreased further by
19–20% in all nutrient treatments. Subsequently, photosynthesis of Nodularia was able to acclimate to the high PAR
on the 14th day without further significant reduction in
DF/Fm 0 regardless of the ‘available’ nutrient in the medium.
When transferred to low PAR, Nodularia was able to increase
photosynthesis (Fig. 2b and c). The increase in DF/Fm 0
occurs during the first hour by 17–32% across the timeseries measurements among the different culture media. An
additional 5–14% increase in DF/Fm 0 was observed after 4 h
in low PAR. This was, however, lower compared with the
initial DF/Fm 0 . In the course of the time-series measurement,
DF/Fm 0 showed a decreasing trend among the different
growth media regardless of whether exposed to high PAR
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234
M.Y. Roleda et al.
6
15
(a)
(d)
Relative ETR
12
4
rETRmax = 3.5
6
2
3
E k = 18
0
rETR max = 9.2
9
α = 0.19
α = 0.05
0
6
15
(b)
(e)
r ETR max = 10.9
12
Relative ETR
E k = 190
rETR max = 3.7
4
9
6
2
3
E k = 18
α=
0.20
0
α = 0.06
0
6
15
(c)
E k = 182
(f)
Relative ETR
12
4
9
rETR max = 3.0
6
2
3
E k = 12
0 α = 0.26
0
50
rETRmax = 4.9
α = 0.05 Ek = 92
0
100
150
200
250
300
Photon fluence rate (µmol photons m–2 s–1)
0
50
100 150 200 250 300 350 400 450 500
Photon fluence rate (µmol photons m–2 s–1)
Fig. 1. Rapid photosynthesis–irradiance curve (P–E) of low-light-acclimated (75 mmol photons m2 s1; left column, a–c) and high-light-acclimated
(300 mmol photons m2 s1; right column, d–f) Nodularia spumigena grown under nitrogen- and phosphorus-enriched (1N/1P) medium (a, d),
phosphorus-enriched ( N/1P) medium (b, e), and nitrogen-enriched (1N/ P) medium (c, f). PFR is the respective photon fluence rate of actinic light
and ETR is the relative electron transport rate. Saturating irradiance (Ek) is estimated as the point at which the initial slope (a) crosses maximum
photosynthesis (rETRmax) using the hyperbolic tangent model of Jassby & Platt (1976).
or allowed to recover in low PAR. Statistical analysis
(RMANOVA, P o 0.001, Table 1) showed a significant effect of
nutrient status in the medium on the photosynthesis of
Nodularia during exposure to high PAR. The effect of
nutrient status was still observed after 1 h incubation under
low light, but not after 4 h. DMRT (P = 0.05) showed
photosynthetic performance of Nodularia in different medium was N/1P 4 1N/1P 4 1N/ P under high PAR
and N/1P 4 1N/1PZ1N/ P after 1-h incubation
under low PAR.
Available phosphorus in phosphorus-replete (1N/1P
and N/1P) and phosphorus-deficient (1N/ P) growth
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media was utilized immediately. After 3 days in culture, up
to 68% of the external replete orthophosphate and 89% of
the natural phosphorus in seawater were consumed by
Nodularia (Fig. 3a). After replacing 50% and 25% volume
(first and second dilution, respectively) of the culture with
corresponding fresh media, available phosphorus was
replenished but was immediately utilized by Nodularia.
There was no further reduction in the available phosphorus
in different media after cessation of dilution from the 9th
day to the last sampling day. The remaining available
phosphorus differ significantly between the different media
used (Table 1, RMANOVA, P o 0.001; DMRT, P = 0.05;
FEMS Microbiol Ecol 66 (2008) 230–242
235
Light and nutrient effects on photosynthesis of Nodularia
(a)
0.4
Semi-continuous culture
Batch culture
+N /+P
–N /+P
0.3
+N / –P
0.2
0.1
0.0
(b)
Effective quantum yield,∆F /Fm’
0.4
0.3
0.2
0.1
0.0
(c)
0.4
0.3
0.2
0.1
0.0
1
4
7
9
Exposure treatment (days)
14
Fig. 2. Mean effective quantum yield (DF/Fm 0 ) of Nodularia spumigena
grown in nitrogen- and phosphorus-enriched (1N/1P), phosphorusenriched ( N/1P), and nitrogen-enriched (1N/-P) media under
300 mmol photons m2 s1 of PAR (a) and after 1-h (b) and 4-h (c)
recovery in low white light (10 mmol photons m2 s1). The culture was
grown under a 16 : 8 h light : dark photoperiod. The growth condition
was changed from semi-continuous (week 1) to batch culture (week 2).
Error bars are SDs (n = 5).
N/1P 4 1N/1P 4 1N/ P). Among phosphorus-replete media, phosphorus absorption was more efficient
under a nitrogen-enriched condition.
Total available nitrogen in the medium was not significantly different between the two nitrogen-replete growth
media (1N/1P and 1N/ P) at anytime during the
different water-sampling periods but significantly higher
FEMS Microbiol Ecol 66 (2008) 230–242
compared with the nitrogen-deficient medium (Table 1,
RMANOVA, P o 0.001; DMRT, P = 0.05, 1N/ PZ1N/
1P 4 N/1P). In the nitrogen-deficient medium, the
increase in total available nitrogen in the medium after every
dilution, while maintaining a semi-continuous culture, was
attributed to nitrogen fixation of Nodularia (Fig. 3b).
The circadian pattern of photosynthesis in Nodularia
during the light phase of the day showed a dynamic
oscillation in response to PAR fluence and spectral composition of irradiation treatments (Fig. 4). The DF/Fm 0 generally
increased with time (days) regardless of radiation and
nutrient treatment except for the slight decrease in DF/Fm 0
on the 7th day under the 1N/ P condition.
The degree of photoinhibition increased significantly
with time under a phosphorus-deficient condition. Under a
phosphorus-replete condition, the percent photoinhibition
of photosynthesis was relatively constant and did not
significantly vary between days of repeated exposure to high
PAR and UVR (Fig. 5a). Unexpectedly, photoinhibition of
photosynthesis under PAB in the phosphorus-deficient
medium was observed to level off from day 3 onward and
was lower on the 7th day compared with PAR and PA
treatment. The average percent photoinhibition over the
7-day period under PAR, PA, and PAB treatment was
43 12%, 50 11%, and 49 6% in 1N/ P, respectively,
and 31 6%, 43 4%, and 45 4% in N/1P, respectively. Statistical analysis showed significant effects of the
main factors (nutrient and radiation) as well as an interaction between the main factors (Table 1). Multiple post hoc
comparison tests showed three significantly different subgroups. Arranged in increasing degree of photoinhibition,
the subgroups are: N/1P (PAR) o N/1P (PA) 1
N/ P (PAR) N/1P (PAB) o 1N/ P (PAB) 1
N/ P (PA).
Photosynthetic recovery under low PAR showed a different trend. Under phosphorus-deficient condition, recovery
of photoinhibition under PAR, PA, and PAB treatments was
on the average maintained across the different sampling
periods (Fig. 5b). Under a phosphorus-enriched condition,
photosynthetic recovery increased on the 3rd sampling day
but was subsequently observed to decrease progressively on
the succeeding sampling days until the end of the experiment (Fig. 5b). To compensate loss of PSII function due to
photoinhibition, Nodularia were able, on average to recover
effective quantum yield of photosynthesis over the 7-day
period: 36 4%, 50 5%, and 46 4% in 1N/ P and
34 7%, 48 6%, and 48 6% in N/1P under PAR, PA,
and PAB treatment, respectively. RMANOVA showed a significant effect of preradiation treatments on the photosynthetic
recovery of Nodularia (Table 1). Cyanobacteria previously
exposed to UVR showed higher photosynthetic recovery
after a 4-h incubation under low PAR (DMRT, P = 0.05;
PAZPAB 4 PAR).
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236
M.Y. Roleda et al.
Table 1. ANOVA (repeated measure, RMANOVA and one-way ANOVA) and significance values for the main effects and interaction of main factors on the
photosynthesis of Nodularia spumigena and on the nutrient status of culture media
Experiment
Variable
Source of variation
df
Nutrient enrichment
Fv/Fm (under high PAR)
Fv/Fm (after 1 h low PAR)
Fv/Fm (after 4 h low PAR)
Nutrient
Total phosphorus
Total nitrogen
Photoinhibition
Medium
Medium
Medium
2
2
2
27.72
13.58
4.13
o 0.001
0.002
0.053NS
Medium
Medium
Medium (A)
Radiation (B)
AB
Medium (A)
Radiation (B)
AB
Medium (A)
Radiation (B)
AB
2
2
1
2
2
1
2
2
1
2
2
7924.42
394.35
51.88
36.97
5.98
1.20
112.71
2.43
5.37
112.71
2.43
o 0.001
o 0.001
o 0.001
o 0.001
0.010
0.288NS
o 0.001
0.116NS
0.033
0.001
0.711NS
Radiation
Radiation
2
2
0.521
302.04
0.611NS
o 0.001
Radiation
Radiation
2
2
0.333
8.841
0.725NS
0.008
Nutrient radiation
Recovery
NPQ
Nutrient
Total phosphorus
1N/ P medium
N/1P medium
Total nitrogen
1N/ P medium
N/1P medium
F-value
P-value
Significant.
NS, not significant.
Induction of nonphotochemical quenching (NPQ) was
significantly higher under the 1N/ P medium than in the
N/1P growth condition (RMANOVA, P = 0.033), and under
PAR1UVR than in PAR1UV-A and PAR treatment alone
(Fig. 6, RMANOVA, P = 0.007; DMRT, P = 0.05, PAB 4 PAZ
PAR). A decline to induce NPQ was observed after prolonged chronic exposure to the irradiation treatments
especially under PAR treatment alone.
Exposure to UVR affected nutrient uptake dynamics.
Under a phosphorus-replete condition, the remaining phosphorus in the medium were significantly higher when
Nodularia was exposed to PAR supplemented with either
UV-A1UV-B or UV-A alone compared with when exposed
to PAR alone (Table 1, ANOVA, P o 0.001; DMRT, P = 0.05,
PAB 4 PA 4 PAR). About 75% of the external replete
orthophosphate was absorbed by Nodularia under PAR
treatment compared with 39% and 20% under PA and PAB
treatments, respectively (Fig. 7a). Under a phosphorusdeficient condition, no significant difference was observed
under different radiation treatments on the absorption of
about 40–63% of the initial available phosphorus content in
the medium.
Conversely, the initial available total nitrogen in a nitrogen-replete medium was minimally consumed by Nodularia,
leaving 83–96% of the external nitrogen (and presumably
also fixed nitrogen) still available in the medium under
different radiation treatments. In the nitrogen-deficient
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
medium, as much as 90% of the initial total available
nitrogen in the medium was consumed under PAR treatments, and 75% and 61% under PA and PAB treatment,
respectively (Fig. 7b; Table 1, ANOVA, P o 0.001; DMRT,
P = 0.05, PABZPA 4 PAR).
Discussion
This study showed no significant nutrient effect on the
photosynthetic capacity of Nodularia in terms of rETRmax,
a, and Ek under low PAR. Under experimental high PAR,
photosynthesis of Nodularia was able to acclimate to the
increase in PFR with a corresponding increase in rETRmax
and Ek, but photosynthetic capacity was significantly lower
under the phosphorus-deficient nutrient condition. Longterm exposure to a high PFR of PAR showed that supplemental nitrogen with or without simultaneous phosphorus
enrichment enhanced photoinhibition of photosynthesis
during the semi-continuous growth. The negative ‘excess’
N effect was, however, dampened when dilution with new
nitrogen-enriched medium was terminated. Nodularia were
also more susceptible to the negative impact of UVR under
nitrogen-replete and phosphorus-deplete nutrient conditions but were capable of dynamic recovery of photosynthesis when UVR was removed.
Changing the growth condition from semi-continuous to
batch culture may potentially affect the physiology of
FEMS Microbiol Ecol 66 (2008) 230–242
237
Light and nutrient effects on photosynthesis of Nodularia
1.0
50
Batch culture
+N /+P 0.8
µM P
40
–N /+P
30
0.6
+N /–P
0.4
10
0.2
0
0.0
(b) 1000
500
800
400
600
300
400
200
200
100
µM N
20
0
µM P (+N / –P)
Semi-continuous culture
µM N (–N /+P)
(a)
0
1
4
After 7 After 9
dilution
dilution
Sampling days
14
Fig. 3. Time course mean phosphorus (a) and nitrogen (b) concentration in different culture media (1N/1P, N/1P, 1N/ P) of Nodularia
spumigena grown in a semi-continuous culture (week 1) and in a batch
culture (week 2). Values of overlapping bars correspond to the secondary
y-axis (right-hand side). After each dilution to maintain semi-continuous
culture, the nutrient content was estimated (column without error bars).
Error bars are SDs (n = 3).
+N/–P
Day 1
Nodularia. The downward trend in DF/Fm 0 was, however,
already observed during semi-continuous culture and
stabilized during batch culture. [Correction to previous
sentence made on 1 September 2008, after first online
publication.] On the other hand, changing growth medium
from semi-continuous to batch culture did not seem to
affect nitrogen fixation (Fig. 3b).
N2 fixation by Nodularia exceeds their own nitrogen
demand by 10–12% and contributes c. 50% of the nitrogen
demand of the total phytoplankton community (Stal &
Walsby, 2000; Stal et al., 2003). Besides being able to fix
molecular nitrogen, Nodularia are known to be able to store
phosphorus. In this species, the cellular phosphorus reserve
is reported to primarily determine growth and bloom
formation rather than the presence of a high nitrogen pool
(Karjalainen et al., 2007). Model simulations showed that
the interannual variation of Nodularia bloom in the Gulf of
Finland was dependent on phosphorus condition, with the
surface layer temperature as the bloom coregulator (Lilover
& Laanemets, 2006). Nodularia is able to sustain physiological activities relying on cellular phosphorus storage and
effective remineralization of organic phosphorus compounds (Vahtera et al., 2007), making diazotrophic cyanobacteria probably more iron limited than thought
previously (Stal et al., 2003) and more competitive under a
nitrogen-limited environment.
Our study showed that phosphorus enrichment in the
absence of additional external nitrogen ( N/1P) induced
lower photoinhibition of photosynthesis under UVR compared with 1N/ P cultures. Although no 1N/1P-enriched culture was exposed to PAR1UVR, the presence of
phosphorus enrichment did not ease the effect of high PAR
Day 3
Day 5
Day 7
Effective quantum yield, ∆F/Fm'
0.5
0.4
0.3
0.2
0.1
0.0
–N/+P
0.5
0.4
0.3
0.2
0.1
0.0
Times of day
Fig. 4. Circadian pattern of the mean effective quantum yield (DF/Fm 0 ) of Nodularia spumigena grown in nitrogen-enriched (1N/ P) and phosphorusenriched ( N/1P) media exposed to different radiation conditions consisting of PAR, PAR1UV-A (PA), and PAR1UV-A1UV-B (PAB) during the light
phase of the 16 : 8 h light : dark photoperiod. Low (early morning and late afternoon) and high (middle of the light phase) PFD was used to broadly
simulate light intensity variation within the day. Error bars are SDs (n = 4).
FEMS Microbiol Ecol 66 (2008) 230–242
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
238
M.Y. Roleda et al.
Day 5
Day 7
µM P (+N/–P)
(a) 70
50
40
30
20
–N /+P
15
0.2
10
0.1
5
0
(b) 750
(b) 70
50
30
20
10
PAR
PA
PAB
PAR
+N/–P
PA
PAB
–N /+P
Fig. 5. Time-series photoinhibition of photosynthesis (a) after a 7-h
exposure to high PAR and UVR in different radiation and nutrient
treatments expressed as percent reduction in DF/Fm 0 relative to the initial
value under low PAR, and photosynthetic recovery (b) expressed as
percent increase in DF/Fm 0 after an 8-h exposure to high PAR and UVR
and after 4 h under low PAR. Error bars are SDs (n = 4).
0.30
Day 1
0.25
Day 3
0.20
Day 5
0.15
0.10
0.05
0.00
PAR
PA
PAB
PAR
+N/–P
PA
PAB
–N /+P
Fig. 6. Nonphotochemical quenching (NPQ) as a function of nutrient
and radiation treatment combinations in Nodularia spumigena. Values
represent NPQ after a 7-h exposure to high PAR and UVR, in the middle
of the light phase of the daily 16 : 8 h light : dark photoperiod. No
induction of NPQ was observed on Day 7. Error bars are SDs (n = 4).
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
+N/–P
µM N (–N /+P)
40
150
initial
40
0
80
300
0
60
120
450
PAB
–N /+P
600
PA
PAB
PAR
+N /–P
PA
PAB
PAR
PA
PAB
PAR
PA
–N /+P
PAR
160
+N /–P
0
% Recovery
0.3
0.0
10
Nonphotochemical quenching
25
+N/–P
20
µM N (+N /–P)
% Photoinhibition
60
c
(a) 0.4
µM P (– /+P)
Day 1
Day 3
0
–N/+P
Fig. 7. Mean phosphorus (a) and nitrogen (b) concentrations in Nodularia spumigena medium grown under 1N/ P (primary y-axis) and
N/1P (secondary y-axis) conditions and exposed to different radiation
conditions consisting of PAR, PAR1UV-A (PA), and PAR1UV-A1UV-B
(PAB). Error bars are SDs (n = 3).
in the presence of external nitrogen (first experiment). The
photoinhibition of photosynthesis can, therefore, be attributed to the excess nitrogen rather than the absence of
phosphorus in the medium. Our materials were previously
grown in a 1N/1P medium before they were subjected to
different combinations of nutrient and radiation treatments.
The probability that Nodularia cells were able to accumulate
enough cellular phosphorus reserved for the duration of the
experiment is highly possible. Phosphorus starvation was
clearly responsible for the higher photoinhibition of photosynthesis in Nodularia during exposure to high PAR and
UVR. But adequate cellular phosphorus reserved and sufficient fixed nitrogen may have contributed to their capacity
for dynamic recovery of photosynthesis when stress (high
PAR and UVR) was removed. This physiological resilience
supports previous studies whereby Nodularia was reported
to be highly tolerant to increased phosphorus starvation
(Degerholm et al., 2006) and the eventual collapse of
biological functions occurred only after the depletion of the
internal phosphorus reserve (Walve & Larsson, 2007).
The results presented here showed that the induction of
NPQ provided some protection to PSII as indicated by the
inverse relationship between photoinhibition of photosynthesis under 1N/ P (Fig. 5a) and the capacity to
induce NPQ (Fig. 6). NPQ is associated with the induction
of several mechanisms that compete with photochemistry
FEMS Microbiol Ecol 66 (2008) 230–242
239
Light and nutrient effects on photosynthesis of Nodularia
for the deactivation of Chl a-excited states when the rate of
captured light exceeds that of the rate of consumption of
NADPH2 and ATP by cellular metabolism (Müller et al.,
2001; Rodrı́guez-Román & Iglesias-Prieto, 2005).
Conversely, the progressive reduction in NPQ under
N/1P (Fig. 6) but without a corresponding increase in the
photoinhibition of photosynthesis under different radiation
treatments (Fig. 5a) was unexpected. In the absence of
empirical data to explain this observation, it is tempting to
speculate that the modification of the acceptor site of PSII
resulting in the development of QB nonreducing PSII might
be responsible for this phenomenon. The QB nonreducing
PSII observed in other cyanobacteria is resistant to photoinhibition (Wykoff et al., 1998; Steglich et al., 2001). This
may have facilitated the dissipation of excess excitation
energy and may have served as an alternate photoprotection
mechanism compensating for the failure to induce NPQ.
The negative ‘excess’ nitrogen effect on the photosynthesis of Nodularia is noteworthy and reported for the first
time. The biochemical process behind this phenomenon is
unknown among cyanobacteria and needs further study.
Among kormophytes, reduction in net photosynthesis under high nitrogen treatment was attributed to the depression
of carboxylation efficiency, coupled by a decrease in Rubisco
concentration and activity (Nakaji et al., 2001; Manter et al.,
2005). Other adverse effects of excess nitrogen are reflected
in terms of growth inhibition, increased hydrogen peroxide
(H2O2) accumulation and superoxide radical (O
2 ) production (Yao & Liu, 2006, 2007). Elevated nitrogen does not
only negatively influence the physiology and growth of trees
(Schulze, 1989) but has also been found to be toxic to
certain species of seagrass (Burkholder et al., 1992, 1994).
In diazotrophic cyanobacteria, photosynthesizing vegetative cells differentiate to form N2-fixing heterocysts upon
nitrogen deprivation (Böhme, 1998). In this study, however,
the increasing total nitrogen content we found in the
medium suggests that regardless of the nitrogen status (e.g.
nitrogen enriched), Nodularia was still actively fixing N2 in
excess of their metabolic requirement. This conforms to a
previous study that showed that cyanobacteria with inactivated genes (sigB and sigC), expressed under nitrogen
deficiency and involved in nitrogen fixation, were still
capable of heterocyst differentiation and nitrogen fixation
(Brahamsha & Haselkorn, 1992). Assimilation of nitrogen
substrates increases energy requirement in the order: ammonium, nitrate and N2. Regulation of nitrogen fixation
is, however, not completely understood. For example,
Nodularia strains M1 and M2 grown with ammonium at a
concentration of 1 mM resulted in the total disappearance of
nitrogenase activity and of heterocyst; heterocyst, however,
persisted in the presence of 20 mM NO
3 at a frequency
similar to that found in the absence of nitrate. Excess NO
3
above 25 mM was consequently observed to inhibit growth
FEMS Microbiol Ecol 66 (2008) 230–242
(Sanz-Alférez & del Campo, 1994). Another Nodularia
spumigena strain AV1 isolated from the Baltic Sea lost
aerobic nitrogen-fixation activity in the presence of ammonium ion and, at the same, time maintained heterocyst
frequency along the filaments (Vintila & El-Shehawy, 2007).
Generally, we found transient experimental irradiation
effects on the photosynthesis of Nodularia regardless of
nutrient status. Long-term diel exposure to PAR and
PAR1UVR showed dynamic recovery of photosynthesis
already 1 h after the exclusion of UVR and under low PAR.
Among other phytoplankton species, nitrogen limitation
significantly increased the sensitivity of photosynthesis of
estuarine dinoflagellates to inhibition by UVR (Litchman
et al., 2002), while the marine unicellular chlorophyte
Dunaliella tertiolecta Butcher also showed the same increased UVR-induced inhibition of photosynthesis but
under both nitrogen-limitation and phosphorus-starvation
conditions (Shelly et al., 2002, 2005; Heraud et al., 2005).
Most studies on the interactive effects of nutrient and
UVR on phytoplankton productivity measured growth as a
response parameter. The results showed contrasting responses between phytoplankton species, community assemblage, lotic systems, and altitude and latitudinal gradients.
In an alpine lake, the effects of UVR on phytoplankton
community depend on temperature and nutrient availability (Doyle et al., 2005). At a low temperature (6 1C), UVR
led to a decrease in growth rates of all phytoplankton species
regardless of the nutrient condition. At a high temperature
(14 1C), no negative UVR effect was observed in the absence
of nutrient addition while addition of nitrogen and phosphorus under UVR increased the growth of one diatom
species Fragilaria crotonensis Kitton and the dinoflagellate
Gymnodinium sp. (Doyle et al., 2005). In a boreal lake,
phytoplankton growth was coregulated by phosphoruslimitation and UVR suppression, with the highest growth
rates found in high phosphorus and low UVR treatments
(Xenopoulos et al., 2002). Phosphorus input is hypothesized
to buffer the harmful UVR effect on algae. However, longterm UVR exposure exerted a significant deleterious effect
on the physiology of a natural pelagic algal community in
the presence of excess phosphorus (Carrillo et al., 2008).
Nutrient limitation was also reported to decrease the
sensitivity of the diatom Chaetoceros brevis Schütt to photoinduced viability loss relative to nutrient-replete conditions
(van de Poll et al., 2005). Conversely, the availability of
inorganic nutrients was reported to mitigate the negative
UV-B radiation effects on the growth of microphytobenthic
communities (Wulff et al., 2000). On the other hand,
variable responses of natural phytoplankton population to
enhanced UV-B radiation and nitrate enrichment were also
reported across a latitudinal gradient (Longhi et al., 2006).
Although our study used only 300 mmol photons m2 s1
of PAR, this was high enough to cause photoinhibition of
2008 Federation of European Microbiological Societies
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c
240
photosynthesis. In nature, wind-induced vertical mixing
could potentially expose microalgal cells to variable light
quantity and quality. Exposure to photoinhibiting PAR
(41000 mmol photons m2 s1) was reported to alter the
phycobiliproteins such as the disappearance of the 31.5kDa linker polypeptide and consequently hinder the energy
transfer process within the phycobilisomes in Spirulina
platensis (Nordstedt) Geitler (Kumar & Murthy, 2007).
H2O2, as a side product of oxygenic photosynthesis exposed
to high irradiance, commonly encountered in an aquatic
environment can present a double jeopardy to cyanobacterial photosynthesis. A generated or an external H2O2 source
does not only inhibit photosynthetic electron transport
(Samuilov et al., 2001; Drábková et al., 2007) but also
degrades D1 protein (Lupı́nková & Komenda, 2004) in cells
of cyanobacteria.
Despite the artificial laboratory condition with a relatively
high UVR : PAR ratio, a probable future scenario under an
ozone-depleted stratosphere, UV irradiances comparable
with those encountered in the field were observed to have a
limited negative impact on the photosynthetic performance
of Nodularia. Reactive oxygen species (ROS) induced by
UV-B affects oxidative damage but may also act as signal
molecules and mediate the genetic regulation of photosynthetic genes and the induction of antioxidant enzymes
(He & Häder, 2002). Removal of ROS by antioxidants is
one of the defense mechanisms providing protection against
excessive light absorption in phototrophs (Logan et al.,
2006). Moreover, protection of photosynthesis against UVR
by carotenoids has been reported in transgenic Synechococcus PCC7942 (Götz et al., 1999). The transgenic cyanobacterium further resists UV-B by exchanging PSII reactioncenter D1 proteins from the psbAI-encoded D1 : 1 to an
alternate psbAII- and psbAIII-encoded D1 : 2 form within
15 min of exposure to moderate UV-B (Campbell et al.,
1998a). The fast and reversible recovery of photoinhibited
photosynthesis in Nodularia can also be attributed to the
carotenoid-controlled effective thermal energy dissipation,
keeping excessive energy flow in control (Logan et al., 2006).
In conclusion, because Nodularia are able to fix nitrogen,
access to available phosphorus (internal input or regenerated source) can render them less susceptible to photoinhibition effectively promoting blooms. In the eventual
depletion of phosphorus in the system, before the collapse
of the bloom, Nodularia might be more susceptible to
radiation but capable of photosynthetic recovery immediately after removal of radiation stress. Whether individual
Nodularia filaments are able to actively sink to escape from
high solar radiation remains to be studied. Other cyanobacteria like Anabaena variabilis and Oscillatoria tenuis are
reported to migrate from the water surface to lower levels
to avoid high solar irradiance (Donkor & Häder, 1995).
Otherwise, in situ dynamic photosynthetic recovery can
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c
M.Y. Roleda et al.
occur when filaments are dispersed from the bloom surface
to depths under the mat by wind-induced vertical mixing
during the day, or eventually during the twilight. Conversely,
alternating periods of calm and deep-mixing events can
increase the photosynthesis of cyanobacteria when buoyed
by gas vacuoles near the water surface (Walsby et al., 1997).
The increasing occurrence and intensity of Nodularia
bloom in the Baltic Sea can be explained by the resilience of
Nodularia to radiation and to their capacity for dynamic
photoinhibition. With the advent of global climate change,
Nodularia can potentially acclimate to increasing UVR.
Moreover, a high summer temperature will not only increase
cyanobacterial growth rate but can also increase water
column stability, reducing vertical mixing. Because the
phosphacline is located in the upper part of the thermocline
(Lilover & Laanemets, 2006), this can give a competitive
advantage to buoyant cyanobacteria (Jöhnk et al., 2008) like
Nodularia to access available phosphorus. Therefore, in the
presence of an internal phosphorus pool that can be
seasonally available to the diazotrophic cyanobacteria, late
spring and summer cyanobacterial blooms in the Baltic Sea
will continue to persist.
Synergistic effects might exist between high PAR and
UVR. Even if strong PAR is missing, this study presents a
piece of the puzzle to explain why extensive summer bloom
formation occurs in the Baltic. Future in situ photosynthetic
studies during the course of bloom initiation and collapse
will yield more ecologically relevant data on the basic
physiological process controlling bloom formation.
Acknowledgements
M.Y.R. thanks Carl Tryggers Stiftelse för Vetenskaplig Forskning for the postdoctoral fellowship. Further financial
support to A.W. was provided by The Swedish Research
Council for Environment, Agricultural Sciences and Spatial
Planning and The Oscar and Lilli Lamm Foundation. Prof.
Christian Wiencke graciously lent the Water PAM unit.
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