Introduction
Sargassum C. Agardh (1820) is the most species-rich genus in the
Phaeophyta and has a global distribution (Mattio and Payri, 2011). The
species of this genus constitutes an important part of the marine flora and
is considered a valuable and unique habitat for a number of highly adapted
marine animal species (Laffoley et al., 2011). Some species of
Sargassum are economically important and are used in animal fodder,
agricultural manure, and alginate production (Ashok-Kumar et al.,
2012; Fenoradosoa et al., 2010; González-López et al., 2012). On the
other hand, Sargassum is an aggressive genus, and it can rapidly
spread and invade new areas (Sfriso and Facca, 2013). The invasion of
Sargassum would accordingly compete with indigenous species for
nutrients and light, leading to the alteration of the macroalgal community
structure (Rueness, 1989; Stæhr et al., 2000). For instance, the
increased abundance of S. muticum in Limfjorden (Denmark) between
1990 and 1997 led to decreased cover of several indigenous species belonging
to the genera Codium, Fucus, and Laminaria, and
thus reduced the species richness and diversity of the macroalgal community
(Stæhr et al., 2000). Recently, species of Sargassum have inundated the coasts along the Gulf of Mexico, West Africa, the Caribbean, and
Brazil in unprecedented biomass, which are termed golden tides (Schell et al., 2015;
Smetacek and Zingone, 2013). Apart from the negative effect on aesthetics and
tourism, the occurrence of golden tides could kill the fish within the algal
mass, mainly due to hypoxia or anoxia in the waters caused by decomposition
of Sargassum thalli (Cruzrivera et al., 2015). In addition, the
dense Sargassum accumulation could clog fishing nets and impede the
passage of boats, leading to food shortages for local people who depend on
artisanal fisheries (Smetacek and Zingone, 2013). The occurrence of golden
tides has been linked to higher nutrient levels in seawater (Lapointe,
1995; Smetacek and Zingone, 2013). The distribution pattern and biomass of
Sargassum spp. are environment-dependent (temperature, light, nutrients,
etc.) (Ang, 2006; Sfriso and Facca, 2013).
Due to the burning of fossil fuels and changes in land use, the atmospheric
concentrations of carbon dioxide increased to the level of 401.72 ppm
in July 2016 (http://www.esrl.noaa.gov/gmd/ccgg/trends/global.html),
which is an unprecedented high over the last 800 000 years (IPCC,
2013). When CO2 dissolves in seawater, it forms carbonic acid, and as more
CO2 is taken up by the ocean's surface, the pH decreases, moving towards
a less alkaline and therefore more acidic state; this is termed ocean acidification.
The mean surface ocean pH has already decreased by 0.1 units since the
beginning of the industrial era, corresponding to a 26 % increase in
hydrogen ion concentration (IPCC, 2013). By 2100, concentrations of CO2
(aq) and HCO3- are predicted to increase by 192 and 14 %,
respectively, and CO32- is predicted to decrease by 56 % with a concomitant
decline in pH to 7.65 (Raven et al., 2005). Increased CO2 could exert
positive, neutral, or negative effects on the physiological properties of macroalgae (Ji
et al., 2016; Wu et al., 2008). In terms of Sargassum species,
increased CO2 (800 ppm) enhanced the photosynthetic rate (based on CO2
uptake) in S. muticum (Longphuirt et al., 2014). On the other hand,
the same level of increased CO2 (750 ppm) did not affect growth,
Rubisco's maximal activity, affinity for CO2, or quantity in S. vulgare (Alvaro and Mazal, 2002). Furthermore, increased CO2 (750 ppm)
significantly decreased the net photosynthetic rate and light saturation point of
S. henslowianum (Chen and Zou, 2014).
Apart from ocean acidification, eutrophication is another environmental
challenge. Eutrophication can occur naturally in lakes through the transfer
of
nutrients from the sediment to the water via living or decomposing macrophytes,
resuspension, diffusion, and bioturbation (Carpenter, 1981). However,
anthropogenic activities have accelerated the rate and extent of
eutrophication (Carpenter et al., 1998). The inevitable urbanization of a growing
human population, the increased use of coastal areas, and rising fertilizer use
for agricultural intensification has led to accelerated nutrient inputs from
land water to coastal waters (Smith et al., 1999). These changes in nutrient
availability result in eutrophication, an increasing threat for coastal
ecosystems (Bricker et al., 2008). One consequence of eutrophication is that
it can lead to algal bloom, such as green tides and golden tides (Smetacek
and Zingone, 2013). There are intensive studies regarding the
effect of nutrients on the physiological properties of Sargassum species
(Hwang et al., 2004; Incera et al., 2009; Lapointe, 1995; Liu and Tan, 2014;
Nakahara, 1990). Enrichment of nutrients can usually enhance the growth and
photosynthetic parameters of Sargassum. For instance, the growth
rate of S. baccularia almost doubled when nutrients increased from
3 µM ammonium plus 0.3 µM phosphate to 5 µM
ammonium plus 0.5 µM phosphate (Schaffelke and Klumpp, 1998), and
the photosynthetic rates of S. fluitans and S. natans were
also 2-fold higher with 0.2 mM PO43- enrichment compared to the
control (Lapointe, 1986). Furthermore, some studies have demonstrated that
macroalgae experience more phosphorus limit than nitrogen limit
(Lapointe, 1986; Lapointe et al., 1987, 1992; Littler et al., 1991). For
instance, nitrogen enrichment did not affect the growth rates of S. fluitans or S. natans, while phosphorus enrichment increased them
from 0.03–0.04 (control) to 0.05–0.08 doublings d-1 (Lapointe, 1986).
Neither ocean acidification nor eutrophication is proceeding in isolation;
rather, they occur simultaneously, particularly in coastal areas. The
interactive effects of the two factors may be completely different or of
greater magnitude compared to the effects of any single stressor. To the best of
our knowledge, no studies have been reported regarding the interactive
effects of ocean acidification and eutrophication on Sargassum. In
this study, we chose the species S. muticum to investigate its
responses to the interaction of ocean acidification and eutrophication.
S. muticum is an invasive macroalga that commonly inhabits rocky
shores (Karlsson and Loo, 1999). It originates from Japan and was imported
to the Northern Pacific coast of the United States in the early 20th century
(Scagel, 1956). It was also introduced to Europe along with the
Japanese oyster in the late 1960s (Jones and Farnham, 1973). Its
distribution is now worldwide due to its introduction and subsequent rapid
expansion (Cheang et al., 2010). Our study supplies insight into how
ocean acidification and eutrophication affect the physiological properties of
S. muticum and thus the development of golden tides.
Materials and methods
Sample collection and experimental design
S. muticum was collected from lower intertidal rocks on the coast of
Lidao, Rongcheng, China (37∘15′ N, 122∘35′ E). The
samples were transported to the laboratory in an insulated polystyrene cooler
(4–6 ∘C) within 3 h. Healthy thalli were selected and rinsed with
sterile seawater to remove sediments, epiphytes, and small grazers. The thalli
were maintained in an intelligent illumination incubator (MGC-250P, Yiheng
Technical Co. Ltd., Shanghai, China) for 24 h before the experiment. The
temperature in the incubator was set at 20 ∘C with a 12 h–12 h
(light–dark) photoperiod of
150 µmol photons m-2 s-1 photosynthetically active
radiation (PAR). After the maintenance, a two-way factorial experiment was
set up to investigate the interactive effects of pCO2 and phosphate on
S. muticum. The thalli were placed in 3 L flasks with 2 L of sterile
seawater (one thallus per flask) and cultured at fully crossed two
pCO2 (400 µatm, lower pCO2, LC; 1000 µatm, higher pCO2, HC) and two
phosphate (0.5 µM, lower phosphate, LP; 40 µM, higher phosphate, HP) levels with continuous
aeration for 13 days. Phosphorus was selected as a nutrient variable, because
some findings have displayed that phosphorus, rather than nitrogen, is the
primary limiting nutrient for macroalgae (Lapointe, 1986; Lapointe et al.,
1987, 1992; Littler et al., 1991). The conditions of natural seawster are 400 µatm pCO2 and
0.5 µM phosphate. The
400 µatm pCO2 was achieved by bubbling ambient air and
1000 µatm pCO2 was obtained through a CO2 plant
chamber (HP1000 G-D, Wuhan Ruihua Instrument & Equipment Ltd, China) with
a CO2 variation of less than 5 %. The higher P level
(40 µM) was achieved by adding NaH2PO4 to natural
seawater, and the nitrate concentration was set at 200 µM for all
treatments to avoid N limit. The media were refreshed every day.
Carbonate chemistry parameters
The seawater pH was recorded with a pH meter (pH 700, Eutech Instruments,
Singapore), and total alkalinity (TA) was measured by titrations. The salinity
of the seawater was 29. Other carbonate system parameters, which were not
directly measured, were calculated via CO2SYS (Pierrot et al., 2006) using
the equilibrium constants of K1 and K2 for carbonic acid
dissociation (Roy et al., 1993).
Measurement of growth
The growth of S. muticum was determined by weighing fresh thalli.
The thalli of S. muticum were blotted gently with tissue paper to
remove water on the surface from the thalli before weighing them. The relative
growth rate (RGR) was estimated as follows: RGR = (lnWt-lnW0)/t×100, where W0 is the initial fresh weight (FW) and
Wt is the weight after t days of culture.
Determination of photosynthesis and respiration
The net photosynthetic rate of the thalli was measured by a Clark-type oxygen
electrode (Chlorolab-3, Hansatech, Norfolk, UK) at the end of the experiment.
Approximately 0.1 g of fresh-weight algae harvested from the culture flask
was transferred to the oxygen electrode cuvette with 8 mL of sterilized
media, and the media were stirred during measurement. The irradiance and
temperature conditions were set the same as in the growth incubators. The
increase of oxygen content in seawater within 5 min was defined as the net
photosynthetic rate, and the decrease of oxygen content in seawater in
darkness within 10 min was defined as the respiration rate. The net
photosynthetic rate (NPR) and respiration rate were presented as
µmol O2 g-1 FW h-1.
Photosynthetic rates at different dissolved inorganic carbon (DIC) levels
were measured under saturating irradiance of
600 µmol photons m-2 s-1 at the end of the experiment.
The various DIC concentrations (0–13.2 mM) were obtained by adding
different amounts of NaHCO3 to the Tris-buffered DIC-free seawater. DIC
was removed from the natural seawater by reducing pH to approximately 4.0
with the addition of 1.0 M HCl and then sparging for 2 h with pure
N2 gas (99.999 %). Finally, Tris buffer (25 mM) was added and the
pH was adjusted to 8.1 with freshly prepared 1 M NaOH and 1 M HCl. The
parameters, which are the maximum photosynthetic rate (Vmax) and the half saturation
constant (K0.5, i.e., the DIC concentration required to give half of
inorganic carbon (Ci)-saturated maximum rate of photosynthetic O2 evolution), were
calculated from the Michaelis–Menten kinetics equation (Caemmerer and
Farquhar, 1981)
V=Vmax×[S]/(K0.5+[S]),
where [S] is the DIC concentration.
Assessment of photosynthetic pigments
At the end of the experiment, approximately 100 mg of fresh-weight thalli from each culture condition were ground thoroughly in 2 mL 80 % acetone
and placed in darkness for 12 h. Then the homogenate was centrifuged for
10 min at 5000 g and the supernatant was used to determine Chl a content
spectrophotometrically according to the equation of Lichtenthaler (1987).
Measurement of nitrate uptake rate
The nitrate uptake rate (NUR) of the thalli was estimated from the decrease of
NO3- concentration in the culture medium over a given time interval
(12 h) during the light period using the following equation: NUR = (N0-Nt) × V/W/12, where N0 is the initial concentration of
NO3-, Nt is the concentration after 12 h, V is the volume of
the culture medium, and W is the fresh weight of the thalli in culture.
NO3- concentration in the seawater was measured according to
Strickland and Parsons (1972).
Estimate of nitrate reductase activity
The nitrate reductase activity of the thalli was assayed according to
modified in situ method of Corzo and Niell (1991). The measurement was
conducted during the local noon period (13:00 UTC + 8 h (Chinese Standard Time)), because the activity of nitrate reductase usually displays circadian
periodicity; a maximum during the light period and a minimum in darkness
(Deng et al., 1991; Velasco and Whitaker, 1989). Approximately 0.3 g (FW) of
thalli from each culture condition was incubated for 1 h at 20 ∘C
in darkness in the reaction solution (10 mL), which contained 0.1 M
phosphate buffer, 0.1 % propanol (v/v), 50 mM KNO3,
0.01 mM glucose, and 0.5 mM EDTA with a pH of 8.0. The mixture was flushed
with pure N2 gas (99.999 %) for 2 min to obtain an anaerobic state
before the incubation. The concentration of nitrite produced was determined
colorimetrically at 540 nm (Zou, 2005). The NRA was expressed as
µmol NO2- g-1 FW h-1.
Analysis of biochemical composition
At the end of the experiment, about 0.2 g of FW thalli from each culture condition were ground in a mortar with distilled water, and soluble
carbohydrates were extracted in a water bath of 80 ∘C for 30 min.
After being centrifuged for 10 min at 5000 g, the supernatant was
volumed to 25 mL with distilled water, and soluble carbohydrate content was
determined by the phenol-sulfuric acid method (Kochert, 1978).
Approximately 0.2 g of FW thalli from each culture condition were ground in a mortar with extraction buffer
(0.1 mol L-1 phosphate buffer, pH 6.8) and then centrifuged for
10 min at 5000 g. Soluble protein was estimated from the
supernatant using the Bradford (1976) assay with bovine serum albumin as a
standard.
Parameters of the seawater carbonate system at different CO2
and phosphate conditions. Measurements and estimation of the parameters are
described in the “Materials and methods” section. Data are reported as means ±SD (n=3).
LCLP is the low pCO2 and low P condition, LCHP is the low pCO2 and
high P condition, HCLP is the high pCO2 and low P condition, and HCHP is the
high pCO2 and P condition. DIC is dissolved inorganic carbon,
and
TA is total alkalinity.
Treatment
pH
pCO2
HCO3-
CO32-
CO2
DIC
TA
(µatm)
(µmol kg-1)
(µmol kg-1)
(µmol kg-1)
(µmol kg-1)
(µmol kg-1)
LCLP
8.07 ± 0.02b
426.9 ± 31.1a
2000.2 ± 51.7a
200.9 ± 5.8b
14.2 ± 1.0a
2215.3 ± 49.7a
2475.2 ± 44.2
LCHP
8.07 ± 0.02b
423.9 ± 21.1a
1987.6 ± 10.9a
199.8 ± 11.4b
14.1 ± 0.7a
2201.5 ± 19.3a
2504.7 ± 33.8
HCLP
7.76 ± 0.02a
1017.2 ± 83.2b
2282.5 ± 27.6b
110.0 ± 10.0a
34.0 ± 2.9b
2426.5 ± 32.5b
2541.5 ± 44.2
HCHP
7.76 ± 0.02a
992.2 ± 44.9b
2261.8 ± 35.9b
110.5 ± 5.9a
33.1 ± 1.5b
2405.4 ± 39.4b
2563.6 ± 44.2
a, b Different superscript letters indicate significant
differences in one parameter between treatments (P<0.05).
Data analysis
Results were expressed as means of replicates ± standard deviation. Data
were analyzed using the software SPSS v.21. The data under every treatment
conformed to a normal distribution (Shapiro–Wilk, P>0.05), and the
variances can be considered equal (Levene's test, P>0.05). Two-way
analysis of variance (ANOVA) was conducted to assess the effects of pCO2 and P on carbonate
parameters, relative growth rate, net photosynthesis rate, Vmax,
K0.5, Chl a, nitrate uptake rate, nitrate reductase activity, soluble
carbohydrates, soluble protein, and dark respiration rate. Tukey's honest significance difference (HSD) was
conducted for a post hoc investigation. A confidence interval of 95 % was
set for all tests.
Results
The effects of ocean acidification and P enrichment on seawater carbonate
parameters were detected (Table 1). Two-way ANOVA analysis (P=0.05)
showed that pCO2 had a main effect on all parameters except TA, while
P did not affect any parameter. A post hoc Tukey's HSD comparison (P=0.05)
showed that elevated pCO2 decreased pH by 0.31 at both LP and HP and
CO32- by 45 % (LP) and 45 % (HP), but it increased DIC by
10 % (LP) and 9 % (HP), HCO3- by 14 % (LP) and 14 %
(HP), and CO2 by 139 % (LP) and 134 % (HP).
The growth of S. muticum cultured at different pCO2 and P
conditions was recorded (Fig. 1). pCO2 and P had an interactive effect
on the relative growth rate of S. muticum (ANOVA, F=5.776,
df=1, 8, P=0.043), and each factor had a main effect (ANOVA, F=19.145, df=1, 8, P=0.002 for pCO2; ANOVA, F=30.592, df=1, 8, P=0.001 for P). A post hoc Tukey's HSD
comparison (P=0.05) showed that the higher levels of pCO2 and
higher P alone increased the relative growth rate by 41 and 48 %,
respectively, compared to the relative growth rate (3.1 ± 0.4 %) at
lower pCO2 and lower P. The combination of the higher
pCO2 and higher P levels did not enhance the relative growth rate as
much as the sum of the higher pCO2 alone plus the higher P alone, with
an increase of 59.66 %. Although the higher P level increased the
relative growth rate at lower pCO2, it did not affect
the relative growth rate at higher pCO2.
Relative growth rate (RGR) of S. muticum grown at different
pCO2 and P conditions for 13 days. Data are reported as means ±SD (n=3). LCLP is the low pCO2 and low P condition, LCHP is the low pCO2
and high P condition, HCLP is the high pCO2 and low P condition, and
HCHP is
the high pCO2 and high P condition. Different letters above the error bars
indicate significant differences between treatments (P<0.05).
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In terms of the net photosynthetic rate (Fig. 2), both pCO2 (ANOVA, F=26.556, df=1, 8, P=0.001) and P had main effects (ANOVA, F=38.963, df=1, 8, P<0.001). A post hoc Tukey's HSD
comparison (P=0.05) showed that the higher pCO2 level increased the net
photosynthetic rates by 46 and 24 % at lower P and
higher P, respectively. The higher P level increased the net photosynthetic
rates by 55 and 31 % at lower pCO2 and higher
pCO2, respectively. The difference in the net photosynthetic rate
between LCHP and HCLP was statistically insignificant.
Net photosynthetic rate (NPR) of S. muticum after being
grown at different pCO2 and P conditions for 13 days. Data are
reported as
means ±SD (n=3). LCLP is the low pCO2 and low P condition,
LCHP is
the low pCO2 and high P condition, HCLP is the high pCO2 and low
P condition, and HCHP is the high pCO2 and high P condition. Different
letters above the error bars indicate significant differences between treatments
(P<0.05).
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The carbon-saturating maximum photosynthetic rate (Vmax,
µmol O2 g-1 FW h-1) and half saturation constant
(K0.5, mM) for S. muticum cultured under different pCO2
and P conditions for 13 days.
LCLP
LCHP
HCLP
HCHP
Vmax
57.00 ± 2.88a
93.99 ± 0.98c
81.18 ± 5.94b
100.67 ± 6.81c
K0.5
0.21 ± 0.02a
0.14 ± 0.05a
0.42 ± 0.08b
0.19 ± 0.05a
a, b, c Different superscript letters indicate
significant differences in one parameter between treatments (P<0.05).
The carbon-saturating maximum photosynthetic rate (Vmax) and the half
saturation constant (K0.5) obtained from the photosynthesis versus DIC
curves (Fig. 3) are shown in Table 2. The pCO2 and P had an
interactive effect on the Vmax of S. muticum (ANOVA, F=10.095,
df=1, 8, P=0.013), and each factor had a main effect (ANOVA, F=31.402, df=1, 8, P=0.001 for pCO2; ANOVA, F=105.116, df=1, 8, P<0.001 for P). A post hoc Tukey's HSD
comparison (P=0.05) showed that the higher pCO2 level increased the
Vmax by 42 % at lower P, while the increase at higher P was statistically insignificant. The higher P level
increased the Vmax at the conditions of both lower pCO2
(65 %) and higher pCO2 (24 %) with a larger promoting
effect at lower pCO2.
The photosynthesis versus DIC curves of S. muticum after
being cultured under pCO2 and P conditions for 13 days. Data are
reported as
means ±SD (n=3). LCLP is the low pCO2 and low P condition,
LCHP is
the low pCO2 and high P condition, HCLP is the high pCO2 and low
P condition, and HCHP is the high pCO2 and high P condition.
DIC is dissolved inorganic carbon.
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pCO2 and P interacted on the K0.5 of S. muticum (ANOVA,
F=5.928, df=1, 8, P=0.041), and each factor had a main
effect (ANOVA, F=14.713, df=1, 8, P=0.005 for pCO2;
ANOVA, F=20.857, df=1, 8, P=0.002 for P). A post hoc
Tukey's
HSD comparison (P=0.05) showed that the higher pCO2 level increased the
K0.5 by 98 % at lower P but did not affect it at
higher P. In contrast, the higher P level decreased the
K0.5 by 55 % at higher pCO2 and the negative
effect of the higher P level at lower pCO2 was
insignificant.
The amounts of the photosynthetic pigment Chl a under various treatments were
also estimated (Fig. 4). pCO2 and P had an interactive effect on the
Chl a content (ANOVA, F=8.184, df=1, 8, P=0.021), and P had
a main effect (ANOVA, F=22.828, df=1, 8, P=0.001), while
pCO2 did not affect it (ANOVA, F=0.676, df=1, 8, P=0.435). A post hoc Tukey's HSD comparison (P=0.05) showed that the higher P level
increased the Chl a content from 0.17 ± 0.00 to
0.25 ± 0.02 mg g-1 FW at lower pCO2,
whereas the difference in the Chl a content between HCLP
(0.21 ± 0.02 mg g-1 FW) and HCHP
(0.23 ± 0.02 mg g-1 FW) was not statistically significant.
Chl a content of S. muticum after being grown at
different pCO2 and P conditions for 13 days. Data are reported as means
±SD (n=3). LCLP is the low pCO2 and low P condition, LCHP is the
low pCO2 and high P condition, HCLP is the high pCO2 and low P
condition, and HCHP is the high pCO2 and high P condition. Different letters
above the error bars indicate significant differences between treatments (P<0.05).
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To assess the effects of ocean acidification and P enrichment on the nitrogen
assimilation in S. muticum, the nitrate uptake rate under various
pCO2 and P treatments was investigated (Fig. 5). Both
pCO2 (ANOVA, F=139.916, df=1, 8, P<0.001) and P
(ANOVA, F=43.923, df=1, 8, P<0.001) had main effects on
the nitrate uptake rate of S. muticum. The nitrate uptake rates at
lower pCO2 were 0.18 ± 0.01 (LP) and
0.25 ± 0.03 µmol NO3- g-1 FW h-1 (HP),
respectively. A post hoc Tukey's HSD comparison (P=0.05) showed that the higher
pCO2 level increased the nitrate uptake rate to
0.31 ± 0.02 µmol NO3- g-1 FW h-1 at lower P and to
0.39 ± 0.01 µmol NO3- g-1 FW h-1 at higher P, compared to the rates at lower
pCO2. The higher P level also increased the nitrate uptake rate by
36 % at lower pCO2 and by 28 % at higher pCO2, compared to the rates at lower P.
Nitrate uptake rate of S. muticum after being grown at
different pCO2 and P conditions for 13 days. Data are reported as means
±SD (n=3). LCLP is the low pCO2 and low P condition, LCHP is the
low pCO2 and high P condition, HCLP is the high pCO2 and low P
condition, and HCHP is the high pCO2 and high P condition. Different letters
above the error bars indicate significant differences between treatments (P<0.05).
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Apart from nitrate uptake, the NRA of S. muticum under various pCO2 and P treatments was also detected
(Fig. 6). pCO2 and P interacted on the NRA of S. muticum (ANOVA,
F=28.435, df=1, 8, P=0.001), and pCO2 had a main
effect (ANOVA, F=59.038, df=1, 8, P<0.001). The NRAs at lower pCO2 were 0.10 ± 0.01 (LP) and
0.14 ± 0.02 µmol NO2- g-1 FW h-1 (HP),
respectively. The higher pCO2 level increased it to
0.19 ± 0.00 µmol NO2- g-1 FW h-1 at lower P and to
0.15 ± 0.02 µmol NO2- g-1 FW h-1 at higher P. The higher P level increased the NRA by 39 % at lower pCO2; however, it decreased the NRA by 18 % at higher pCO2.
Nitrate reductase activity (NRA) of S. muticum after being
grown at different pCO2 and P conditions for 13 days. Data are
reported as means
means ±SD (n=3). LCLP is the low pCO2 and low P condition,
LCHP is
the low pCO2 and high P condition, HCLP is the high pCO2 and low
P condition, and HCHP is the high pCO2 and high P condition. Different
letters above the error bars indicate significant differences between treatments
(P<0.05).
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The soluble carbohydrates (Fig. 7a) and protein (Fig. 7b) were estimated to
understand the effects of ocean acidification and P enrichment on the
products of carbon and nitrogen assimilation in S. muticum.
pCO2 and P had an interactive effect on the soluble carbohydrates
(ANOVA, F=18.294, df=1, 8, P=0.003), and P had a main
effect (ANOVA, F=23.129, df=1, 8, P=0.001). The higher P
level increased the soluble carbohydrates from 25.40 ± 1.66 to
41.10 ± 1.74 mg g-1 FW at lower pCO2
but did not alter them at higher pCO2. The higher
pCO2 level increased the soluble carbohydrates to
33.72 ± 3.31 mg g-1 FW at lower P, while the
decrease of soluble carbohydrates caused by the higher pCO2 level was
not statistically significant at higher P.
The contents of soluble carbohydrates (a) and
protein (b) of S. muticum after being grown at different
pCO2 and P conditions for 13 days. Data are reported as means ±SD (n=3). LCLP is the low pCO2 and low P condition, LCHP is the low pCO2
and high P condition, HCLP is the high pCO2 and low P condition, and
HCHP is
the high pCO2 and high P condition. Different letters above the error bars
indicate significant differences between treatments (P<0.05).
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Both pCO2 (ANOVA, F=106.663, df=1, 8, P<0.001) and
P (ANOVA, F=75.003, df=1, 8, P<0.001) had main effects on
the soluble protein of S. muticum, and an interactive effect of the
two factors was not detected (ANOVA, F=4.961, df=1, 8, P=0.057). The soluble protein contents at lower pCO2
were 8.49 ± 0.49 (LP) and 9.77 ± 0.14 mg g-1 FW (HP),
respectively. The higher pCO2 level increased it to
10.11 ± 0.16 mg g-1 FW at lower P and to
12.28 ± 0.44 mg g-1 FW at higher P. The higher
P level also increased the soluble protein contents by 15 % at lower pCO2 and by 21 % at higher
pCO2.
Finally, the effects of ocean acidification and P enrichment on the dark
respiration rate of S. muticum were investigated (Fig. 8).
pCO2 and P had an interactive effect on the dark respiration rate
(ANOVA, F=19.584, df=1, 8, P=0.002), and each factor had a
main effect (ANOVA, F=6.428, df=1, 8, P=0.035 for
pCO2; ANOVA, F=6.754, df=1, 8, P=0.032 for P). The
higher pCO2 level increased the dark respiration rate from
14.21 ± 1.94 to
21.24 ± 1.28 µmol O2 g-1 FW h-1 at higher P but did not affect it at lower P.
Likewise, the higher P level increased the respiration rate from
14.15 ± 0.65 to
21.24 ± 1.28 µmol O2 g-1 FW h-1 at higher pCO2 but did not change it at
lower pCO2.
Dark respiration rate of S. muticum after being grown at
different pCO2 and P conditions for 13 days. Data are reported as means
±SD (n=3). LCLP is the low pCO2 and low P condition, LCHP is the
low pCO2 and high P condition, HCLP is the high pCO2 and low P
condition, and HCHP is the high pCO2 and high P condition. Different letters
above the error bars indicate significant differences between treatments (P<0.05).
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Discussion
Effects of <inline-formula><mml:math id="M624" display="inline"><mml:mi>p</mml:mi></mml:math></inline-formula>CO<inline-formula><mml:math id="M625" display="inline"><mml:msub><mml:mi/><mml:mn mathvariant="normal">2</mml:mn></mml:msub></mml:math></inline-formula> and P on carbon assimilation
The higher pCO2 level increased the net photosynthetic rate in
S. muticum at lower P in the present study.
Although the dissolved inorganic carbon in seawater is around 2 mM, the
dominant form is HCO3- with CO2 typically accounting for less
than 1 % (Dickson, 2010). In addition, CO2 in seawater diffuses
∼ 8000 times more slowly than in air (Gao and Campbell, 2014). Furthermore,
marine macroalgae have high K0.5 values
(40–70 µM CO2) for Rubisco, the carbon assimilating enzyme
(Ji et al., 2016). The evidence above indicates that the CO2 in seawater
should be carbon limited for marine macroalgae. The promoting effect of
elevated CO2 on photosynthesis was also reported in other macroalgae
species, such as the green algae Ulva linza (Gao et al., 1999), the red
algae Pyropia haitanensis (Zou and Gao, 2002), and the brown algae
Petalonia binghamiae (Zou and Gao, 2010). The higher
pCO2 level increased K0.5 of S. muticum at lower P in the present study, which indicates that a plant grown
under
conditions of higher pCO2 reduces its photosynthetic affinity for DIC.
This phenomenon is commonly found in both microalgae and macroalgae (Gao and
Campbell, 2014; Ji et al., 2016; Wu et al., 2008) and is considered a sign
of downregulated CCMs at high CO2 conditions (Gao and Campbell, 2014).
However, this decrease of photosynthetic affinity for DIC did not lead to reduced
photosynthesis in S. muticum compared to that at the lower
pCO2 in the present study, mainly because of increased CO2
availability for Rubisco and depressed photorespiration at the elevated ratio
of CO2 to O2, which has been confirmed in the red seaweed
Lomentaria articulata (Kübler et al., 1999).
The higher P level also increased the net photosynthetic rate of S. muticum in the present study, which can be partially explained by the
decreased K0.5 at higher P. The decreased K0.5 is
an indication of increased photosynthetic carbon-use capability. Phosphorus
is a key macronutrient component for organisms, and high levels of P
availability are not only essential for chloroplast DNA and RNA synthesis
(Vered and Shlomit, 2008), but are also required for various chloroplast functions
referring to the phosphorylation of photosynthetic proteins, the synthesis of
phospholipids, and the generation of adenosine triphosphate (ATP; Zer and Ohad, 2003). Therefore, high P
levels could speed up the transport of Ci from media to the site of Rubisco
by supplying necessary energy. In addition, P enrichment can increase both
the activity and the amount of Rubisco (Lauer et al., 1989). Phosphorus,
with low concentrations in seawater, is generally considered to be limiting
for marine primary producers (Elser et al., 2007; Howarth, 1988; Müller
and Mitrovic, 2015). Therefore, adding extra phosphorus to natural seawater
can stimulate the photosynthesis of algae. For instance, the midday (12:00)
photosynthetic rates increased from 1.3 to 2.3 mg C g-1 DW h-1
for S. natans and from 0.9 to 2.1 mg C g-1 DW h-1 for
S. fluitans when 0.2 mM P was added (Lapointe, 1986). In the
present study, the addition of 40 µmol P also resulted in a nearly
2-fold increase of the net photosynthetic rate and the Vmax, which
suggests the importance of P in the photosynthesis of this alga. In
addition, the higher P level promoted the synthesis of Chl a at the
condition of lower pCO2, which may also contribute to the increased
net photosynthetic rate in S. muticum at higher P.
Although P is not a component constituting Chl a, a higher P supply may
stimulate the content of Chl a synthesis-related enzymes and thus the
production of Chl a. The positive effect of P on Chl a was also reported
in S. thunbergii (Nakahara, 1990). On the other hand, the higher P
level did not increase the Chl a content at higher
pCO2 in the present study. A possible reason is that there is more
ATP available at higher pCO2 due to the
downregulation of CCMs, and thus there is no need to synthesize more Chl a
to capture more light for cells, as excessive energy can harm the
photosynthesis and growth of algae (Gao et al., 2012; Xu and Gao, 2012).
Effect of <inline-formula><mml:math id="M669" display="inline"><mml:mi>p</mml:mi></mml:math></inline-formula>CO<inline-formula><mml:math id="M670" display="inline"><mml:msub><mml:mi/><mml:mn mathvariant="normal">2</mml:mn></mml:msub></mml:math></inline-formula> and P on nitrogen
assimilation
The higher pCO2 level noticeably enhanced the nitrate uptake rate in
S. muticum regardless of P concentration in the present study. This
could be attributed to the increased NRA at the
condition of higher pCO2. The enhanced NRA at the conditions of high
CO2 was also reported in U. rigida (Gordillo et al., 2001),
Hizikia fusiforme (Zou, 2005), P. haitanensis (Liu and Zou,
2015), and Corallina officinalis (Hofmann et al., 2013), as well as in the
higher plants Plantago major (Fonseca et al., 1997) and tomatoes (Yelle
et al., 1987). Taken together, these findings indicate that the response
of NRA in plants to elevated CO2 may be homogeneous.
The higher P level also enhanced the nitrate uptake in S. muticum
regardless of the pCO2 level, which could be partially due to the increased
NRA at higher P. This is very evident at lower pCO2. However, the higher P level decreased the NRA at higher pCO2, which did not lead to reduced nitrate
uptake. This indicates that there should be other mechanisms to account for the
promoting effect of the higher P level on the nitrate uptake. One possible
mechanism is the higher P level increasing the availability of ATP
required for the active uptake of nitrate across the plasma membrane.
The phenomenon of ATP concentration increasing with P level has been found
in higher plants (Olivera et al., 2004; Rychter et al., 2006). Apart from
S. muticum, the positive effect of a higher P level on nitrate uptake
was also reported in the red macroalgae Gracilaria lemaneiformis (Xu et
al., 2010) and the higher plant Phaseolus vulgaris (Gniazdowska and
Rychter, 2000). The increased nitrate uptake, NRA, and soluble protein at higher P in the present study suggest that high P availability
promoted nitrogen assimilation in S. muticum. It is worth noting
that the nitrate uptake rates were commonly higher than the corresponding
reduction rates of NO3- to nitrite NO2- by nitrate reductase
in the present study, which might be due to the intercellular nitrate storage
(Collos, 1982; Lartigue and Sherman, 2005) and the underestimation of RNA
measured by the in situ assay (Lartigue and Sherman, 2002). The higher P
level increased the nitrate uptake rate and soluble protein at both lower pCO2 and higher pCO2, but it only increased the
NRA in S. muticum at lower pCO2 in the
present study. Surprisingly, it decreased the NRA at higher
pCO2. There may be more than one reason related to
interaction of pCO2 and P. High pCO2, on the one hand, could
enhance photosynthetic carbon fixation and thus growth by supplying
sufficient CO2. On the other hand, it also results in the decrease of pH
and the increase of seawater acidity, which can disturb the acid–base balance
on the
cell surface of algae (Flynn et al., 2012). Algae may accordingly allocate
additional energy to act against the acid–base perturbation in some way.
This hypothesis is supported by increased respiration at higher pCO2 and higher P in the present study. The increased soluble
protein and decreased NRA at higher pCO2 and higher P
suggest that some H+ transport-related protein, such as plasma membrane
H+-ATPase, might be synthesized to counteract the acid–base
perturbation caused by increased pCO2 and H+. The additional
production of an H+ transport-related protein, like plasma membrane
H+-ATPase, could competitively decrease the synthesis of nitrate
reductase. This hypothesis needs further experimental evidence to confirm, even
though it could explain the results in the present study.
Connection between carbon and nitrogen assimilation
The increased net photosynthetic rate at higher
pCO2 and higher P did not result in higher soluble
carbohydrates compared to higher pCO2 and
lower P. The additional ATP produced by photosynthetic electron transport higher pCO2 and higher P may be drawn to
nitrogen assimilation as more soluble protein was synthesized at higher pCO2 and higher P. The additional energy
allocation to protein synthesis, possibly an H+ transport-related
protein,
to maintain the balance of acid–base hindered the increase of growth, which
may be the reason that the higher P increased the net photosynthetic rate
but not the growth rate at higher pCO2.
Although synthesized protein can also contribute to the increase of thalli
weight, it is not as energy-effective as carbohydrates (Norici et al., 2011;
Raven, 1982). It seems that S. muticum tends to maintain a steady
state in vivo, even if it can sacrifice growth to some extent, considering
that the regulation of the intracellular acid–base balance is crucial for organismal
homoeostasis (Flynn et al., 2012; Smith and Raven, 1979). The increased
respiration at HC was also demonstrated in G. lemaneiformis (Xu et
al., 2010) and U. prolifera (Xu and Gao, 2012). The respiration at
higher pCO2 and lower P did not increase
compared to at lower pCO2 and lower P in
the present study, suggesting that action against acid–base perturbation did
not commence. The acid–base perturbation at higher
pCO2 and lower P may lead to the decreased photosynthetic
rate compared to that at lower pCO2 and
lower P.