Ocean warming alters the responses to eutrophication in a commercially farmed seaweed, Gracilariopsis lemaneiformis

Abstract

The economically important marine red macroalga Gracilariopsis lemaneiformis was cultured for 2 weeks at two different temperatures, ambient and elevated CO₂ levels, and two fertilizer levels. After the experimental period, growth, photosynthesis, and biochemical components were determined. Higher pCO₂ significantly increased relative growth rates and nitrate reductase activity with the combination of higher temperature (HT) and/or N enrichment, whereas photosynthetic rates were rapidly reduced at the same condition. Both Q₁₀ values for photosynthetic- (P_m) and respiratory (R_d) rates were higher in algae grown at 24 than at 20°C. This enhancement of P_m rate by HT was much more pronounced than that of the R_d rate. As a result, soluble carbohydrates produced by photosynthesis accumulated to higher levels at the higher temperature. Most of fluorescence variables and antioxidant enzyme activities (super oxide dismutase, catalase, peroxidase, malondialdehyde) did not differ among treatments. Additionally, higher pCO₂ reduced chlorophyll a, carotenoids, phycoerythrin, and phycocyanin contents by 48.6, 41.7, 24.7, and 47.8% under LTLN condition, respectively, compared with the control treatment. We propose that high pCO₂ would depress the carbon acquisition but increase N assimilation with higher temperature and/or coastal eutrophication, thereby exerting a positive effect on algal growth.

Introduction

The atmospheric and oceanic concentrations of carbon dioxide have rapidly increased recently at an approximate rate of 2 ppm per year (Moreira & Pires, 2016), mainly due to the burning of fossil fuels combined with changes in land use. Our CO₂-enriched world has significantly contributed to global warming and ocean acidification (OA), both of which have directly and indirectly had a strong impact on a wide range of organisms within the biosphere (Joos, 2015). Moreover, the global mean sea-surface temperature had already increased since the beginning of the industrial era, and has been projected to increase by 1.9°C by the end of the 21st century, and even by as much as 4°C in high northern latitudes (Bartsch et al., 2012; Alexander et al., 2013). It is necessary for the proposition of the Paris Agreement to set the goal of maintaining global temperature rise within 2°C above preindustrial levels, which is considered to be a sweeping-program of CO₂ capture and storage (Archer et al., 2009; Schreurs, 2016), while biological approaches to CO₂ capture remain research and policy priorities.

Apart from the immense challenges posed by rising global CO₂ and temperature levels, eutrophication is another serious threat to environmental equilibrium. The rate and extent of eutrophication have been accelerated by anthropogenic activities (Taranu et al., 2015). In fact, several reasons (the expansion of urban populations and the increasing use of fertilizers in agriculture, etc.) ultimately enhance nutrient availability in coastal waters, which have rapidly developed contraries with increasing coastal populations (Liu & Wang, 2016). Excessive nutrient enrichment causes alternating cycles of algal flourishing and collapsing (i.e., red and green tides) (Farmaki et al., 2014). Moreover, changes in nutrient availability can alter the physiological properties of macroalgae (Hernández et al., 2006; Xu et al., 2010; Kang et al., 2017), even affect the production of primary and secondary metabolites. For instance, enrichment of the growth medium with nutrients effectively promoted the growth and photosynthesis in Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik (Xu et al., 2010), or changed the flow of C among compounds from carbohydrates to organic N compounds (Vergara et al., 1995). In addition, the carbohydrates of macroalgae have been displayed to be inversely related to nutrient concentrations (Marinho-Soriano et al., 2006), on account of carbohydrates from storage reserves toward the production of energy and carbon skeletons necessary to reduce nitrate and form amino acids, which both can cause starch degradation (Gouvêa et al., 2017).

Despite algae proliferation (microalgae and macroalgae) being a symptom of the eutrophication dilemma, algae have gained increasing attention as CO₂ and nutrient bio-capture candidates. Due to their large biomass and relatively long turnover time, marine macroalgae are more effective carbon sinks than microalgae (Smith, 1981). Species of G. lemaneiformis exhibited high growth rates with strong CO₂ capture abilities and a high affinity for nutrients (Xu et al., 2010). However, the responses of Gracilariopsis to a sustained increase of carbon and nutrients in the ocean in the future are not well known.

In coastal areas, global change (an interactive effect of elevated CO₂ and temperature) and eutrophication may occur simultaneously, rather than individually. The interactive effects of two or three separate factors were possibly quite strong as compared to the independent effects of any separate factor by itself. To the best of our knowledge, no study has yet investigated the interactive effects of ocean warming and eutrophication on G. lemaneiformis from the perspective of their potential for carbon and nutrient capture for PSII, as well as their antioxidant enzyme activities and metabolic production but on other macroalga species (Binzer et al., 2016; Ontoria et al., 2019; Schulhof et al., 2019). Therefore, in this study we cultured G. lemaneiformis at current and simulated future conditions in the ocean. The objectives of the present study were to investigate: (1) whether there is an acclimation of photosynthesis in response to higher pCO₂ and temperature, and if so, whether the degree of such acclimation is affected by N availability; (2) whether short-term (or long-term) exposure to higher temperature inhibits photosynthetic- and respiratory rates, and if so, whether this direct (or indirect) effect varies with growth, CO₂ and N availability.

Materials and methods

Sample collection and culture conditions

Gracilariopsis lemaneiformis was collected in November 2017 from the cultivation field at Shenao Bay, Nanao Island, Shantou, in southern China (23.5° N, 117.1° E). Thalli were placed in a zip-lock plastic bag and transported to the laboratory within 1 h, where they were gently rinsed in one-micron filtered natural seawater to remove visible epiphytes and any accumulated sediments. Healthy thalli were randomly assigned to nine plastic tanks (10 L) for maintenance. Each tank contained 8 L of natural seawater. Thalli in the tanks were irradiated with cool-white light at 150 μmol photons m⁻² s⁻¹, under a 12/12 h light/dark regime, at 15°C for 2 days. The interactive effects of ocean warming, elevated CO₂ and eutrophication were investigated using a fully crossed factorial design. Thalli were incubated under combinations of two pCO₂ levels (400, 1000 μl L⁻¹; designated as lower pCO₂, LC and higher pCO₂, HC, respectively), two temperatures (20, 24°C; designated as LT and HT, respectively), and two nutrient conditions (25 μM N as nitrate and 2.5 μM P as phosphate, or 500 μM N and 50 μM P; designated as LN and HN, respectively). These treatments under which G. lemaneiformis was grown were representative of current ambient and simulated, future ocean conditions. Experimental treatments started when 2.5 g fresh weight (FW) thalli were introduced into each of 24 flasks containing 2 L of filtered seawater. Three replicates were set up for each treatment. The flasks were placed in two temperature-controlled chambers; temperature inside the chambers was controlled at either 20 or 24°C, respectively. In each chamber, six flasks were aerated with atmospheric CO₂, and the remaining flasks were aerated with elevated CO₂. Three of the six flasks under either CO₂ treatment were incubated under the HN condition, and the other three were incubated under the LN condition. Before measurement of experimental indictors, total alkalinity (TA) was measured by titration. Carbonated system values not directly measured were calculated using CO₂SYS with the carbonic equilibrium constants (K₁, K₂) and KSO₄⁻ dissociation constant (Dickson, 1990; Pierrot et al., 2006). Carbonate chemical changes are shown in Table 1. Nutrient concentrations were maintained relatively constant by changing fresh seawater every 2 days. Culture temperatures and CO₂ levels were controlled using a microcomputer system embedded in the laboratory incubators. The thalli were illuminated with a light intensity of 150 μmol photons m⁻² s⁻¹ under a 12/12 h light/dark photoperiod. Algae were cultured for 2 weeks during which seawater was fully renewed every 2 days.

Table 1 Seawater carbonate physicochemical values under the different treatments

Relative growth rate (RGR)

Biomass of thalli was measured in each treatment to estimate growth. The RGR was estimated as follows: RGR (% d⁻¹) = (lnW_t − lnW₀) × t⁻¹ × 100%, where W₀ was the initial fresh weight, and W_t was the culture weight after t days of cultivation. Thalli were gently blotted on filter paper before FW weighing.

Determination of respiration and photosynthesis

Net photosynthetic rate (P_n) of the thalli was measured by a Clark-type oxygen electrode (YSI 5300, USA) held in a circulating water-bath to keep the desired temperature. Approximately 0.05 g FW algae sampled from each flask was introduced into the electrode chamber together with 8 mL of reaction seawater, same as their respective culture medium, and magnetically stirred. Algae were allowed to equilibrate in darkness until the rate of oxygen consumption was constant, usually for 4–6 min, prior to determination of respiration rate (R_d). Sequentially, oxygen fluxes were measured at light irradiances from 0 to 700 μmol photons m⁻² s⁻¹ to ensure active photosynthesis. R_d and P_n (P_g) rates were recorded to examine the instantaneous effect of temperature (measured temperature: 20, 24 and 28°C) on the algae with their own grown culture medium. Photosynthetic efficiency (α) was estimated from the slope of the initial linear region of photosynthesis-irradiation (P–I) curves. Light saturation (E_k) was calculated as E_k = P_m/α (Henley, 2010), where P_m was the maximum net photosynthesis. Gross photosynthetic rate (P_g), was obtained from the sum of R_d plus P_n. In turn, R_d and P_n (P_g) rates were measured at different temperatures (20, 24 and 28°C) to examine temperature effects on photosynthetic and respiratory flux. Different measured temperatures were adjusted in the oxygen electrode chamber.

Q₁₀ values, which were termed as the proportional change in photosynthesis and respiration per 10°C rise, were calculated for photosynthesis and respiration over the measured temperature interval of 20–28°C using the following equation according to Atkin et al. (2010): Q₁₀ = 10^(slope·10), where the slope was the regression slope of log₁₀-transformed photosynthetic- and respiratory rates verse temperature plot.

Measurement of fluorescence variables

Approximately 0.03 g FW thalli sampled from each culture flask was transferred to the cuvette with 2 mL of sterilized seawater. Thalli were firstly dark-adapted for 10 min and then the index of chlorophyll a fluorescence transient was measured using an Aqua Pen fluorometer-100-PC (PSI, Photon Systems Instruments, Czech Republic), which supplied a saturating light pulse of 3000 μmol m⁻² s⁻¹ during the measurement. Fluorescent transients were recorded over the time-span from 10 μs to 1 s, at 10-μs intervals. The resultant O-J-I-P transients were analyzed according to the JIP-test. Meanwhile, several relative variables were also calculated to explain PSII behavior, such as phenomenological fluxes/activities (effective antenna size of an active RC, ABS/RC; maximal trapping rate of PSII, TR_o/RC; electron transport in an active RC, ET_o/RC; effective dissipation of an active RC, DI_o/RC), flux ratios/yields (maximum quantum yield of primary photochemistry, ψ_Po; electron transport probability, ψ_o; and quantum yield of electron transport, ψ_Eo), and density of reaction centers (RC/CS).

With the Junior-PAM, rapid light curves (RLCs; so-called “short-term” (rapid) photosynthesis-irradiance curves) were measured yielding E_k (light saturation value) and rETR_m (relative maximum electron transport rate) values. These were measured with light over the 400–700 nm wavelength interval while increasing from 0 to 820 μmol photons m⁻² s⁻¹, with an exposure time of 20 s at each photon flux density (PFD). Total in vivo Chl fluorescence signal was also recorded from PAM.

Physiological and biochemical variables

Estimation of antioxidant characteristics

Antioxidant activities, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are important. We used the SOD assay kit, the CAT kit and POD kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for each enzyme assay, respectively. The SOD assay was based on SOD ability to oxidize hydroxylamine by the xanthine–xanthine oxidase system (Oyanagui, 1984). One unit (U) of SOD activity was calculated as the inhibition by 50% of the oxidation of hydroxylamine without an enzyme source. The CAT assay was according to the method of Góth (1991). The POD assay was based on the method of Lawrence et al. (1974), after minor changes. In addition, we used a malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the method of Ohkawa et al. (1979).

Biochemical composition

Approximately 0.2 g FW thalli from each treatment were ground thoroughly in 10 mL methanol and placed in a refrigerator at 4°C for 24 h in total darkness. Then, the homogenates were centrifuged for 10 min at 5000 g and the supernatants were used to determine chlorophyll a (Chl a) and carotenoids (Car) content spectrophotometrically, according to the method of Wellburn (1994). The concentrations of phycobiliprotein (PB) in the supernatants were determined spectrophotometrically according to Beer and Eshel (1985). For extraction of soluble carbohydrates (SC) and soluble protein (SP), about 0.2 g of FW thalli from each treatment were ground in a mortar with distilled water and extraction buffer (0.1 M phosphate buffer, pH 6.8); SC were determined as sucrose equivalents, by the phenol–sulphuric acid method. SP was estimated from the supernatants using the Bradford (1976) assay with bovine serum albumin as a standard.

Nitrate reductase activity (NRA)

The NRA in the thalli was assayed according a modified in situ method (Corzo & Niell, 1991). Due to a circadian periodicity of the NRA, such as a maximum during the light period and a minimum in darkness (Whitaker, 1989; Deng et al., 1991), here the measurement was conducted during local noon. Approximately 0.1 g FW thalli from each treatment was incubated for 30 min at 30°C in darkness in 10 mL of a buffer (pH 8.1) containing 0.1 M phosphate buffer, 50 mM KNO₃, 10 μM glucose, and 0.5 mM EDTA. The mixture was flushed with pure N₂ gas (99.999%) for 2 min to attain anaerobic conditions before incubation. The concentration of NO₂⁻ produced from NO₃⁻ was determined colorimetrically at 543 nm; NRA was expressed as μmol NO₂⁻ g⁻¹ FW h⁻¹.

Data analysis

Data were expressed as means of three replicates ± standard deviation (SD). The statistical (multifactorial ANOVAs and complemented with Duncan test) was performed to assess the effects of pCO₂ (400 μatm, LC; 1000 μatm, HC), temperature (20°C, LT; 24°C, HT) and nutrients (low (LN)-25 μM N as nitrate and 2.5 μM P as phosphate; high (HN)-500 μM N and 50 μM P) on physiologic and biochemical characteristics of G. lemaneiformis, using IBM SPSS Statistics 23.0 software with a significance level of 5% (P < 0.05). The pCO₂, temperature, and nutrient levels were considered as fixed factors. The normality and assumption of homogeneity of variances were checked during the process of multifactor ANOVA. Where necessary, data were transformed in order to reduce structure in analytical residuals and homogenize variances. The graphs in present study were made using Origin 8.5 software and Microsoft Office 2016.

Results

Relative growth rates (RGR)

The temperature and pCO₂ (T× C) showed interactive effects (double effect) on RGR of G. lemaneiformis (ANOVA, F_(1,16) = 11.560, P = 0.004). The pCO₂ also showed a main effect (ANOVA, F_(1,16) = 44.737, P = 0.000) on RGR. At HTLN condition, higher pCO₂ increased RGR by 31.3% (Fig. 1, P < 0.05). Furthermore, with the combination of higher T and N-enriched conditions, higher pCO₂ enhanced RGR by 58.1% (F_(1,16) = 11.828, P = 0.003).

Fig. 1

The relative growth rates (RGR) of Gracilariopsis lemaneiformis grown at the experimental conditions during the cultivation. The error bars indicated the standard deviations (n = 3). LT lower temperature (20°C); HT higher temperature (24°C); LC lower pCO₂ (400 μl L⁻¹ CO₂); HC higher pCO₂ (1000 μl L⁻¹ CO₂); LN lower nutrients (25 μM N as nitrate and 2.5 μM P as phosphate); HN higher nutrients (500 μM N and 50 μM P)

Respiration and photosynthesis

The fitting values (P_m and α) derived from gross photosynthetic rates (P_g; i.e., P_n plus R_d, shown in Fig. 2a–f) are displayed in Table 2. All factors (temperature, T; pCO₂ level, C; nutrient supply, N; measuring temperature, MT), as well as the interactive effects among these factors, almost had main effects on irradiance-saturated maximum photosynthetic rates (P_m) and apparent photosynthetic efficiency (α) (Table 3, P < 0.05). A Duncan comparison (P = 0.05) showed that higher pCO₂ did not always have an impact on P_m at the lower temperature (20°C), while it reduced P_m rates by 20.0% (30.7%) under HTLN condition (or HTHN condition, 24°C) (Table 3, P < 0.05). In contrast, higher pCO₂ increased the α by 41.5% under LTLN condition (F_(1,48) = 21.40, P = 0.000), while it decreased the α by 17.5% with increased growth temperature (HTHCLN, F_(1,48) = 49.72, P = 0.000). As for respiratory rates (R_d, Fig. 3), both T (ANOVA, F_(1,48) = 23.799, P = 0.000) and pCO₂ (ANOVA, F_(1,48) = 9.434, P = 0.004) had main effects on them. However, there seemed no significant changes of R_d rates together with the interaction of higher T and N or enriched C and N (Fig. 3a, b).

Fig. 2

The fitting curves of gross photosynthetic rates (P_g) verse irradiances for Gracilariopsis lemaneiformis grown at the experimental conditions during the cultivation. LT lower temperature (20°C); HT higher temperature (24°C); LC lower pCO₂ (400 μl L⁻¹ CO₂); HC higher pCO₂ (1000 μl L⁻¹ CO₂); LN lower nutrients (25 μM N as nitrate and 2.5 μM P as phosphate); HN higher nutrients (500 μM N and 50 μM P). The P_g rates were measured at 20°C, 24°C and 28°C, respectively. Vertical bar represented ± SD of the means (n = 3)

Table 2 Maximum photosynthetic rate (P_m) and apparent photosynthetic efficiency (α) in G. lemaneiformis grown under saturating irradiance levels under different temperatures (20°C, LT; 24°C, HT), different CO₂ levels (400 μl L⁻¹ CO₂, LC; 1000 μl L⁻¹ CO₂, HC) and different nutrient supply (25 μM N as nitrate and 2.5 μM P as phosphate, 500 μM N and 50 μM P; coded as LN and HN, respectively)

Table 3 F- and P-values from analysis of variance (ANOVA) for the assessment of single and interactive effects to P_m, α and R_d of growth and measuring conditions: temperature (20°C, LT; 24°C, HT), CO₂ levels (400 μl L⁻¹ CO₂, LC; 1000 μl L⁻¹ CO₂, HC), nutrient supply (25 μM N as nitrate and 2.5 μM P as phosphate, 500 μM N and 50 μM P; coded as LN and HN, respectively), and measuring temperatures (20°C, 24°C, 28°C, MT)

Fig. 3

Respiratory rates (R_d) of Gracilariopsis lemaneiformis exposure to combined conditions of growth temperature (20 and 24°C, T), CO₂ levels (400 and 1000 μl L⁻¹), nutrient levels (25 μM N as nitrate and 2.5 μM P as phosphate, LN; 500 μM N and 50 μM P, HN), and measured temperature (20, 24 and 28°C). The approaches were conducted with samples for measuring at three temperatures. Data were pooled means ± SD, in accordance with significant effects obtained by ANOVA: interactive effects of T and N (a, n = 18), interactive effects of C and N (b, n = 18), interactive effects of T and MT (c, n = 12). Different letters above the histograms indicate significant difference (P < 0.05, Duncan post hoc test)

The instantaneous response of photosynthesis to changes in temperature showed that P_m rates increased with increasing measurement temperature up to 28°C (Table 2). The Q₁₀ values of P_m rates indeed increased with an enhanced temperature interval (24–28°C, Table 4, P < 0.05). For 24°C-grown G. lemaneiformis, high pCO₂ significantly (P < 0.05) reduced the Q₁₀ values of P_m rates when measured at the higher temperature interval under evaluation. The MT and T had an interactive effect on R_d rates of G. lemaneiformis (ANOVA, F_(1,48) = 4.584, P = 0.037). A Duncan comparison (P = 0.05) showed that higher measured temperature significantly decreased R_d rates of algae grown at low temperature (20 ^oC, Fig. 3c, P < 0.05), together with the higher Q₁₀ values at two temperature intervals (20–24°C and 24–28°C, Table 4, P < 0.05). Moreover, the Q₁₀ values of the R_d rates were much higher in low-C and low-N (LCLN) than high-C and/or high-N conditions, regardless of temperature (P < 0.05).

Table 4 Values of Q₁₀ (the rate increase caused by raising temperature 10°C) for the gross photosynthetic- (P_m) and respiratory rates (R_d) of Gracilariopsis lemaneiformis grown under different temperatures (20°C, LT; 24°C, HT), different CO₂ levels (400 μl L⁻¹ CO₂, LC; 1000 μl L⁻¹ CO₂, HC) and different nutrients (25 μM N as nitrate and 2.5 μM P as phosphate, 500 μM N and 50 μM P; coded as LN and HN, respectively)

OJIP fluorescence curves and rapid light curves (RLCs)

Figures showed fluorescence induction curves for dark-adapted G. lemaneiformis (see supplementary materials). The highest polyphasic chlorophyll fluorescence transients (OJIP) intensities showed for the control treatment (LTLCLN). High culture temperature (24°C) increased OJIP intensities of G. lemaneiformis during the whole duration of the experiment. Selected variables of Chl a fluorescence and variables derived by the JIP-test are shown in Fig. 4a–h. All factors (T, C, N), as well as the interactive effects among these factors, almost had no significant effects on those variables (Table 5). The differences in patterns displayed by these variables indicated the extent of the impact of elevated temperature/CO₂/nutrients in G. lemaneiformis. The ABS/RC (Fig. 4a), TR_o/RC (Fig. 4b), ET_o/RC (Fig. 4c), DI_o/RC (Fig. 4d), ψ_Po (Fig. 4e), ψ_Eo (Fig. 4f), and ψ_o (Fig. 4g) did not increase at high temperature, pCO₂, and nutrient level (P > 0.05). In contrast, compared to low CO₂, higher pCO₂ reduced RC/CS by 84.6% (80.9%) at 20°C (Fig. 4h, P < 0.05), and this decrease was alleviated when temperature increased (24°C), for example, higher pCO₂ reduced RC/CS by 24.8% with higher T and N enrichment, compared to the control.

Fig. 4

The phenomenological fluxes of log scale OJIP chlorophyll fluorescence transients (ABS/RC = effective antenna size of an active RC, aTR_o/RC maximal trapping rate of PSII, bET_o/RC electron transport in an active RC, cDI_o/RC effective dissipation of an active RC, d flux ratios or yields (ψ_Po = maximum quantum yield of primary photochemistry, eψ_Eo = the quantum yield of electron transport, f and ψ_o = electron transport probability, g and density of reaction centers (RC/CS, h and RLCs variables (maximum relative transport rates, rETR_m, i the light saturation point, E_k, μmol photos m⁻² s⁻¹, j and the initial photosynthetic efficiency, α, k of Gracilariopsis lemaneiformis grown at the experimental conditions during the cultivation. The error bars indicated the standard deviations (n = 3). LT = lower temperature (20 ^oC); HT higher temperature (24°C); LC lower pCO₂ (400 μl L⁻¹ CO₂); HC higher pCO₂ (1000 μl L⁻¹ CO₂); LN lower nutrients (25 μM N as nitrate and 2.5 μM P as phosphate); HN higher nutrients (500 μM N and 50 μM P)

Table 5 ANOVA results for experiment with Gracilariopsis lemaneiformis after being exposed to combined conditions of temperatures (20°C, LT; 24°C, HT), CO₂ levels (400 μl L⁻¹ CO₂, LC; 1000 μl L⁻¹ CO₂, HC) and nutrient supplies (25 μM N as nitrate and 2.5 μM P as phosphate, 500 μM N and 50 μM P; coded as LN and HN, respectively) on fluorescence variables (n = 3)

Maximum relative electron transport rates (rETR_m), half-light saturation (E_k) and photochemical yield (α) obtained from the RLCs of G. lemaneiformis are shown in Fig. 4i–k. The T and N had an interactive effect (ANOVA, F_{(1, 80)} = 5.814, P = 0.018) on rETR_m of G. lemaneiformis. Likewise, T × C (ANOVA, F_(1,80) = 8.250, P = 0.005), T × N (ANOVA, F_(1,80) = 11.528, P = 0.001) and C × N (ANOVA, F_(1,80) = 8.789, P = 0.004) had the most double interactive effect on E_k. In contrast, no separate factor had a main effect on α (P > 0.05). A Duncan comparison (P = 0.05) showed that the rETR_m (Fig. 4i), E_k (Fig. 4j) and α (Fig. 4k) remained constant under all treatments in the present study (P > 0.05), with the exception of reduction of rETR_m and E_k at higher T and N conditions.

Physiological and biochemical variables

Antioxidant characteristics

The extent of SOD (Fig. 5a), CAT (Fig. 5b), and POD (Fig. 5c) activities was generally not affected by higher temperature, elevated CO₂, and nutrient levels (P > 0.05). Likewise, significant differences in MDA concentration were not found among treatments (Fig. 5d, P > 0.05).

Fig. 5

The antioxidant characteristics (activities of SOD, a CAT, b POD, c MDA contents, d biochemical compositions (the contents of chlorophyll a, Chl a, e carotenoids, Car, e phycoerythrin, PE, f phycocyanin, PC, f soluble carbohydrates, SC, g soluble proteins, SP, h, and nitrate reductase activity (NRA, i of Gracilariopsis lemaneiformis grown at the experimental conditions during the cultivation. The error bars indicated the standard deviations (n = 3). LT lower temperature (20°C); HT higher temperature (24°C); LC lower pCO₂ (400 μl L⁻¹ CO₂); HC higher pCO₂ (1000 μl L⁻¹ CO₂); LN lower nutrients (25 μM N as nitrate and 2.5 μM P as phosphate); HN higher nutrients (500 μM N and 50 μM P)

Biochemical composition

Higher pCO₂ decreased Chl a and Car contents by 48.6% and 41.7% under LTLN condition, respectively (Fig. 5e). Likewise, higher pCO₂ reduced PE and PC contents by 24.7% and 47.8% under LTLN condition, respectively (Fig. 5f). There were no obvious variations in PC among other treatments. Soluble carbohydrates (SC, Fig. 5g) and protein (SP, Fig. 5h) were estimated to understand the effects of ocean warming and eutrophication on the products of carbon and nitrogen assimilation in G. lemaneiformis. The T × C had an interactive effect on the SC (ANOVA, F_(1,16) = 7.333, P = 0.016). Both T and C had a main effect on the SC (ANOVA, F_(1,16) = 135.076, P = 0.000 for T; ANOVA, F_(1,16) = 52.274, P = 0.000 for C). At 20°C, thalli under high pCO₂ showed a slight change of SC compared with the control. However, high pCO₂ combined with high T exhibited a positive effect on SC (P < 0.05, Fig. 5g). Additionally, the interactive effect of the three factors under study (T × C × N) was detected on SP content in G. lemaneiformis (ANOVA, F_(1,16) = 4.657, P = 0.046). The higher N level increased SP content by 19.4% at HTLC, and increased it by 20.4% at HTHC, respectively, compared with the control (Fig. 5h).

Nitrate reductase activity (NRA)

The three factors showed an interaction effect on NRA of G. lemaneiformis (ANOVA, F_(1,16) = 15.386, P = 0.001) and T/pCO₂/N had a main effect (ANOVA, F_(1,16) = 7.902, P = 0.013 for T; ANOVA, F_(1,16) = 11.675, P = 0.004 for C; ANOVA, F_(1,16) = 31.816, P = 0.000 for N). Higher pCO₂ increased NRA by 145.6% under HTLN (compared with that at ambient CO₂ level), while higher T decreased NRA by 53.1% under LCLN (compared with that at low temperature) and higher N decreased NRA by 44.7% (59.1%) under LTLC (under HTHC, compared with that at low nutrient level), respectively (Fig. 5i, P < 0.01).

Discussion

In the present study, higher pCO₂ reduced gross photosynthetic rate in Gracilariopsis lemaneiformis at higher temperature and nutrient level. This observation is consistent with our previous study (Liu et al., 2016). The negative effects or the absence of any effects of elevated CO₂ on photosynthesis were also reported in other macroalgal species, such as Ulva conglobate Kjellman (Deng & Zou, 2014), and Phaeocystis spp (Keys et al., 2018). In contrast, a promoting effect of elevated CO₂ on photosynthesis was demonstrated in Ulva linza Linnaeus [Enteromorpha linza (L.) J. Ag.] (Gao et al. 1999), Pyropia haitanensis Chang et Zheng (Zou & Gao, 2002), Petalonia binghamiae (J. Agaradh) Vinogradova (Zou & Gao, 2010), and Sargassum muticum (Yendo) Fensholt (Xu et al., 2017).

Generally, the long-term response of photosynthesis to CO₂ enrichment shows as a decreasing or even an inhibitory tendency in most plants (Porras et al., 2017). In the present study, the possible explanation for a decrease in photosynthesis in G. lemaneiformis might involve an excess accumulation of carbohydrates or the decline of NR activity (responsible for the reduction in N content), together with a negligible consumption of photosynthetic products through respiration. However, the obvious enhancement of growth was shown in G. lemaneiformis as a result of an interaction between higher pCO₂ and higher temperature. This indicted that elevated CO₂ might stimulate other assimilation processes that support growth. In this study, elevated CO₂ did enhance N assimilation of the algae under higher temperature and lower nutrient levels.

Photosynthetic rates of G. lemaneiformis were significantly affected by the instantaneous change in temperature, termed temperature sensitivity, which could be described by the Q₁₀ value. Our results showed that the values of Q₁₀ for photosynthesis significantly increased with higher temperature. However, they were reduced when the temperature raised to 33°C (Zou & Gao, 2013). Indeed, previous studies demonstrated that the Q₁₀ response of gross photosynthetic capacity was heterogeneous among terrestrial species and other macroalgae, upon exposure to high temperature (Gerard, 1997; Tjoelker et al., 2001; Atkin & Tjoelker, 2003). Likewise, changes in temperature also resulted in an immediate alternation in the rate of R_d. The calculated Q₁₀ value for R_d was generally greater when temperature increased, and the value of Q₁₀ increased significantly under HTLCLN (resulting in an increased temperature sensitivity for R_d at ambient CO₂ level), which was higher than that often assumed in plants (Tjoelker et al., 2001). These results also exhibited similar Q₁₀ values for respiration under two different culture temperatures at elevated CO₂. Furthermore, the extent of acclimation of respiration and photosynthesis of G. lemaneiformis to a new culture temperature had been confirmed in previous reports, which might suggest possible large variations in acclimation potential to thermal environment (Zou & Gao, 2013; Liu et al., 2016). Additionally, our results showed that Q₁₀ values were higher for photosynthesis than for respiration, implying that photosynthetic processes in G. lemaneiformis had a higher degree of temperature dependence than respiratory processes.

The effect of interaction of environmental factors on PSII photosynthetic performance in G. lemaneiformis, are just obtained from the JIP-test. The ψ_Po was used to evaluate PSII photosynthetic performance of G. lemaneiformis mainly due to a good correlation between this method and results obtained by the Junior-PAM. Similar studies have reported a high correlation between flash fluorescence induction and Phyto-PAM measurements (Pierangelini et al., 2014a, b). The ABS/RC, which refers to the photons absorbed per active reaction center, responded to different environmental factors similarly in G. lemaneiformis, indicting the quantity of antenna chlorophyll played similar functions regardless of treatment, so that light was harvested to support the subsequent photochemical process based on the integrity of the photosynthetic apparatus of the thalli (the antioxidant defense system functioned well and resulted in no lipid peroxidation events). The main photosynthetic inhibition targets by high CO₂ on G. lemaneiformis were a decrease in the number of active reaction centers (RC/CS) at lower temperature (20°C). This implied that the density of photosynthetic units was reduced, thereby reducing photochemical transfer of harvested and excited photons. However, at higher nutrient level and higher temperature, increased excitation energy was transferred to the reaction centers (RCs) and converted in the RCs to redox energy by reducing the electron acceptor Q_A to Q ⁻_A , which was then re-oxidized to Q_A leading to electron transport. Nitrogen assimilation was a competing sink for electrons to CO₂ fixation. The lower proportion of electrons distributed to NRA ultimately resulted in lower nitrogen metabolism (Figueroa et al., 2003; Liu & Zou, 2015). Our observations about NRA also confirmed this point. Ultimately, as the photochemical flux of photons and further metabolic events in the photosystem of G. lemaneiformis were maintained, the growth increase of the algae was expressed.

It has been suggested that CO₂-induced global warming will lead to increases in mean global sea-surface temperatures by 1.0–4.4°C by the end of this century (Solomon et al., 2007); further, there is increasing concern over the potential consequences of anthropogenic climate change on marine systems and organisms (Helmuth et al., 2013). In order to predict the effects of climate change on mariculture systems of seaweeds in a warmer and eutrophicated ocean, it is imperative to understand the performance of algal photosynthesis, respiration and biochemical components during the period of algal cultivations at sea. Our results suggest that photosynthesis of G. lemaneiformis was favored over respiration as temperatures increased, yet nutrient assimilation decreased as a result of elevated CO₂ and temperature. Therefore, seemingly, ocean warming may benefit photosynthetic carbon metabolism and may in fact enhance mariculture productivity of G. lemaneiformis. An increased capacity for carbon sequestration may be a negative feedback reaction to the environmental changes caused by human activities and would thus alleviate some aspects of global climate change.

Conclusions

It is essential to further investigate the antioxidant and gene expression characteristics of G. lemaneiformis in order to evaluate the possible consequences of ocean warming, increased atmospheric CO₂ level, and increased eutrophication. Our results suggest that photosynthesis of G. lemaneiformis had a higher degree of temperature dependence than respiratory processes. Furthermore, high pCO₂ would depress the carbon acquisition but increase N assimilation with higher temperature and/or coastal eutrophication, thereby exerting a positive effect on algal growth. Anyway, G. lemaneiformis could optimize metabolic balance by adjustment of carbon and nutrient capture in harmony with future global climate changes, which would have important ecophysiological implications for sea cultivation of this species.