effects of elevated p CO 2 on physiological performance of marine microalgae Dunaliella salina (Chlorophyta,Chlorophyceae)*

HU Shunxin (胡顺鑫) , WANG You (王悠) , WANG Ying (王影) , ZHAO Yan (赵妍) ,ZHANG Xinxin (张鑫鑫) , ZHANG Yongsheng (张永生) , , JIANG Ming (姜铭) ,TANG Xuexi (唐学玺) ,

1 College of Marine Life Science, Ocean University of China, Qingdao 266003, China

2 Rongcheng Ocean and Fisheries Bureau, Weihai 264300, China

1 INTRODUCTION

The concentration of atmospheric CO2has increased from ～280 to 395 μatm since the Industrial Revolution because of human activities such as deforestation, cement manufacture and burning of fossil fuels (Caldeira and Wickett, 2003). As humans continue to burn fossil fuels and biomass, atmosphericpCO2is predicted to continue to increase by a minimum of 0.5% per year in the next centuries,reaching 1 000 and 2 000 μatm by 2100 and 2300,respectively (Caldeira and Wickett, 2003; IPCC,2013). Approximately 1/3 of this atmosphericpCO2will be dissolved in the surface ocean, increasing CO2(aq) and decreasing the pH, thereby changing the seawater carbonate chemistry in a process called ocean acidification (Guinotte and Fabry, 2008). By 2100 and 2300, oceanic absorption of CO2will lead to a decrease in pH of 0.4 and 0.7 pH units, respectively(Caldeira and Wickett, 2003).

Marine algae fix inorganic carbon via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase(RubisCO), which utilizes CO2exclusively as the substrate for the carboxylase reaction. The concentration of CO2(aq)(～10 μmol/L) in oceans at present is far less than the half-saturation constant of Rubisco (～20–200 μmol/L) (Badger et al., 1998).Consequently, most marine algae have developed CO2concentrating mechanisms (CCMs) to overcome the limitations of Rubisco and compensate for the low CO2(aq)(Rost et al., 2006). Marine algae have evolved diverse types of CCMs, two of which are widely found. In the first CCM, the reversible dehydration of HCO3ˉ to CO2is catalyzed by extracellular carbonic anhydrase at the cell surface, then taken into the cell by passive diffusion, while in the second, HCO3ˉ is transported across cell membranes via anion exchange(AE), after which CO2is produced through the dehydration of HCO3ˉ catalyzed by intracellular carbonic anhydrase (Reinfelder, 2011). The types and energy costs of CCMs will largely determine the sensitivity of marine algae to ocean acidification(Reinfelder, 2011).

Functioning of CCMs has been widely investigated in different algal species. In diatoms, the CCMs ofPseudo-nitzschiamultiseries,Stellarimastellaris,PhaeodactylumtricornutumandThalassiosira pseudonanawere down-regulated with elevatedpCO2, as indicted by reduced photosynthetic affinities for dissolved inorganic carbon (DIC) and CO2(Trimborn et al., 2008; Wu et al., 2010; Yang and Gao, 2012). Furthermore, these responses were generally accompanied by reduced active transport of HCO3ˉ or lowered extracellular carbonic anhydrase activities. The affinities for CO2and DIC were also lower under highpCO2conditions in the coccolithophoreEmilianiahuxleyiand the cyanobacteriumTrichodesmium, while CAextactivity appeared to play a minor role in CCMs, and was not affected by elevatedpCO2(Rost et al., 2003, Kranz et al., 2009). Furthermore, the relative expression of genes associated with carbonic anhydrases and aquaporins in the dinoflagellateThoracosphaera heimiidecreased with increasingpCO2(Van de Waal et al., 2013). Nevertheless, Zou and Gao (2009) and Zou et al. (2011) reported thatHizikiafusiformisandGracilarialemaneiformisshowed no down-regulation of CCMs with elevatedpCO2. These different types of CCMs and their responses to ocean acidification have deepened our knowledge of species-specific responses to ocean acidification in marine algae.

Inevitably, the expression and operation of CCMs involve an energetic investment. Down-regulation under high CO2conditions could reduce the energetic requirement for photosynthesis. Consequently, the energy saved from down-regulated CCMs could be invested in other physiological processes, such as assimilation of other nutrients, leading to stimulated growth and photosynthesis (Giordano et al., 2005;Riebesell et al., 2007; Wu et al., 2010). Nevertheless,elevatedpCO2is not always beneficial to marine algae. It has been widely reported that calcifying organisms are more sensitive to ocean acidification due to the negative effects they have on the formation of aragonite or calcite armor (Langer et al., 2006;Kurihara et al., 2008; Van de Waal et al., 2013), while negative effects on growth and photosynthesis have been observed in non-calcifying species (Mercado et al., 1999; Iñíguez et al., 2015), likely due to the negative effects on the physiological processes caused by reduced external pH.

Dunaliellasalinais a unicellular and halotolerant biflagellate marine microalgal species that has been widely applied as an important model organism to evaluate physiological responses to environmental changes and fundamental molecular mechanisms because of its tolerance to hyper salinity and the simplicity of its cytoarchitecture (Booth and Beardall,1991; Zhang et al., 2015). However, comprehensive studies of the effects of elevatedpCO2on the physiological performance of this species remain scare. Nevertheless, it is essential to investigate the long-term acclimation of physiological activities such as photosynthesis and respiration under stable carbonate chemistry. Therefore, our study was conducted using semi-continuous cultures to maintain stable carbonate chemistry systems while keeping the microalgae in an exponential stage for a long period of time. The specific goal of this study was to improve our understanding of the physiological responses of the marine microalgaeD.salinato elevatedpCO2.This was achieved by evaluating the growth,photosynthesis, dark respiration and CCM modes ofD.salinaunder three differentpCO2levels in semicontinuous cultures. The threepCO2levels included:390 μatm (pHNBS: 8.10), which is the present pH value, as well as 1 000 μatm (pHNBS: 7.78) andpCO2:2 000 μatm (pHNBS: 7.49), which are the pH values predicted for 2100 and 2300, respectively. We hypothesized that CO2-induced ocean acidification will change the metabolic energy requirement to balance the reduced external pH, and that elevated CO2availability and changes in seawater carbonate chemistry may reduce their ability to actively transport CO2and HCO3ˉ, thereby reducing the energetic costs of CCMs. Consequently, these effects are predicted to lead to different physiological sensitivities to CO2-induced ocean acidification.

2 MATERIAL AND METHOD

2.1 Culture conditions and experimental design

D.salinawas obtained from the Culture Collection of Algae at the Ocean University of China. The cells were cultured in 0.45 μm-filtered natural seawater collected from Lu Xun seaside Park (Qingdao), which had been autoclaved (30 min, 121°C) and enriched with modified f/2 medium (Guillard, 1975). All cultures were incubated at 20±1°C and illuminated with 80 μmol photon/(m2∙s) (a sub-saturating light intensity) under a 12 h:12 h light: dark cycle. The salinity of the culture medium was adjusted to 30.

Experiments were conducted in triplicate in 500 mL sterilized and acid-washed Erlenmeyer flasks containing 300 mL medium. Prior to inoculation, the cultures were aerated with three different CO2levels:390, 1 000 and 2 000 μatm, corresponding to approximately present-day levels and those predicted for 2100 and 2300, respectively. different air/CO2mixtures were generated by plant CO2chambers(HP400G-D, Ruihua Instrument & Equipment Ltd.,Wuhan, China) with a variation of less than 5%.Semi-continuous cultures used to measure the physiological responses ofD.salinato elevatedpCO2in the present study have been widely applied in other relevant studies (Fu et al., 2007; Hutchins et al., 2007;Wu et al., 2010). In the present study the culture medium was renewed every 24 h to ensure that the cell concentration remained within a range of 2×104to 5×104cells/mL (the dilution rate is about 40%) at their exponential growth phase so that the pH fluctuations during growth were less than 0.06.Cultures were harvested following 4–6 weeks of semi-continuous incubation when their growth rates did not fluctuate significantly for three or more consecutive days, at which time they were considered fully acclimated to their respective experimental treatments.

2.2 Seawater carbonate chemistry

The concentrations of dissolved inorganic carbon(DIC) and pH were measured before and after diluting the culture, as well as during the middle of the light period to ensure stability of the carbonate system in culture. The DIC was determined using a total organic carbon analyzer (TOC-VCPN, Shimadzu) following the method described by Liu et al. (2014). pH values were determined using a pH meter (SevenCompactTMS210k, METTLER TOLEDO) calibrated with the standard National Bureau of Standards (NBS) buff er system in a three-point calibration. The other relevant parameters of the seawater carbonate system were computed according to the known values of pH, DIC,salinity, temperature andpCO2using the CO2SYS software (Lewis et al., 1998).

2.3 Growth and photosynthetic pigment

The growth rate of microalgae was monitored daily using a hemocytometer before and after the medium was renewed. The specific growth rate (μ) was calculated from the equation:μ=(lnN1–lnN0)/(t1–t0),whereN0andN1represent the average cell numbers at timest0(initial or just after the dilution) andt1(before the dilution), respectively.

Chlorophylla(Chla), chlorophyllb(Chlb) and carotenoid (Car) concentrations were measured according to Wellburn (1994). Briefly, 60 mL of culture were filtered onto glass microfiber filters(GF/F, Whatman), then extracted with 10 mL of methanol overnight at 4°C. Concentrations were calculated according to the following equations:

where,A470,A652andA665represent absorbance values of the acetone extracts at 470 nm, 652 nm and 665 nm,respectively.

2.4 Chlorophyll fluorescence measurements

Fluorescence induction curves and rapid light curves (RLCs) were applied to evaluate the changes in photosynthetic performance of microalgae under different levels ofpCO2using a pulse amplitude modulated fluorometer (Water-PAM fluorometer,Walz, Eff eltrich, Germany).

The RLCs were determined at eight different PAR levels (83, 123, 188, 282, 400, 556, 991 and 1 332 μmol photon/(m2∙s)), each of which lasted 10s.The RLCs were fitted with the empirical equation proposed by Platt et al. (1980) to determine the relevant parameters; namely, rETRmax(maximum relative electron transport rate),α(photosynthetic efficiency), andEk(light saturation point). The minimum saturating irradiation was derived from rETRmaxandαaccording to the following equation:The following settings ensured convergence of the regression model: iterations=100,step size=100, tolerance=0.000 1, and initial seed value forP=5,α=0.05 andβ=0 (Ralph and Gademann,2005).

For fluorescence induction curves, all of the samples were dark-adapted for 20 min before measurement. The dark-adapted induction curves were then measured with a delay of 40 s betweenFv/Fmmeasurements. The actinic light was set at 188 μmol photon/(m2∙s) to measure the value of effective quantum yield (ΔF/F'm) and nonphotochemical quenching (NPQ).

2.5 Photosynthetic oxygen evolution and respiration

Photosynthetic oxygen evolution and dark respiration were measured using a Clark-type oxygen electrode (Chlorolab 3, Hansatech, UK). Light was supplied by a halogen lamp, and temperature was maintained using a water bath circulator at 20°C.Prior to the determinations, the cells were allowed to acclimate to the light or dark conditions in the reaction chamber for 15 min. The 5 mL reaction medium was continuously magnetically stirred during the measurement.

The evolution of photosynthetic oxygen ofD.salinaunder differentpCO2values with the addition of inhibitors was measured to determine the mechanism of inorganic carbon acquisition. The inhibitors included acetazolamide (AZ), which is an impermeant CA inhibitor and thus inhibits only extracellular CA, ethoxyzolamide (EZ), which is a membrane-permeable carbonic anhydrase inhibitor that can inhibit both extracellular and intracellular CA, and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate), which inhibits direct HCO3ˉ uptake by means of the anion-exchange protein. These inhibitors have been widely used to determine the contribution of external CA, internal CA and anion-exchange protein to photosynthetic inorganic carbon uptake(Moroney et al., 1985; Axelsson et al., 1995; Ihnken et al., 2011).

2.6 Determination of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities

The cells were collected by centrifugation at 3 000×gand 4°C for 15 min. After removing the supernatant, 1 mL buff er solution (40 mmol/L Tris-HCl, 5 mmol/L glutathione, 10 mmol/L MgCl2and 0.25 mmol/L EDTA, pH 7.6) was added, and the cells were ground on ice. The liquid was subsequently concentrated, after which the supernatant was used for further assays. The Rubisco activity in the supernatant was generally determined following the methods described by Gerard and Driscoll (1996).Briefly, the assay mixture contained 5 mmol/L NADH, 50 mmol/L ATP, 50 mmol/L phosphocreatine,0.2 mmol/L NaHCO3, 160 U/mL creatine phosphokinase, 160 U/mL phosphoglycerate kinase,160 U/mL glyceraldehyde-3-phosphate dehydrogenase and reaction buff er (0.1 mol/L Tris-HCl, 12 mmol/L MgCl2and 0.4 mmol/L EDTA, pH 7.8). The absorbance values at 340 nm (A340) were measured every 20 s for 3 min to obtain the background NADH oxidation rate. Next, 0.05 mL RuBP (final concentration of 25 mmol/L) was added into the assay mixture, and theA340was recorded every 20 s for 3 min. The activities of Rubisco were computed by subtracting the background rate of decrease inA340from the rate determined in the three minutes following RuBP addition and then converting the corrected rate ofA340decrease to a rate of NADH oxidation.

2.7 Measurement of carbonic anhydrase activity

Cells grown under three differentpCO2levels were collected to determine the internal carbonic anhydrase and external carbonic anhydrase activity according to an electrometric method (Giordano and Maberly,1989). Briefly, cells were harvested by centrifugation at 4 000×gand 4°C for 10 min, then re-suspended in Veronal buff er (20 mmol/L, pH 8.2) adjusted to the salinity of the culture medium with NaCl. The cell suspension was initially analyzed for CAextactivity,after which it was used for CAintdetermination. The CAextwas determined based on the time taken for the pH to decrease from 8.2 to 7.2 following the addition of 2 mL CO2-saturated distilled water (also adjusted to the salinity of the culture medium with NaCl) to a 5 mL cell suspension. For measurements of CAintactivity, the same method as described above was used; however, the cell suspension was disrupted by a sonicator and the disruption of cells was confirmed by microscopic observation. Enzyme activity was calculated using the following equation:

where,T0is the uncatalyzed reaction andTis the time of the catalyzed reaction.

Table 1 Parameters of the seawater carbonate chemistry system at different p CO 2 levels prior to and after dilution

Fig.1 The growth rate (a) and pigment contents (chlorophyll a, chlorophyll b and carotenoids) (b) of D. salina acclimated to different p CO 2 levels

3 STATISTICAL ANALYSIS

One-way ANOVA was conducted to identify significant differences among treatments using the SPSS software (20.0). The LSD (Least Significant difference) post-hoc comparison test was used if ANOVA indicated a significant difference. Prior to analysis, data were initially examined for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene’s test. Dates were presented as the means±SE and the significance was set toP＜0.05.All figures were prepared with Sigmaplot 12.5.

4 RESULT

4.1 Carbonate system

Under the simulated laboratory conditions of ocean acidification, the seawater carbonate chemistry system under highpCO2(1 000 μatm and 2 000 μatm)levels showed significant changes compared to under 390 μatm CO2conditions (Table 1). Throughout the semi-continuous culture systems, the pH varied by less than 0.04 before and after diluting the culture medium.

4.2 Growth and photosynthetic pigment

The growth rates ofD.salinaat three differentpCO2treatments were 0.56±0.02, 0.57±0.02 and 0.59±0.03, respectively (Fig.1a). However, the growth rates ofD.salinadid not vary significantly(P＞0.05) amongpCO2treatments. Similar to the growth rates, elevatedpCO2had no significant(P＞0.05) effect on the concentrations of chlorophylla, chlorophyllband carotenoids (Fig.1b).

4.3 Chlorophyll fluorescence

The rapid light curves (RLCs) ofD.salinaacclimated to differentpCO2levels showed a classical pattern of rETR as a function of PAR (Fig.2). With regard to the parameters derived from the RLCs(Table 2), theαvalue increased significantly by 13.4%(P＜0.01) and 10.5% (P＜0.01) when exposed to 1 000 and 2 000 μatmpCO2, respectively. Similar to the trend inα, the rETRmaxincreased significantly by 20.4% (P＜0.05) and 27.5% (P＜0.01) when exposed to 1 000 and 2 000 μatm CO2, respectively.In addition,theEkvalue under 2 000 μatm CO2was significantly higher than that of the control (P＜0.01), while there was no significant difference between 390 μatm and 1 000 μatm CO2(P＞0.05). Neither elevatedpCO2conditions significantly influenced the value ofβ(P＞0.05).

The ΔF/F'mvalue during the induction curves(Fig.3a) showed that elevatedpCO2increased the effective quantum yield ofD.salina. The ΔF/F'mvalue was significantly stimulated by 2.2% (P＞0.05)and 6.6% (P＜0.01) under 1 000 and 2 000 μatm CO2relative to the control. Non-photochemical quenching(NPQ) showed a linear increase, then reached a plateau after 140 s. The cells acclimated to 1 000 and 2 000 μatm CO2showed a lower NPQ of about 86.6%(P＜0.01) and 70.4% (P＜0.01) of that in 390 μatm CO2(Fig.3b).

Table 2 Photosynthetic parameters derived from the rapid light curves of D. salina acclimated to different p CO 2 levels

Fig.2 The rapid light curves of D. salina acclimated to different p CO 2 levels

Fig.3 effective quantum yield (yield) (a) and non-photochemical quenching (NPQ) (b) of D. salina acclimated to different p CO 2 levels

4.4 Photosynthetic oxygen evolution, dark respiration and Rubisco activities

The gross photosynthetic O2evolution (Fig.4b)was significantly enhanced by 14.65% (P＜0.05) and 25.73% (P＜0.01) under 1 000 and 2 000 μatmpCO2,while elevatedpCO2levels did not significantly influence the net photosynthetic O2evolution (Fig.4a)ofD.salina(P＞0.05). Similar to gross photosynthetic O2evolution, Rubisco activity (Fig.4d) was significantly stimulated by 23.86% (P＜0.05) and 29.95% (P＜0.01) under 1 000 and 2 000 μatmpCO2.Cells acclimated to 2 000 μatmpCO2treatments showed a higher dark respiration rate than that of the control group (P＜0.05), while there was no significant difference between the control group and the 1 000 μatmpCO2group (P＞0.05) (Fig.4c).

The inhibitors AZ and EZ had significant effects on the photosynthetic O2evolution (Fig.5) ofD.salinaunder differentpCO2levels (P＜0.05). The inhibitory effect was more pronounced at 390 μatmpCO2than at 1 000 and 2 000 μatmpCO2. The inhibitory effect of AZ on the photosynthetic O2evolution ofD.salinawas 18.67% and 12.67% under 1 000 and 2 000 μatmpCO2, respectively, which was significantly lower than that of 390 μatmpCO2(P＜0.05), for which the inhibitory rate was 26.33%. The inhibitory rates of EZ were 24.67% and 19.10% under the two highpCO2treatments, respectively, while they were significantly lower than those obtained under 390 μatmpCO2(P＜0.05). The inhibitory effects of DIDS on photosynthetic O2evolution ofD.salinadid not differ significantly between the 390 and 1 000 μatmpCO2treatments (P＞0.05), while the inhibitory effect of DIDS under 2 000 μatmpCO2was significantly lower than that of 390 μatmpCO2.

Fig.4 Net photosynthetic oxygen evolution (a), gross photosynthetic oxygen evolution (b), dark respiration (c) and Rubisco activity (d) of D. salina acclimated to different p CO 2 levels

Fig.5 Inhibition rate of inhibitors (AZ, EZ and DIDS) on the photosynthetic O 2 evolution of D. salina under different p CO 2 levels

Fig.6 Carbonic anhydrase activity of D. salina acclimated to different p CO 2 levels

4.5 Carbonic anhydrase

The carbonic anhydrase activities ofD.salinaunder differentpCO2levels are shown in Fig.6.Relative to the control conditions, both the 1 000 μatm and 2 000 μatmpCO2groups showed significantly reduced internal carbonic anhydrase activity (CAint),which decreased by 25.7% (P＜0.05) and 34.6%(P＜0.01), respectively. The external carbonic anhydrase activity (CAext) decreased significantly by 27.7% (P＜0.05) when exposed to 2 000 μatmpCO2levels, while there was no significant difference between the 390 μatm and 1 000 μatmpCO2groups(P＞0.05).

5 DISCUSSION

different species of algae show different types of CCMs. The diatomThalassiosirapseudonana(Yang and Gao, 2012), the diatomPhaeodactylum tricornutum(Burkhardt et al., 2001) and the chlorophyteChlorellaellipsoidea(Matsuda and Colman, 1995) can use both CO2and HCO3ˉ as a source of inorganic carbon, while the chlorophyteNannochlorisatomus(Huertas and Lubián, 1998) and the raphidophyceaeHeterosigmaakashiwo(Nimer et al., 1997) only use CO2. It is generally believed that CAextfunctions to increase the CO2concentration in the boundary layer by converting HCO3ˉ to CO2,thereby facilitating CO2uptake. The presence of CCMs inD.salinais well established, and CAextplays an important role in the operation of CCMs (Booth and Beardall, 1991). In the present study, the net photosynthetic oxygen evolution ofD.salinawas significantly inhibited by acetazolamide (AZ) and 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS),indicating thatD.salinacan use HCO3ˉ via CAextand anion exchange (AE) protein. Furthermore, the operation of CCMs was down-regulated under highpCO2conditions, as indicated by the lower CA activity(CAextand CAint) and the lower inhibition of the photosynthetic rate by AZ, EZ and DIDS (Fig.5) at highpCO2. Such down-regulation of CCMs might be attributed to the elevated availability of CO2and HCO3ˉ, as well as the increased entry of inorganic carbon into the cell by passive diffusion. Mercado et al. (1997) found that the CAintactivity ofPorphyra leucostictewas significantly reduced at elevatedpCO2levels, while the CAextactivity was unaff ected.Conversely, only the CAextactivity ofMacrocystis pyriferawas reduced when acclimated to 1 200 μatm CO2for 7 days, while the CAintshowed no significant changes (Fernández et al., 2015). These different responses to elevated CO2might be due to the different types of inorganic carbon uptake mechanisms in different species and the contribution of CCMs to photosynthetic carbon fixation. Trimborn et al. (2009)showed that both CAextand CAintactivities ofT.pseudonanaare unaff ected when acclimated to 800 μatm CO2for three days, while the CAintactivity and the photosynthetic affinity for CO2were lowered when the same strain was acclimated to 1 000 μatm CO2for 15 days (more than 20 generations) (Yang and Gao, 2012; Wu et al., 2015). The inconsistent results of the responses to elevatedpCO2in the same strain might be due to the acclimation span or a different seawater carbonate system.

The long term cultivation experiment ofD.salinaunder present and predicted futurepCO2conditions showed that photosynthesis ofD.salinawas significantly influenced by CO2-induced ocean acidification in terms of rETRmax, ΔF/F'm,αand Rubisco activities. Moreover, the results indicated that the photosynthetic rate in this species is not saturated at present seawater inorganic carbon concentrations. The ΔF/F'mofD.salinaat highpCO2levels was significantly higher than that observed at the presentpCO2levels. These results indicate that cells grown at highpCO2can use light more efficiently.The positive response of ΔF/F'mto ocean acidification has also been reported for the rhodophyteNeosiphonia harveyiand the diatomNaviculadirecta(Torstensson et al., 2012; Olischläger and Wiencke, 2013).Generally, photosynthetic efficiency (α) represents light use efficiency (Fu et al., 2007, 2008). InD.salina,αin the control was significantly lower than in the highpCO2treatments, suggesting that future highpCO2levels increased the light use efficiency inD.salina. Moreover, Fu et al. (2007) proposed that an increasedαat highpCO2is probably due to reduced energy allocation to the concentration CO2by the cell and for more efficient light use. The results of the present study support the conclusions of Fu et al.(2007), who found that elevatedpCO2down-regulated the CCMs inD.salina, as indicated by decreased carbonic anhydrase activity and lowered inhibition of photosynthetic O2evolution by AZ, EZ and DIDS at highpCO2.Ek(rETRmax/α) represents the optimum light of the photosynthetic apparatus to maintain a balance between photosynthetic energy capture and the capacity to process this energy (Falkowski and Raven, 1997). Liu et al. (2012) reported that CO2-induced seawater acidification down-regulated CCMs in the green alga,Ulvaprolifera, and that the subsequent energy savings contributed to a lowerEk.In the present study, although CCMs were also down-regulated at highpCO2inD.salina, the energy saved did not lead to a lowerEk. On the contrary, increasedEkvalues at highpCO2were found inD.salina,probably due to stimulation of the maximal photosynthetic capacity (rETRmax). Notably, the growth illumination ofD.salinain our experiment was far below theEkvalue, suggesting thatD.salinawas light-limited. The higherEkat highpCO2indicates that the growth ofD.salinaunder future conditions may be closely related to the availability of light, and that light is more likely to be a limiting factor forD.salinain the future. Conversely, the higherEkat highpCO2also indicated that elevatedpCO2led to a higher light threshold at which light becomes excessive, so that algae are less likely to experience light stress than under the present conditions.

Non-photochemical quenching (NPQ) is composed of energy-dependent quenching (qE), which is induced by acidification of the thylakoid lumen, state transition quenching (qT), which is concerned with the balance in the distribution of excitation energy between the two photosystems, and photoinhibitory quenching (qI), which is related to photo-inhibition of photosynthesis (Krause and Jahns, 2004). In the present study, CO2-induced ocean acidification significantly decreased the NPQ ofD.salina(Fig.3b).The synthesis of ATP will lead to translocation of the hydrogen ion from the thylakoid lumen to the stroma,thus weakening the acidification of the thylakoid lumen. Because enhanced carboxylation (Fig.4d) at highpCO2requires more ATP, more H+ions are transported out of the thylakoid lumen, leading to decreased qE. However, cyclic electron transports,which play an important role in photo-protection by producing and maintaining the ΔpH, can be accelerated by the operation of CCMs (Heimann and Schreiber 1999). Down-regulated CCMs ofD.salinacan reduce cyclic electron transport (Moroney and Somanchi,1999), leading to increased qI. Thus, the response of NPQ to elevatedpCO2appears to reflect the net effects of a decreased qE and an increased qI.

Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco) catalyzes the first step in photosynthetic carbon fixation and is the rate-limiting reaction of the Calvin cycle (Spreitzer and Salvucci, 2002). In the present study, increased Rubisco activity at highpCO2indicated that elevatedpCO2enhanced the photosynthetic carbon fixation inD.salina(Fig.4d).Similar to the trend in rubisco activity, highpCO2conditions enhanced dark respiration inD.salina(Fig.4c), indicating a higher energy requirement due to either enhanced biosynthesis in response to increased photosynthetic carbon fixation or increased energy requirements to maintain the intracellular acid-base stability (Geider and Osborne, 1989). The enhanced dark respiration would consume more gross photosynthetic production under future highpCO2and low pH conditions (del Giorgio and Duarte,2002). Therefore, the unchanged growth ofD.salinacould be attributed to the balance of the stimulated carbon assimilation and carbon loss. Similar toD.salina, enhanced photosynthesis and respiration were found in the diatomThalassiosirapseudonana, while growth was not significantly affected by highpCO2(Yang and Gao, 2012). Stimulated photosynthesis and growth were found inPhaeodactylumtricornutum,UlvaproliferaandHeterosigmaakashiwo(Fu et al.,2008; Wu et al., 2010; Xu and Gao, 2012) under highpCO2conditions, since energy was saved in downregulated CCMs or when there was enhanced availability of CO2. However, many other studies have shown no significant effects (Fu et al., 2008;Fernández et al., 2015), and even negative eff ects(Gao et al., 2012; Iñiguez et al., 2016) on the growth and photosynthesis of marine phytoplankton. These studies, together with our results, indicate that the different responses of marine phytoplankton to CO2-induced ocean acidification might be related to the net outcome of the positive and negative (extra carbon loss) eff ects, although the diverse mechanisms of inorganic carbon acquisition and other environmental factors might affect the responses of marine algae to elevatedpCO2.

It has been widely reported that other environmental factors, such as light intensity, temperature and nutrient supply, might modulate the response of algae to increasedpCO2. Gao et al. (2012) found that stimulated growth ofP.tricornutum,T.pseudonanaandSkeletonemacostatumunder highpCO2levels was observed under low light levels, whereas a reverse trend was observed at high light levels.Similarly, different light intensity and nitrogen levels modulated the effects ofpCO2onGracilaria lemaneiformis(Zou and Gao, 2009; Zou et al., 2011).The PSP toxin content ofAlexandriumfundyensedecreased with elevatedpCO2under N-limited conditions, while it showed an adverse trend under N-replete conditions (Eberlein et al., 2016). ForH.akashiwo, the effects ofpCO2on the growth rate were dependent on temperature, with a positive effect observed at 20°C and no effect observed at 24°C (Fu et al., 2008). For the red algaeChondruscrispus, a significant effect of elevatedpCO2on photosynthesis and growth was only observed in interactions with either high temperature or reduced light levels (Sarker et al., 2013). Overall, the results of these studies indicate how changeable and complicated the response ofD.salinato the predicted future ocean might be, and that this response might be modulated by experimental conditions.

6 CONCLUSION

Based on the results of the present study, CO2-induced ocean acidification might leadD.salinato enhance photosynthesis, down-regulate their CCMs,and stimulate dark respiration, resulting in an unchanged growth rate. The net effect of ocean acidification on microalgae will be determined by the balance of these positive and negative eff ects.However, further studies are needed to evaluate the interactive effects ofpCO2, light, nutrients and temperature on growth, photosynthesis and other physiological processes to determine how this species might respond to future ocean conditions.

7 ACKNOWLEDGMENT

We thank all of the members of the laboratory and Dr. LIANG in the College of Fisheries of Ocean University of China for their help.

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