Effect of CO2-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi

Shunxin Hu; Bin Zhou; You Wang; Ying Wang; Xinxin Zhang; Yan Zhao; Xinyu Zhao; Xuexi Tang

doi:10.1371/journal.pone.0183289

Abstract

Karenia mikimotoi is a widespread, toxic and non-calcifying dinoflagellate, which can release and produce ichthyotoxins and hemolytic toxins affecting the food web within the area of its bloom. Shifts in the physiological characteristics of K. mikimotoi due to CO₂-induced seawater acidification could alter the occurrence, severity and impacts of harmful algal blooms (HABs). Here, we investigated the effects of elevated pCO₂ on the physiology of K. mikimotoi. Using semi-continuous cultures under controlled laboratory conditions, growth, photosynthesis and inorganic carbon acquisition were determined over 4–6 week incubations at ambient (390ppmv) and elevated pCO₂ levels (1000 ppmv and 2000 ppmv). pH-drift and inhibitor-experiments suggested that K. mikimotoi was capable of acquiring HCO₃^-, and that the utilization of HCO₃^- was predominantly mediated by anion-exchange proteins, but that HCO₃^- dehydration catalyzed by external carbonic anhydrase (CA_ext) only played a minor role in K. mikimotoi. Even though down-regulated CO₂ concentrating mechanisms (CCMs) and enhanced gross photosynthetic O₂ evolution were observed under 1000 ppmv CO₂ conditions, the saved energy did not stimulate growth of K. mikimotoi under 1000 ppmv CO₂, probably due to the increased dark respiration. However, significantly higher growth and photosynthesis [in terms of photosynthetic oxygen evolution, effective quantum Yield (Yield), photosynthetic efficiency (α), light saturation point (E_k) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity] were observed under 2000 ppmv CO₂ conditions. Furthermore, elevated pCO₂ increased the photo-inhibition rate of photosystem II (β) and non-photochemical quenching (NPQ) at high light. We suggest that the energy saved through the down-regulation of CCMs might lead to the additional light stress and photo-damage. Therefore, the response of this species to elevated CO₂ conditions will be determined by more than regulation and efficiency of CCMs.

Citation: Hu S, Zhou B, Wang Y, Wang Y, Zhang X, Zhao Y, et al. (2017) Effect of CO₂-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi. PLoS ONE 12(8): e0183289. https://doi.org/10.1371/journal.pone.0183289

Editor: Hans G. Dam, University of Connecticut, UNITED STATES

Received: July 14, 2016; Accepted: August 2, 2017; Published: August 16, 2017

Copyright: © 2017 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This work was supported by the Natural Science Foundation of China (41476091) and NSFC-Shangdong Joint Fund (U1406403).

Competing interests: The authors have declared that no competing interests exist.

Introduction

Ocean acidification refers to the ongoing reduction in the ocean pH over an extended period of time, which is primarily caused by the uptake of anthropogenic CO₂ from the atmosphere [1, 2]. Industrialization and fossil fuel combustion have increased the atmospheric CO₂ concentrations from pre-industrial levels of approximately 280 ppmv to the current level of approximately 390 ppmv [2, 3]. The atmospheric CO₂ concentrations are predicted to increase to 1000 ppmv and 2000 ppmv by the years 2100 and 2300, respectively, if the present energy utilization structure persists [1]. Such increases in CO₂ would lead to a reduction in pH (0.4 and 0.77 units, respectively) and cause substantial chemical changes in seawater carbonate systems, including increases in pCO₂, HCO₃^- and DIC and decreases in H⁺ and CO₃^2- [4, 5].

Marine phytoplankton assimilates inorganic carbon and fixes CO₂ into carbohydrates through the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which can only use CO₂ as substrate for the carboxylase reaction. Rubisco is generally characterized by low affinities for CO₂ (K_M of 20–70 μmol L^-1), and a competitive reaction with O₂ further reduces its efficiency [6]. Therefore, photosynthesis of some marine phytoplankton might suffer from CO₂ limitation, due to the present concentration of aqueous CO₂ in seawater ranging from 8 to 20 μmol L^-1. Most marine phytoplankton have developed so-called CO₂ concentrating mechanisms (CCMs) to overcome the deficiencies of Rubisco. Two main types of CCMs are suggested to be involved in marine phytoplankton. Firstly, the dehydration of HCO₃^- is catalyzed by external carbonic anhydrase, facilitating the supply of CO₂ at plasma membrane and improving the potential for CO₂ uptake, and then CO₂ accumulation is achieved by the active transport of HCO₃^- or CO₂ at the chloroplast envelope. Secondly, HCO₃^- is transported across the plasmalemma and/or chloroplast envelope and then converted to CO₂, catalyzed by one of several types of internal carbonic anhydrase [7]. However, the expression and operation of CCMs require energetic investment. Therefore, marine phytoplankton could benefit from elevated pCO₂ in two synergetic ways: one is that the increased dissolved carbon dioxide (CO₂aq) would supply additional substrate for photosynthetic carbon fixation and reduce the oxygenase reaction of Rubisco, alleviating the carbon limitations of species without a CCM [8]. The other is that, increased pCO₂ could down-regulate the energetically costly operation of CCMs.

Many marine phytoplankton species down-regulate their operation of CCMs at high pCO₂ conditions, as revealed by a lower photosynthetic affinity for CO₂, decreased activities of carbonic anhydrase and/or a lower contribution of HCO₃^-assimilation [9–12]. This is taken as evidence that elevated pCO₂ exerts positive effects on the growth and photosynthesis of species bearing CCMs[9, 13–15]. Some species (Macrocystis pyrifera and Gracilaria lemaneiformis), however, do not show any deactivation of CCMs or changes in growth and photosynthesis in response to elevated pCO₂ [16, 17]. In addition, increased pCO₂ might exert negative effects on calcifying species because of a lowering of saturation of CaCO₃, which might make calcfication more difficult [18,19]. Deleterious effects of elevated pCO₂, however, also occur on non-calcifying species [20–23], probably due to the negative effects on physiological processes caused by reduced external pH. [24]. Altogether, these findings havedeepened our understandings of differential physiological responses to elevated pCO₂ among various marine phytoplankton species, such as diatoms and coccolithophores.

Karenia mikimotoi is a widespread, toxic and non-calcifying dinoflagellate, which can have deleterious effects on other marine phytoplankton, fish and shellfish through the release of ichthyotoxins and hemolytic toxins [25]. K. mikimotoi toxic blooms have been reported in Japan, Ireland, England, France, India and China [26–30]. At least 103 harmful algal blooms (HABs) have been reported between 2004 and 2014 caused by K. mikimotoi in eastern and southern Chinese seas, affecting about 37527 km² (according to China Marine Disaster Bulletin). Owing to its ecological and environmental implications, it is necessary to study the physiological responses of this species to elevated pCO₂ in order to predict the occurrence, severity and impacts of blooms of this species in the future. In the present study, we evaluated growth, photosynthesis, dark respiration and the CCMs modes of K. mikimotoi exposed to three different pCO₂ levels: 390 ppmv (pH_NBS: 8.10) which is the present pH value, as well as 1000 ppmv (pH_NBS: 7.78) and pCO₂: 2000 ppmv (pH_NBS: 7.49), which are predicted to be possible conditions in 2100 and 2300, respectively. We hypothesized that (1) the operation of CCMs will be down-regulated with elevated pCO₂ and (2) the reduction in the energy costs of CCMs will benefit growth and photosynthesis of K. mikimotoi.

Materials and methods

Culture conditions and experimental design

Karenia mikimotoi (strain OUC151001) was obtained from the Algal Culture Collection at the Ocean University of China. Cells were cultured in 0.45μm-filtered natural seawater, collected from Luxun Seaside Park (Qingdao), which had been autoclaved (30min, 121°C) and enriched with f/2 medium [31]. All cultures were incubated at 20±1°C and illuminated with 80 μmol photon m^-2 s^-1 under a 12:12 light: dark cycle.

Experiments were conducted in triplicate 1000 ml sterilized and acid-washed Erlenmeyer flask containing 600 ml of medium. Prior to inoculation, the cultures were equilibrated at three different CO₂ levels: 390 ppmv CO₂ (~present-day), 1000 and 2000 ppmv CO₂ (predicted CO₂ levels in 2100 and 2300, respectively), obtained by gentle bubbling with 0.22 μm-filtered ambient air and air/CO₂ mixtures. The air/CO₂ mixtures were generated by plant CO₂ chambers (HP400G-D, Ruihua Instrument & Equipment Ltd, Wuhan, China) with a variation of less than 5%. Semi-continuous cultures were used to measure the effects of CO₂-induced seawater acidification on the growth and physiology of K. mikimotoi in the present study, similar to previous ocean acidification research [9, 32–34]. All cultures were diluted to 800 cells mL^-1 with fresh medium pre-acclimated to the desired CO₂ level every 24h to maintain cells in exponential growth phase, and to minimize pH fluctuations. Cultures were harvested following 4–6 weeks of semi-continuous incubation when the growth rates were not significantly different for three or more consecutive days, which was considered fully acclimated to their respective experimental treatments.

Seawater carbonate chemistry

The pH value and dissolved inorganic carbon (DIC) were determined prior to and after the daily dilution. The concentration of DIC in the culture medium was measured using a total organic carbon analyzer (TOC-V_CPN, Shimadzu). The samples were filtered onto brown glasses via 0.45 μm cellulose acetate membranes and stored in a refrigerator (4°C). The pH was measured using a pH meter (SevenCompact^™ S210k, Mettler Toledo, Switzerland), which was calibrated daily with standard National Bureau of Standards (NBS) buffer system. The other relevant parameters of carbonate system were determined with the CO₂SYS software [35], based on the known parameters (pH, DIC, salinity and temperature).

Growth and elemental analysis

Cell growth rate was monitored daily using a plankton counting chamber (0.1 mL) before and after the medium was diluted. The specific growth rate (μ) was calculated using the equation:

where N₀ and N₁ represent the average cell numbers at times t₀ (after the dilution) and t₁ (before the dilution), respectively.

Samples for measurements of cellular carbon (C), nitrogen (N) and phosphorus (P) were filtered onto glass microfiber membranes (GF/F, Whatman), which were pre-combusted at 500°C for 4 h, and then stored at -20°C in a refrigerator before analysis. Cellular C and N content were determined using a Perkin-Elmer 2400 CHNS analyzer following the method of Zhao et al. [36]. Cellular P content was measured as in Fourqurean et al. [37].

Chlorophyll a

Chlorophyll a (Chl a) samples from the cultures were filtered onto glass microfiber filters (GF/F, Whatman), and extracted with 10 mL of methanol overnight at 4°C. Samples were then analyzed in a spectrophotomer, and the Chl a concentration was calculated according to the following equation [38]: where A_652, A₆₆₅ and A₇₅₀ denoted the absorbance values of the methanol extracts at 652nm, 665nm and 750nm, respectively.

Photosynthetic oxygen evolution and respiration

Photosynthetic oxygen evolution was measured under the same light intensities used for growing cultures, with lighting provided by a halogen lamp. The dark respiration rate was determined using a Clark-type oxygen electrode (Chlorolab 3, Hansatech, UK). Experimental temperature was maintained at 20°C using a water bath circulator. Before the determination, cells were acclimated to light or dark conditions in the reaction chamber for 20 min. The 5ml-reaction media was continuously stirred with a magnetic stirrer during treatment. DIC concentrations were consistent with their culture conditions, which were nominally 390ppmv: 1919–1924 μmol/kg; 1000ppmv: 2059–2067 μmol/kg; 2000 ppmv:2152–2156 μmol/kg, respectively.

Chlorophyll fluorescence measurements

Fluorescence induction curves and rapid light curves (RLCs) were applied to evaluate the photosynthetic performance of K. mikimotoi acclimated to different pCO₂, using a Water-PAM fluorometer.

The RLCs were measured at 8 different actinic irradiance levels (80, 119, 184, 276, 393, 546, 897 and 1315 μmol photon m^-2 s^-1), each of which lasted 10s. To quantitatively compare the RLCs of K. mikimotoi acclimated to different pCO₂, the equation of Platt et al. [39] was applied to derive characteristic parameters: photosynthetic efficiency (α), light saturation point (E_k), photo-inhibition rate of photosystemII(β) and maximum relative electron transport rate (rETR_max). The light saturation point was determined from: E_k = rETR_max/α [40]. Related parameters were applied to determine the convergence of the regression mode according to Ralph and Gademann [40].

For the fluorescence induction curves, all of the samples were dark-acclimated for 20 mins before determination. The dark-acclimation induction curves were measured with a delay of 40 s between the determinations of F_v / F_m. The actinic light was set at 80, 276 and 897 μmol photon m^-2 s^-1, respectively, to measure the value of effective quantum yield (Yield) and non-photochemical quenching (NPQ)

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

Cells were collected by centrifugation at a rotating speed of 3000g at 4°C for 15 min. After removing the supernatant, cells were grinded on ice with the addition of 1mL buffer solution (40 mM Tris-HCl, 5 mM Glutathione, 10 mM MgCl₂ and 0.25 mM EDTA, pH 7.6). The liquid was concentrated, and the supernatant was used for further assays. Rubisco activity in the supernatant was generally determined following the methods described by Gerard and Driscoll [41]. The assay mixture contained 5 mM NADH, 50 mM ATP, 50 mM phosphocreatine, 0.2 mM NaHCO₃, 160 U/mL creatine phosphokinase, 160 U/mL Phosphoglycerate kinase, 160 U/mL glyceraldehyde-3-phosphate dehydrogenase and reaction buffer (0.1M Tris-HCl, 12 mM MgCl₂ and 0.4 mM EDTA, pH 7.8). Absorbance values at 340 nm (A₃₄₀) were measured every 20s for 3 min to obtain the background NADH oxidation rate. A volume of 0.05 mL of RuBP (final concentrations of 25 mM) was added into the assay mixture, and the A₃₄₀ was then recorded every 20 s for 3 min. The activities of Rubisco were computed by subtracting the background rate of decrease in A₃₄₀ from the rate determined during the three minutes following RuBP addition, and then converting the corrected rate of A₃₄₀ decrease to a rate of NADH oxidation.

pH drift experiment

A pH drift experiment was applied to determine whether K. mikimotoi can utilize HCO₃^- as an inorganic carbon source; the ability of algae to raise the medium pH to higher than 9.0 is considered as evidence of its capacity to utilize HCO₃^-. The experiment was performed in sterilized glasses containing 10 mL samples (cell concentrations of 10×10⁴ mL^-1) at 20°C and 80 μmol photon m^-2 s^-1. The pH was measured every hour and the final pH values were obtained once no further pH increases were detected.

Determination of carbonic anhydrase activity

Cells were collected by centrifugation at a rotating speed of 3000g at 4°C for 15 min and re-suspended in 20mM barbitone (pH 8.2). The total carbonic anhydrase (CA_tot) and external carbonic anhydrase (CA_ext) activities were measured using an electrometric method [42]. For determination of CA_tot, cells were disrupted with a sonicator, and cell brakage confirmed under a microscope. The reaction was begun by adding 2 mL ice-cold CO₂ to saturated Milli-Q water, and the time it took for the pH to decrease from 8.2 to 7.2 was recorded. The temperature was controlled at 4°C.

Effect of inhibitors on chlorophyll fluorescence

The RLCs of K. mikimotoi acclimated to different pCO₂ with addition of inhibitors were obtained to determine the mechanism of inorganic carbon acquisition. The inhibitors included acetazolamide (AZ), which inhibits only extracellular CA, ethoxyzolamide (EZ), which inhibits both extracellular and intracellular CA, and DIDS (4,4’-diisothiocyanostilbene-2,2’-disulfonate), which inhibits direct HCO₃^- 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 [21, 43, 44].

Statistical analysis

One-way ANOVA was used to analyze the significance of the differences between treatments using the SPSS software (20.0), and the significant difference level was set to P < 0.05. All figures were prepared with Sigmaplot 12.5.

Results

Seawater carbonate chemistry

Under the simulated laboratory conditions of ocean acidification, the seawater carbonates chemistry system at elevated pCO₂ (1000 ppmv and 2000 ppmv) levels significantly differed from that of the control group (Table 1). The DIC, CO₂ and HCO₃^- concentrations in the 1000 ppmv-treated system were increased by 7.0%, 125.9% and 10.1%, respectively, whereas in the 2000 ppmv-treated system, these values increased by 12.1%, 361.4% and 15.5%, respectively. The CO₃^2- concentrations were decreased by 46.5% and 71.1% in the 1000 ppmv- and 2000 ppmv-treated systems, respectively, and the difference in total alkalinity (TA) was insignificant. The fluctuations of pH prior and after the dilution of the culture medium were <0.03.

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Table 1. Parameters of the seawater carbonate chemistry system at different pCO₂ levels prior and after the dilution. The dissolved inorganic carbon (DIC) concentration, pH_NBS, temperature and salinity were used to compute other parameters with a CO₂ system analyzing software (CO₂SYS). Data are shown as the mean ± SE (n = 9). Different letters represent significant difference between variables (P < 0.05).

https://doi.org/10.1371/journal.pone.0183289.t001

Growth and elemental composition

The growth rates at the three pCO₂ levels are shown in Fig 1. The growth rate of K. mikimotoi was significantly stimulated by 16.84% (P<0.05) after exposure to 2000 ppmv pCO₂; although growth was also enhanced under 1000 ppmv pCO₂, the increase was not statistically significant (P>0.05).

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Fig 1. Growth rate of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 9).

https://doi.org/10.1371/journal.pone.0183289.g001

The total cellular C,N,P and their elemental ratio in K. mikimotoi are shown in Table 2. The cellular C and P concentrations of K. mikimotoi exposed to 2000 ppmv pCO₂ levels were significantly (P<0.05) higher than those of the control, whereas there was no significant difference between the control and 1000 ppmv pCO₂ (P>0.05). Furthermore, elevated pCO₂ exerted no significant effects on the cellular N, C: N, C: P and N: P ratios (P>0.05).

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Table 2. Elemental composition (total C, N and P) and elemental ratio (C:N, C:P and N:P) of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 9).

Different letters represent significant difference between variables (P < 0.05).

https://doi.org/10.1371/journal.pone.0183289.t002

Chlorophyll a

Cells acclimated to the three pCO₂ levels showed the same Chl a content (Fig 2) of about 2.0 pg cell^-1, with no significant differences among treatments.

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Fig 2. Chlorophyll a content of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 9).

https://doi.org/10.1371/journal.pone.0183289.g002

Photosynthetic oxygen evolution, dark respiration and Rubisco activities

Results of the determinations of the net photosynthetic oxygen evolution, gross photosynthetic oxygen evolution, dark respiration, and Rubisco activity are shown in Fig 3. Net photosynthetic oxygen evolution (Fig 3A) and Rubisco activity (Fig 3D) were significantly enhanced by 22.86% (P<0.05) and 31.99% (P<0.05) under 2000 ppmv pCO₂, whereas there were no significant difference between the control and 1000 ppmv pCO₂ (P>0.05). Cells acclimated to both 1000 ppmv and 2000 ppmv pCO₂ treatments showed higher gross photosynthetic oxygen evolution (Fig 3B) and dark respiration (Fig 3C) than those of the control (P<0.05).

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Fig 3. Net photosynthetic oxygen evolution (A), gross photosynthetic oxygen evolution (B), dark respiration (C) and Rubisco activity (D) of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 9).

https://doi.org/10.1371/journal.pone.0183289.g003

Chlorophyll fluorescence

Rapid light curves (RLCs) were determined at the three levels of pCO₂ (control, 1000 ppmv and 2000 ppmv). The three treatments all exhibited a classical pattern of rETR as a function of PAR (Fig 4A), with a rapid increase under light-limited conditions followed by a plateau, at which the photosynthetic pathway was saturated.

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Fig 4. The rapid light curves of K. mikimotoi without and with the addition of inhibitors (AZ, EZ and DIDS) acclimated to different pCO₂. Data are shown as the mean ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0183289.g004

The parameters derived from the RLCs are shown in Table 3. The photosynthetic efficiency (α) of K. mikimotoi was significantly stimulated by 23.1% (P<0.05) under 2000 ppmv pCO_2. Cells acclimated to 1000 ppmv pCO₂ also showed higher α than that of control, but the increase was not statistically significant (P>0.05). Contrary to the trend observed in α, elevated pCO₂ significantly decreased the light saturation point (E_k) of K. mikimotoi by 7.5% (P<0.05) and 10.2% (P<0.05) under 1000 and 2000 ppmv CO₂ compared with the control, respectively. The maximum relative electron transport rates (rETR_max) were approximately 10% higher in both elevated pCO₂ levels compared with the control, but the differences were not significant (P > 0.05). Moreover, the results showed that increased pCO₂ levels had no significant effect on the F_v / F_m of K. mikimotoi (P > 0.05).

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Table 3. Photosynthetic parameters derived from the rapid light curves of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 3).

Different letters represent significant difference between variables (P < 0.05).

https://doi.org/10.1371/journal.pone.0183289.t003

The inhibition of rETR was obtained from the rapid light curves in all three different pCO₂ levels when exposed to the actinic irradiance of 1315 μmol photon m^-2 s^-1. β values (relative inhibition), characterizing the photo-inhibition rate of PSII exposed to high actinic irradiance, were significantly increased by 126.7% (P<0.01) and 194.3% (P<0.001), respectively, in 1000 ppmv and 2000 ppmv pCO₂ compared with the control (Table 3).

The non-photochemical quenching (NPQ) and effective quantum yield of PS II (Yield) values derived from the induction light curve (IC) are shown in Figs 5 and 6, respectively. The results indicated that response of NPQ to higher CO₂ concentrations depended on actinic irradiances. Under 80 μmol photon m^-2 s^-1, the NPQ values in the 390ppmv pCO₂ were 15.4% (P<0.05) and 43.9% (P<0.01) higher than those observed in the 1000 ppmv and 2000 ppmv pCO₂, respectively. By contrast, NPQ values at 276 and 897 μmol photon m^-2 s^-1 were significantly increased in the two high pCO₂ groups (P < 0.01), but there was no significant difference between 1000 ppmv and the control when exposed to 276 μmol photon m^-2 s^-1 (P>0.05). Relative to the control conditions, both the 1000 ppmv and 2000 ppmv pCO₂ groups significantly stimulated the Yield, which was increased by 4.0% (P<0.05) and 4.9% (P<0.01) at 80 μmol photon m^-2 s^-1, and increased by 11.8% (P<0.05) and 11.8% (P<0.05) at 897 μmol photon m^-2 s^-1.

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Fig 5. Non-photochemical quenching (NPQ) of K. mikimotoi acclimated to different pCO₂ levels at an actinic irradiance of 80, 276 and 897 photon m^-2 s^-1. Data are shown as the mean ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0183289.g005

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Fig 6. Effective quantum yield (Yield) of K. mikimotoi acclimated to different pCO₂ levels at an actinic irradiance 80, 276 and 897 photon m^-2 s^-1. Data are shown as the mean ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0183289.g006

At an actinic of 276 μmol photon m^-2 s^-1, the 2000 ppmv pCO₂ groups exhibited a significant (P<0.01) increase, but not the 1000 ppmv pCO₂ groups.

pH drift experiment and carbonic anhydrase activity

The final pH value obtained in the pH drift experiment was 9.8±0.1. Furthermore, the total carbonic anhydrase activity (CA_tot) and internal anhydrase activity (CA_int) (Fig 7) of K. mikimotoi were significantly decreased by 50.6% (P<0.01) and 55.5% (P<0.05) after exposure to 2000 ppmv pCO₂, and no significant changes were observed when exposed to 1000 ppmv pCO₂ (P>0.05). The external carbonic anhydrase activity (CA_ext) (Fig 7) of K. mikimotoi was significantly lower than the CA_int, and no significant changes were observed when exposed to 1000 ppmv (P>0.05) and 2000 ppmv (P>0.05) pCO₂.

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Fig 7. Total, external and internal carbonic anhydrase activity of K. mikimotoi acclimated to different pCO₂ levels. Data are shown as the mean ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0183289.g007

Effect of inhibitors on chlorophyll fluorescence

The rapid light curves of K. mikimotoi acclimated to different pCO₂ levels with and without the addition of AZ, EZ and DIDS are shown in Fig 4. The results indicated that the rETR values of K. mikimotoi were significantly inhibited by the addition of EZ and DIDS, whereas the inhibition of rETR by the addition of AZ was significantly less than that obtained with EZ and DIDS.

The inhibition rates of rETR at different pCO₂ levels obtained with the addition of AZ, EZ and DIDS are shown in Table 4. The results indicated that the inhibitions of rETRs by the addition of EZ and DIDS were significantly (P<0.01) higher in the controls than those obtained at the high pCO₂ groups (1000 ppmv and 2000 ppmv). Furthermore, the results from all three pCO₂ levels indicated that the inhibition of rETR was significantly increased by the increasing PAR. in the presence of AZ, rETR inhibition was only observed in the 390 ppmv pCO₂.

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Table 4. Percent inhibition of rETR acclimated to different pCO₂ with the addition of AZ, EZ and DIDS within a PAR range of 0 to 1315 μmol photon m^-2 s^-1, “—” represents no inhibition of rETR. Data are shown as the mean ± SE (n = 3).

https://doi.org/10.1371/journal.pone.0183289.t004

Discussion

Inorganic carbon acquisition

Dinoflagellates are abundant and ecologically important in marine ecosystems, and morphologically and physiologically diverse [45]. They are also the only oxygenic photoautotrophs with type II Rubisco, the enzyme with the lowest affinity for CO₂ among eukaryotic phytoplankton. Thus, dinoflagellates are at a disadvantage with regard to photosynthetic carbon fixation under the present ocean conditions of low CO₂, and high O₂ [6]. Consequently, dinoflagellates probably require an efficient CCM to compete with other phytoplankton that have higher photosynthetic and growth rates. It is a common notion that the ability of algae to raise the final pH of the medium to higher than 9.0 is an indicator of HCO₃^- utilized by the species [46, 47]. The pH-drift experiments conducted in our study indicated that the final pH value in the medium of K. mikimotoi was 9.8±0.1, suggesting that K. mikimotoi could use HCO₃^- in seawater. Although most phytoplankton species possess CCMs, large differences exist in their efficiencies. Owing to their rather inefficient CCMs (strongly dependent on CO₂ as inorganic carbon source), the photosynthetic carbon fixation of the coccolithophorid Emiliania huxleyi and the raphidophyceae Heterosigma akashiwo are well below saturation at present CO₂ levels, and therefore are more CO₂-sensitive than species with highly efficient CCMs (which rely heavily on HCO₃^- as inorganic carbon source), such as Skeletonema costatum and Phaeocystis globose [32, 48]. In the present study, Rubisco activity of K. mikimotoi did not change between 390 and 1000 ppmv, and ETR values only increase slightly (~10%), which is strong evidence that K. mikimotoi, similar to S. costatum and P. globose, relies heavily on HCO₃^- as an inorganic carbon source.

CA_ext, which catalyzes the dehydration of HCO₃^- to CO₂ at the cell surface [47–49], was found to decrease at high pCO₂ conditions in other species such as Phaeocystis globosa and S. costatum, underlining the important role of CA_ext in inorganic carbon acquisition [48]. In the present study, such a HCO₃^- dehydration mechanism was likely to contribute very little to the inorganic carbon acquisition in K. mikimotoi, because the activities of CA_ext were very low in all treatments (Fig 7). Furthermore, the minor role of CA_ext was also indicated by the addition of membrane impermeable CA inhibitor AZ, which only slightly inhibited the rETR of K. mikimotoi (Fig 4 and Table 4). The low CA_ext activity observed in K. mikimotoi was consistent with most other tested dinoflagellate species. Rost et al. [50] investigated CA_ext activities in Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum, and found relatively low or negligible activities in all three species. Eberlein et al. [51] showed that CA_ext activities of the dinoflagellate Alexandrium tamarense acclimated to a range of pCO₂ from 180 to 1200 μatm were close to detection limits, and thus only played a minor role. However, low CA_ext activity is not universal in all dinoflagellate species. High activity of CA_ext was found in Scrippsiella trochoidea, probably to convert the effluxing CO₂ to HCO₃^-, and then utilized via the HCO₃^- transporter by the cells [51]. This ‘CO₂ recirculation mechanism’ might be especially beneficial for species with high dark respiration rates.

Furthermore, direct HCO₃^- uptake via the anion-exchange (AE) protein has also been observed in other species, which suggests that HCO₃^- utilization could be inhibited by the AE protein inhibitor, DIDS [52–53]. From our results, such a direct HCO₃^- uptake was likely to be present in K. mikimotoi, and it was significantly reduced when the cells acclimated to 1000 and 2000 ppmv pCO₂, because the rETR of K. mikimotoi was drastically depressed by the addition of DIDS, and the inhibition decreased at high pCO₂ as compared with the control (Fig 4 and Table 4). Down-regulated CCMs at high pCO₂ have also been found in widely distributed species such as Skeletonema costatum, Emiliania huxleyi, Thalassionema nitzschioides and Pseudo-nitzschia multiseries [48,54,55]. This down-regulation might result from the increasing diffusive CO₂ uptake at high pCO₂ conditions, since CO₂ uptake is considered to be less energetically costly than HCO₃^- uptake. Consequently, cells can optimize their allocation of energy and apportion more energy for photosynthetic carbon fixation. Marine phytoplankton productivity based on energy or carbon content might thus increase under typically resource-limited conditions in the ocean. From this point of view, species with regulated CCMs, as shown for K. mikimotoi, might have a competitive advantage in the future compared to species that do not react to high pCO₂ such as Phaeocystis globosa, Thalassiosira pseudonana, Eucampia zodiacus and Nitzschia navis-varingica [48,54,55]. As a consequence, these different responses of CCMs to elevated pCO₂ might change the fitness of the different group and possibly alter the distribution and succession of marine algae in natural systems.

Growth, photosynthesis, respiration and photosynthetic electron transport

Growth is a comprehensive parameter integrating all physiological processes in marine phytoplankton, and different responses of growth to increased pCO₂ have been reported among different marine phytoplankton species, with positive, negative and no significant responses. According to our experimental results, the growth rate of K. mikimotoi was not significantly affected under 1000 ppmv pCO₂ conditions, while the stimulated growth rate was observed under 2000 ppmv pCO₂ conditions. Enhanced growth and photosynthesis by elevated pCO₂ have been reported in species such as the diatoms Navicula pelliculosa [56] and Phaeodactylum tricornutum [9], the raphidophyceae Heterosigma akashiwo [33] and the chlorophyte Ulva rigida [57]. However, other studies found no significant effects [32, 58, 59], or even negative effects [22, 60, 61] on the growth, photosynthesis or primary productivity of marine phytoplankton. With regard to the species with highly efficient and strongly regulated CCMs, stimulated growth by elevated pCO₂ is generally attributed to decreased energetic cost of CCMs and HCO₃^- uptake, with the saved energy allocated to support growth [62]. For K. mikimotoi, the operation of CCMs were downregulated under both 1000 and 2000 ppmv pCO₂ conditions, but the saved energy from the downregulated CCMs did not stimulate growth under 1000 ppmv pCO₂, probably because of enhanced respiratory carbon loss. Even though gross photosynthesis of K. mikimotoi was enhanced under 1000 ppmv pCO₂ (Fig 3B), net photosynthesis was not significantly affected (Fig 3A), which may be largely caused by the enhanced dark respiration. Consequently, the growth of K. mikimotoi grown in 1000 ppmv pCO₂ was not significantly stimulated, similarly to what was reported for the diatom Thalassiosira pseudonana [10]. When the cells acclimated to 2000 ppmv pCO₂, photosynthesis of K. mikimotoi significantly increased. The explanation might be that energy was saved from down-regulation of CCM_S, and thus resulted in increased growth, Results in the present study suggest that the responses of marine phytoplankton to future CO₂-driven seawater acidification are not only determined by the efficiency and regulation of CCMs, but also controlled by the balance of the positive and negative effects associated with increased pCO₂ and seawater acidity.

Enhanced dark respiration rates by elevated pCO₂ have been reported in other species such as the diatom Thalassiosira pseudonana, the dinoflagellate Alexandrium tamarense and the diatom Phaeodactylum tricornutum [9, 10, 52], but not in the dinoflagellate Scrippsiella trochoidea and the rhodophyte Porphyra leucosticte [51, 63]. Conversely, a decrease in dark respiration was observed in the chlorophyte Ulva rigida and the diatom T. pseudonana [64, 65]. Stimulation of dark respiration under high pCO₂ condition could reflect higher energy requirement due to either enhanced biosynthesis in response to increased carbon fixation, more energy demand to counteract external pH reduction and to maintain intracellular acid-base stability [66], or pH-dependent changes in the function of respiratory enzymes and altered proton gradient across the mitochondrial membrane [67]. Alternatively, decreased dark respiration by elevated pCO₂ has been ascribed to the down-regulation of CCMs in order to prevent oxidative damage from excess energy [65].

Comparing the parameters derived from rapid light curves (RLCs) and induction curves, elevated pCO₂ appeared to have a positive impact on the efficiency of PSII, indicated by stimulated α, Yield and decreased E_k at high pCO₂. Generally, photosynthetic efficiency (α) represents the energetic costs of photosynthesis. Accordingly, the present study indicated that future increased pCO₂ reduces the costs of photosynthesis in K. mikimotoi. Fu et al. (2007) suggested that a stimulated α at high pCO₂ is attributed to the decreased energetic cost of CCMs and more efficient light use [32]. This suggestion is supported by our findings because the lower contribution of HCO₃^- to inorganic acquisition was also observed in K. mikimotoi. The E_k (rETR_max / α) represents the optimum light of the photosynthetic apparatus to maintain a balance between photosynthetic energy capture and the capacity to process this energy [68]. For K. mikimotoi, an increase in α, and no significant changes in ETR_max at high pCO₂ resulted in a decrease in the light saturation point (E_k). This indicates that elevated pCO₂ can stimulate the efficiency of light harvesting and processing of PSII, and thus fewer photons are demanded to reach the E_k at high pCO₂. Furthermore, the lower E_k of K. mikimotoi in future CO₂-induced ocean acidification also suggests that light is less likely to be a limiting factor, thus making it more competitive in light-limited conditions. By contrast, the lower E_k also indicated that high pCO₂ lowered the light threshold at which light became excessive in K. mikimotoi, and thus the cells easily became photo-inhibited at high light conditions, an inference supported by the increased NPQ (Fig 5) and β (Table 3) at high pCO₂ conditions. The operation of CCMs serves as the pathway for alleviating photo-damage through the dissipation of excess light energy [69, 70]. For K. mikimotoi, the more active CCMs in the control would consume more energy and drain more hydrogen ion out of the thylakoid lumen to the stroma, which results in a lower NPQ (Fig 5). Therefore, elevated pCO₂ diminished the energy-dissipation via the down-regulated CCMs, leading to increased NPQ (Fig 5) and enhancing photo-inhibition (Table 3) at high light conditions. Wu et al. (2010) also found that elevated pCO₂ enhanced the photo-inhibition of rETR in Pheodactylum tricornutum when exposed to high PAR, but the NPQ decreased at high pCO₂ [9]. In contrast, Thalsassiosira pseudonana acclimated to 390 and 1000 ppmv CO₂ showed identical photo-inhibition and NPQ when exposed to high PAR, indicating that high light tolerance was not altered by high pCO₂ [10]. Such different responses among K. mikimotoi, P. tricornutum and T. pseudonana suggest that species-specific metabolic pathways might be involved in coping with elevated pCO₂ and high light stress.

Elemental composition

The elemental composition of marine phytoplankton differs intra and interspecifically. It has been hypothesized that elevated pCO₂ could increase carbon assimilation, and thereby alter the elemental composition of marine phytoplankton. In this study, the similar activities of Rubisco (Fig 3D) and growth rate (Fig 1) under 390 and 1000 ppmv CO₂ treatments suggest that photosynthetic carbon fixation did not differ between these conditions, which could explain why the total cellular carbon, nitrogen and phosphorus contents of K. mikimotoi were unaffected by the 1000 ppmv CO₂ conditions. However, the total cellular carbon and phosphorus contents of K. mikimotoi increased under 2000 ppmv CO₂, likely due to either enhancement of photosynthetic carbon fixation and elevated uptake of PO₄^3-, or to increased activities of phosphatase. Furthermore, enhancement of protein and carbohydrate synthesis also likely contributed to the increased cellular C and P. The unchanged cellular nitrogen contents under 2000 ppmv CO₂ were probably determined by the balance between enhanced nitrogen accumulation and nitrogen loss.

Ecological and environmental implications

It has been proposed that the dominance of bloom-forming species might be dependent on their ability to operate a regulated and efficient CCM [48,54]. In the current study, K. mikimotoi was found to have an efficient CCM, and the operation of CCM was down-regulated at high pCO₂ (1000 ppmv and 2000 ppmv) conditions. However, the growth of K. mikimotoi in 1000 ppmv pCO₂ was not stimulated by the reduced energetic costs of the CCM, probably due to additional carbon loss caused by enhanced dark respiration. Gao et al. [71] conducted an experiment to investigate the responses of natural phytoplankton assemblages in the South China Sea grown, over a range of light, to elevated pCO₂. The results showed that growth rates of three diatom species (Thalassiosira pseudonana, Phaeodactylum tricornutum, and Skeletonema costatum) under 1000 ppmv pCO₂ were significantly stimulated at low light levels. The different responses between the dinoflagellate K. mikimotoi and the diatoms T. pseudonana, P. tricornutum and S. costatum suggest that ongoing CO₂-related changes could affect their dominance and succession in the future, possibly not favoring K. mikimotoi in inter-specific competitions.

Global changes not only involve increasing CO₂ levels, but also other environmental factors such as shifts in light availability, nutrient supplies and temperature. Further studies will need to investigate whether species response to elevated pCO₂ might be modulated by other interactive environmental factors. It will be critical to establish the environmental, ecological and economic consequences of K. mikimotoi blooms in a future changing ocean.

Acknowledgments

We are thankful to all of the members of the laboratory and Dr. Liang in the College of Fisheries in Ocean University of China for help. This study was supported by the Natural Science Foundation of China (41476091) and NSFC-Shangdong Joint Fund (U1406403)

References

1. Caldeira K, Wickett ME. Oceanography: anthropogenic carbon and ocean pH. Nature. 2003;425(6956): 365–365. pmid:14508477
- View Article
- PubMed/NCBI
- Google Scholar
2. Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO₂ problem. Mar Sci 1. 2009.
- View Article
- Google Scholar
3. Griggs DJ, Noguer M. Climate change 2001: the scientific basis. Contribution of working group I to the third assessment report of the intergovernmental panel on climate change. Weather. 2002;57(8): 267–269.
- View Article
- Google Scholar
4. Gattuso JP, Allemand D, Frankignoulle M. Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: a review on interactions and control by carbonate chemistry. Am Zool. 1999;39(1): 160–183.
- View Article
- Google Scholar
5. Raven J, Ball L, Beardall J, Giordano M, Maberly SC. Algae lacking carbon-concentrating mechanisms. Can J Botany 2005;83(7): 879–890.
- View Article
- Google Scholar
6. Badger MR, Andrews TJ, Whitney SM, Ludwig M, Yellowlees DC, Leggat W, et al. The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO₂-concentrating mechanisms in algae. Can J Botany.1998;76(6): 1052–1071.
- View Article
- Google Scholar
7. Nimer NA, Iglesias-Rodriguez MD, Merrett MJ. Bicarbonate utilization by marine phytoplankton species. J Phycol.1997; 33(4): 625–631.
- View Article
- Google Scholar
8. Raven JA, Giordano M, Beardall J, Maberly SC. Algal evolution in relation to atmospheric CO₂: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles. Philos T Roy Soc Lon B. 2012; 367(1588): 493–507.
- View Article
- Google Scholar
9. Wu Y, Gao K, Riebesell U. CO₂-induced seawater acidification affects physiological performance of the marine diatom Phaeodactylum tricornutum. Biogeosciences.2010; 7(9): 2915–2923
- View Article
- Google Scholar
10. Yang G, Gao K. Physiological responses of the marine diatom Thalassiosira pseudonana to increased pCO₂ and seawater acidity. Mar Environ Res. 2012; 79: 142–151 pmid:22770534
- View Article
- PubMed/NCBI
- Google Scholar
11. Rost B, Riebesell U, Burkhardt S, Sültemeyer D. Carbon acquisition of bloom-forming marine phytoplankton. Limnol Oceanogr. 2003; 48(1): 55–67.
- View Article
- Google Scholar
12. Kranz SA, Dieter S, Richter KU, Rost B. Carbon acquisition by Trichodesmium: the effect of pCO₂ and diurnal changes. Limnol Oceanogr. 2009;54(2): 548–559.
- View Article
- Google Scholar
13. Kim JM, Lee K, Shin K, Kang J H, Lee HW, Kim M, et al. The effect of seawater CO₂ concentration on growth of a natural phytoplankton assemblage in a controlled mesocosm experiment. Limnol oceanogr.2006;51(4): 1629–1636.
- View Article
- Google Scholar
14. Olischläger M, Bartsch I, Gutow L, Wiencke C. Effects of ocean acidification on different life-cycle stages of the kelp Laminaria hyperborea (Phaeophyceae) Bot Mar. 2012; 55(5): 511–525.
- View Article
- Google Scholar
15. Olischläger M, Bartsch I, Gutow L, Wiencke C. Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool-scenario. Phycol Res. 2013; 61(3): 180–190.
- View Article
- Google Scholar
16. Zou D, Gao K. Effects of elevated CO₂ on the red seaweed Gracilaria lemaneiformis (Gigartinales, Rhodophyta) grown at different irradiance levels. Phycologia. 2009; 48(6):510–517.
- View Article
- Google Scholar
17. Fernández P A, Roleda M Y, Hurd C L. Effects of ocean acidification on the photosynthetic performance, carbonic anhydrase activity and growth of the giant kelp Macrocystis pyrifera. Photosynth Res. 2015; 124(3):293–304. pmid:25869634
- View Article
- PubMed/NCBI
- Google Scholar
18. Kurihara H, Asai T, Kato S, Ishimatsu A. Effects of elevated pCO₂ on early development in the mussel Mytilus galloprovincialis. Aquat. Biol. 2008; 4(3): 225–233.
- View Article
- Google Scholar
19. Van de Waal DB, John U, Ziveri P, Reichart G-J, Hoins M, Sluijs A, et al. Ocean Acidification Reduces Growth and Calcification in a Marine Dinoflagellate. Plos one. 2013; 8 (6): e65987. pmid:23776586
- View Article
- PubMed/NCBI
- Google Scholar
20. Mercado J M, Javier F, Gordillo L, Niell F X, Figueroa F L. Effects of different levels of CO₂ on photosynthesis and cell components of the red alga Porphyra leucosticta. 1999; J Appl Phycol 11(5): 455–461.
- View Article
- Google Scholar
21. Ihnken S, Roberts S, Beardall J. Differential responses of growth and photosynthesis in the marine diatom Chaetoceros muelleri to CO₂ and light availability. Phycologia. 2011; 50(2): 182–193
- View Article
- Google Scholar
22. Gao K, Helbling EW, Häder DP, Hutchins DA. Ocean acidification and marine primary producers under the sun: interactions between CO₂, warming, and solar radiation. Mar. Ecol. Prog. Ser. 2012; 470: 167–189.
- View Article
- Google Scholar
23. Torstensson A, Chierici M, Wulff A. The influence of increased temperature and carbon dioxide levels on the benthic/sea ice diatom Navicula directa. Polar Biol. 2012; 35(2): 205–214
- View Article
- Google Scholar
24. Flynn KJ, Blackford JC, Baird ME, Raven JA, Clark DR. Beardall J, et al. Changes in pH at the exterior surface of plankton with ocean acidification. Nature climate change. 2012;2(7): 510–513
- View Article
- Google Scholar
25. Yang Z B, Hodgkiss I J. Hong Kong’s worst “red tide”—causative factors reflected in a phytoplankton study at Port Shelter station in 1998. Harmful Algae. 2004; 3(2):149–161.
- View Article
- Google Scholar
26. Tangen K. Blooms of Gyrodinium aureolum (Dinophygeae) in North European waters, accompanied by mortality in marine organisms. Sarsia. 1977; 63(2): 123–133.
- View Article
- Google Scholar
27. Vanhoutte-Brunier A, Fernand L, Ménesguen A, Lyons S, Gohin F, Cugier P. Modelling the Karenia mikimotoi bloom that occurred in the western English Channel during summer 2003. Ecol Model. 2008; 210(4): 351–376.
- View Article
- Google Scholar
28. Raine R, McMahon T. Physical dynamics on the continental shelf off southwestern Ireland and their influence on coastal phytoplankton blooms. Cont Shelf Res. 1998; 18(8): 883–914.
- View Article
- Google Scholar
29. Godhe A, Otta S K, Rehnstam-Holm A S, Karunasagar I, Karunasagar I. Polymerase Chain Reaction in Detection of Gymnodinium mikimotoi and Alexandrium minutum in Field Samples from Southwest India. Mar Biotechnol. 2001; 3(2):152–62. pmid:14961378
- View Article
- PubMed/NCBI
- Google Scholar
30. Yao W, Li C, Gao J. Red tide plankton along the south coastal area in Zhejiang province. Marine Science Bulletin. 2006; 25(3):87–91.
- View Article
- Google Scholar
31. Guillard R R L. Culture of phytoplankton for feeding marine invertebrates. In:Culture of marine invertebrate animals. Springer.1975; 29–60.
32. Fu FX, Warner ME, Zhang Y, Feng Y, Hutchins DA. Effects of increased temperature and CO₂ on photosynthesis, growth, and elemental ratios in marine Synechococcus and Prochlorococcus (Cyanobacteria) J Phycol. 2007;43(3): 485–496.
- View Article
- Google Scholar
33. Fu F X, Zhang Y, Warner M E, Feng Y, Sun J, Hutchins DA. A comparison of future increased CO₂ and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum. Harmful Algae. 2008;7(1): 76–90.
- View Article
- Google Scholar
34. Hutchins D A, Fu F X, Zhang Y, Warner M E, Feng Y, Portune K, et al. CO₂ control of Trichodesmium N₂ fixation, photosynthesis, growth rates, and elemental ratios: Implications for past, present, and future ocean biogeochemistry. Limnology and Oceanography.2007;52(4): 1293–1304.
- View Article
- Google Scholar
35. Lewis E, Wallace D, Allison LJ. Program developed for CO₂ system calculations. Tennessee: Carbon Dioxide Information Analysis Center, managed by Lockheed Martin Energy Research Corporation for the US Department of Energy,1998.
36. Zhao Y, Wang Y, Quigg A. The 24 hour recovery kinetics from N starvation in Phaeodactylum tricornutum and Emiliania huxleyi. J phycol. 2015; 51(4): 726–738. pmid:26986793
- View Article
- PubMed/NCBI
- Google Scholar
37. Fourqurean J W, Zieman J C, Powell G V N. Phosphorus limitation of primary production in Florida Bay: evidence from C: N: P ratios of the dominant seagrass Thalassia testudinum. Limnol Oceanogr. 1992; 37(1): 162–171.
- View Article
- Google Scholar
38. Porra R J. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res. 2002; 73: 149–156. pmid:16245116
- View Article
- PubMed/NCBI
- Google Scholar
39. Platt T, Gallegos C L, Harrison W G. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res. 1980; 38: 687–701.
- View Article
- Google Scholar
40. Ralph PJ, Gademann R. Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot. 2005; 82(3): 222–237.
- View Article
- Google Scholar
41. Gerard V A, Driscoll T. A spectrophotometric assay for rubisco activity: application to the kelp laminaria saccharina and implications for radiometric assays1. J phycol. 1996; 32(5): 880–884
- View Article
- Google Scholar
42. Wilbur K M, Anderson N G. Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem. 1948; 176(1): 147–154. pmid:18886152
- View Article
- PubMed/NCBI
- Google Scholar
43. Moroney JV, Husic HD, Tolbert NE. Effect of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii. Plant Physiol. 1985;79(1): 177–183. pmid:16664365
- View Article
- PubMed/NCBI
- Google Scholar
44. Axelsson L, Ryberg H, Beer S. Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca. Plant Cell Environ. 1995;18(4): 439–445.
- View Article
- Google Scholar
45. Raven J A, Johnston A M. Mechanisms of inorganic-carbon acquisition in marine phytoplankton and their implications for the use of other resources. Limnology and Oceanography. 1991; 36(8):1701–1714.
- View Article
- Google Scholar
46. Maberly SC. Exogenous sources of inorganic carbon for photosynthesis by marine macroalgae 1. J Phycol. 1999; 26(3): 439–449.
- View Article
- Google Scholar
47. Johnston A M, Maberly S C, Raven J A. The acquisition of inorganic carbon by four red macroalgae. Oecologia.1992; 92(3): 317–326. pmid:28312597
- View Article
- PubMed/NCBI
- Google Scholar
48. Rost B, Riebesell U, Burkhardt S, Sültemeyer D. Carbon Acquisition of Bloom-Forming Marine Phytoplankton. Limnol and Oceanogr. 2003; 48(1):55–67.
- View Article
- Google Scholar
49. Mercado J M, Niell F X. Carbonic anhydrase activity and use of HCO₃^- in Bostrychia scorpioides (Ceramiales, Rhodophyceae). Eur J Phycol. 1999; 34:13–19
- View Article
- Google Scholar
50. Rost B, Richter K U, Riebesell U, Hansen P J. Inorganic carbon acquisition in red tide dinoflagellates. Plant Cell Environ. 2006; 29(5): 810–822. pmid:17087465
- View Article
- PubMed/NCBI
- Google Scholar
51. Eberlein T, Van de Waal DB, Rost B. Differential effects of ocean acidification on carbon acquisition in two bloom-forming dinoflagellate species. Physiol plantarum. 2014;151(4): 468–479.
- View Article
- Google Scholar
52. Drechsler Z, Sharkia R, Cabantchik Z I, Beer S. Bicarbonate uptake in the marine maxroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells. Planta. 1993; 191:34–40
- View Article
- Google Scholar
53. Larsson C, Axelsson L, Ryberg H, Beer S. Photosynthetic carbon utilization by Enteromorpha intestinalis (Chorophyta) form a Swedish rockpool. Eur J Phycol. 1997; 32:49–54
- View Article
- Google Scholar
54. Trimborn S, Lundholm N, Thoms S, Richter K U, Krock B, Hansen P J, et al. Inorganic carbon acquisition in potentially toxic and non-toxic diatoms: the effect of pH-induced changes in seawater carbonate chemistry. Physiol Plantarum. 2008; 133(1):92–105.
- View Article
- Google Scholar
55. Trimborn S, Wolf-Gladrow D, Richter K U, Rost B. The effect of pCO₂, on carbon acquisition and intracellular assimilation in four marine diatoms. J Exp Mar Biol Ecol. 2009; 376(1):26–36.
- View Article
- Google Scholar
56. LOW-DÉCARIE E, Fussmann GF, Bell G. The effect of elevated CO₂ on growth and competition in experimental phytoplankton communities. Global Change Biology. 2011;17(8): 2525–2535.58.
- View Article
- Google Scholar
57. Gordillo F J L, Niell F X, Figueroa F L. Non-photosyntheticenhancement of growth by high CO₂ level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta). Planta.2001;213:64–70 pmid:11523657
- View Article
- PubMed/NCBI
- Google Scholar
58. Chen X, Gao K. Effect of CO₂ concentrations on the activity of photosynthetic CO₂ fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum. Chinese Sci Bull. 2003;48(23): 2616–2620.
- View Article
- Google Scholar
59. Nielsen L T, Hallegraeff G M, Wright S W, Hansen P J. Effects of experimental seawater acidification on an estuarine plankton community. Aquat Microb Ecol. 2011; 65(3): 271–285.
- View Article
- Google Scholar
60. Gao K, Ruan Z, Villafane V E, Gattuso J P, Helbling E W. Ocean acidification exacerbates the effect of UV radiation on the calcifying phytoplankter Emiliania huxleyi. Limnol Oceanogr. 2009; 54(6): 1855–1862.
- View Article
- Google Scholar
61. Montechiaro F, Giordano M. Compositional homeostasis of the dinoflagellate Protoceratium reticulatum grown at three different pCO₂. J Plant Physiol. 2010; 167(2): 110–113. pmid:19740567
- View Article
- PubMed/NCBI
- Google Scholar
62. Beardall J, Giordano M. Ecological implications of microalgal and cyanobacterial CO₂ concentrating mechanisms, and their regulation. Funct Plant Biol. 2002;29(3): 335–347.
- View Article
- Google Scholar
63. Mercado J M, Javier F, Gordillo L, Niell F X, Figueroa F L. Effects of different levels of CO₂ on photosynthesis and cell components of the red alga Porphyra leucosticta. J Appl Phycol. 1999; 11(5):455–461.
- View Article
- Google Scholar
64. Gordillo F J L, Niell F X, Figueroa F L. Non-photosynthetic enhancement of growth by high CO₂ level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta). Planta. 2001; 213(1):64–70. pmid:11523657
- View Article
- PubMed/NCBI
- Google Scholar
65. Hennon G M M, Quay P, Morales R, Swanson L M, Armbrust E V. Acclimation conditions modify physiological response of the diatom Thalassiosira pseudonana to elevated CO₂ concentrations in a nitrate-limited chemostat. J phycol. 2014; 50(2): 243–253. pmid:26988182
- View Article
- PubMed/NCBI
- Google Scholar
66. Geider R, Osborne B A. Respiration and microalgal growth: a review of the quantitative relationship between dark respiration and growth. New Phytologist. 1989; 12(3): 327–341.
- View Article
- Google Scholar
67. Amthor J S. Respiration in a future, higher CO₂ world. Plant Cell Environ. 1991; 14:13–20
- View Article
- Google Scholar
68. Falkowski P G, Raven J A. Aquatic photosynthesis. Princeton University Press. 2013.
69. Li Q, Canvin DT. Energy Sources for HCO3− and CO₂ Transport in Air-Grown Cells of Synechococcus UTEX 625. Plant physiol. 1998;116(3): 1125–1132. pmid:9501145
- View Article
- PubMed/NCBI
- Google Scholar
70. Tchernov D, Helman Y, Keren N, Luz B, Ohad I, Reinhold L, et al. Passive entry of CO₂ and its energy-dependent intracellular conversion to HCO3− in Cyanobacteria are driven by a photosystem I-generated ΔμH+. Biol Chem. 2001; 276(26): 23450–23455.
- View Article
- Google Scholar
71. Gao K, Xu J, Gao G, et al. Rising CO₂ and increased light exposure synergistically reduce marine primary productivity. Nature Climate Change. 2012; 2(7):519–523.
- View Article
- Google Scholar

[ref1] 1. Caldeira K, Wickett ME. Oceanography: anthropogenic carbon and ocean pH. Nature. 2003;425(6956): 365–365. pmid:14508477
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Doney SC, Fabry VJ, Feely RA, Kleypas JA (2009) Ocean acidification: the other CO₂ problem. Mar Sci 1. 2009.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref3] 3. Griggs DJ, Noguer M. Climate change 2001: the scientific basis. Contribution of working group I to the third assessment report of the intergovernmental panel on climate change. Weather. 2002;57(8): 267–269.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref4] 4. Gattuso JP, Allemand D, Frankignoulle M. Photosynthesis and calcification at cellular, organismal and community levels in coral reefs: a review on interactions and control by carbonate chemistry. Am Zool. 1999;39(1): 160–183.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref5] 5. Raven J, Ball L, Beardall J, Giordano M, Maberly SC. Algae lacking carbon-concentrating mechanisms. Can J Botany 2005;83(7): 879–890.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref6] 6. Badger MR, Andrews TJ, Whitney SM, Ludwig M, Yellowlees DC, Leggat W, et al. The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO₂-concentrating mechanisms in algae. Can J Botany.1998;76(6): 1052–1071.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref7] 7. Nimer NA, Iglesias-Rodriguez MD, Merrett MJ. Bicarbonate utilization by marine phytoplankton species. J Phycol.1997; 33(4): 625–631.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref8] 8. Raven JA, Giordano M, Beardall J, Maberly SC. Algal evolution in relation to atmospheric CO₂: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles. Philos T Roy Soc Lon B. 2012; 367(1588): 493–507.
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref9] 9. Wu Y, Gao K, Riebesell U. CO₂-induced seawater acidification affects physiological performance of the marine diatom Phaeodactylum tricornutum. Biogeosciences.2010; 7(9): 2915–2923
View Article
Google Scholar

[27] View Article

[28] Google Scholar

[ref10] 10. Yang G, Gao K. Physiological responses of the marine diatom Thalassiosira pseudonana to increased pCO₂ and seawater acidity. Mar Environ Res. 2012; 79: 142–151 pmid:22770534
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref11] 11. Rost B, Riebesell U, Burkhardt S, Sültemeyer D. Carbon acquisition of bloom-forming marine phytoplankton. Limnol Oceanogr. 2003; 48(1): 55–67.
View Article
Google Scholar

[34] View Article

[35] Google Scholar

[ref12] 12. Kranz SA, Dieter S, Richter KU, Rost B. Carbon acquisition by Trichodesmium: the effect of pCO₂ and diurnal changes. Limnol Oceanogr. 2009;54(2): 548–559.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref13] 13. Kim JM, Lee K, Shin K, Kang J H, Lee HW, Kim M, et al. The effect of seawater CO₂ concentration on growth of a natural phytoplankton assemblage in a controlled mesocosm experiment. Limnol oceanogr.2006;51(4): 1629–1636.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref14] 14. Olischläger M, Bartsch I, Gutow L, Wiencke C. Effects of ocean acidification on different life-cycle stages of the kelp Laminaria hyperborea (Phaeophyceae) Bot Mar. 2012; 55(5): 511–525.
View Article
Google Scholar

[43] View Article

[44] Google Scholar

[ref15] 15. Olischläger M, Bartsch I, Gutow L, Wiencke C. Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool-scenario. Phycol Res. 2013; 61(3): 180–190.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref16] 16. Zou D, Gao K. Effects of elevated CO₂ on the red seaweed Gracilaria lemaneiformis (Gigartinales, Rhodophyta) grown at different irradiance levels. Phycologia. 2009; 48(6):510–517.
View Article
Google Scholar

[49] View Article

[50] Google Scholar

[ref17] 17. Fernández P A, Roleda M Y, Hurd C L. Effects of ocean acidification on the photosynthetic performance, carbonic anhydrase activity and growth of the giant kelp Macrocystis pyrifera. Photosynth Res. 2015; 124(3):293–304. pmid:25869634
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref18] 18. Kurihara H, Asai T, Kato S, Ishimatsu A. Effects of elevated pCO₂ on early development in the mussel Mytilus galloprovincialis. Aquat. Biol. 2008; 4(3): 225–233.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref19] 19. Van de Waal DB, John U, Ziveri P, Reichart G-J, Hoins M, Sluijs A, et al. Ocean Acidification Reduces Growth and Calcification in a Marine Dinoflagellate. Plos one. 2013; 8 (6): e65987. pmid:23776586
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref20] 20. Mercado J M, Javier F, Gordillo L, Niell F X, Figueroa F L. Effects of different levels of CO₂ on photosynthesis and cell components of the red alga Porphyra leucosticta. 1999; J Appl Phycol 11(5): 455–461.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref21] 21. Ihnken S, Roberts S, Beardall J. Differential responses of growth and photosynthesis in the marine diatom Chaetoceros muelleri to CO₂ and light availability. Phycologia. 2011; 50(2): 182–193
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref22] 22. Gao K, Helbling EW, Häder DP, Hutchins DA. Ocean acidification and marine primary producers under the sun: interactions between CO₂, warming, and solar radiation. Mar. Ecol. Prog. Ser. 2012; 470: 167–189.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref23] 23. Torstensson A, Chierici M, Wulff A. The influence of increased temperature and carbon dioxide levels on the benthic/sea ice diatom Navicula directa. Polar Biol. 2012; 35(2): 205–214
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref24] 24. Flynn KJ, Blackford JC, Baird ME, Raven JA, Clark DR. Beardall J, et al. Changes in pH at the exterior surface of plankton with ocean acidification. Nature climate change. 2012;2(7): 510–513
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref25] 25. Yang Z B, Hodgkiss I J. Hong Kong’s worst “red tide”—causative factors reflected in a phytoplankton study at Port Shelter station in 1998. Harmful Algae. 2004; 3(2):149–161.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref26] 26. Tangen K. Blooms of Gyrodinium aureolum (Dinophygeae) in North European waters, accompanied by mortality in marine organisms. Sarsia. 1977; 63(2): 123–133.
View Article
Google Scholar

[81] View Article

[82] Google Scholar

[ref27] 27. Vanhoutte-Brunier A, Fernand L, Ménesguen A, Lyons S, Gohin F, Cugier P. Modelling the Karenia mikimotoi bloom that occurred in the western English Channel during summer 2003. Ecol Model. 2008; 210(4): 351–376.
View Article
Google Scholar

[84] View Article

[85] Google Scholar

[ref28] 28. Raine R, McMahon T. Physical dynamics on the continental shelf off southwestern Ireland and their influence on coastal phytoplankton blooms. Cont Shelf Res. 1998; 18(8): 883–914.
View Article
Google Scholar

[87] View Article

[88] Google Scholar

[ref29] 29. Godhe A, Otta S K, Rehnstam-Holm A S, Karunasagar I, Karunasagar I. Polymerase Chain Reaction in Detection of Gymnodinium mikimotoi and Alexandrium minutum in Field Samples from Southwest India. Mar Biotechnol. 2001; 3(2):152–62. pmid:14961378
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref30] 30. Yao W, Li C, Gao J. Red tide plankton along the south coastal area in Zhejiang province. Marine Science Bulletin. 2006; 25(3):87–91.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref31] 31. Guillard R R L. Culture of phytoplankton for feeding marine invertebrates. In:Culture of marine invertebrate animals. Springer.1975; 29–60.

[ref32] 32. Fu FX, Warner ME, Zhang Y, Feng Y, Hutchins DA. Effects of increased temperature and CO₂ on photosynthesis, growth, and elemental ratios in marine Synechococcus and Prochlorococcus (Cyanobacteria) J Phycol. 2007;43(3): 485–496.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref33] 33. Fu F X, Zhang Y, Warner M E, Feng Y, Sun J, Hutchins DA. A comparison of future increased CO₂ and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum. Harmful Algae. 2008;7(1): 76–90.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref34] 34. Hutchins D A, Fu F X, Zhang Y, Warner M E, Feng Y, Portune K, et al. CO₂ control of Trichodesmium N₂ fixation, photosynthesis, growth rates, and elemental ratios: Implications for past, present, and future ocean biogeochemistry. Limnology and Oceanography.2007;52(4): 1293–1304.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref35] 35. Lewis E, Wallace D, Allison LJ. Program developed for CO₂ system calculations. Tennessee: Carbon Dioxide Information Analysis Center, managed by Lockheed Martin Energy Research Corporation for the US Department of Energy,1998.

[ref36] 36. Zhao Y, Wang Y, Quigg A. The 24 hour recovery kinetics from N starvation in Phaeodactylum tricornutum and Emiliania huxleyi. J phycol. 2015; 51(4): 726–738. pmid:26986793
View Article
PubMed/NCBI
Google Scholar

[108] View Article

[109] PubMed/NCBI

[110] Google Scholar

[ref37] 37. Fourqurean J W, Zieman J C, Powell G V N. Phosphorus limitation of primary production in Florida Bay: evidence from C: N: P ratios of the dominant seagrass Thalassia testudinum. Limnol Oceanogr. 1992; 37(1): 162–171.
View Article
Google Scholar

[112] View Article

[113] Google Scholar

[ref38] 38. Porra R J. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res. 2002; 73: 149–156. pmid:16245116
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref39] 39. Platt T, Gallegos C L, Harrison W G. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res. 1980; 38: 687–701.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref40] 40. Ralph PJ, Gademann R. Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot. 2005; 82(3): 222–237.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref41] 41. Gerard V A, Driscoll T. A spectrophotometric assay for rubisco activity: application to the kelp laminaria saccharina and implications for radiometric assays1. J phycol. 1996; 32(5): 880–884
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref42] 42. Wilbur K M, Anderson N G. Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem. 1948; 176(1): 147–154. pmid:18886152
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref43] 43. Moroney JV, Husic HD, Tolbert NE. Effect of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii. Plant Physiol. 1985;79(1): 177–183. pmid:16664365
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref44] 44. Axelsson L, Ryberg H, Beer S. Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca. Plant Cell Environ. 1995;18(4): 439–445.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref45] 45. Raven J A, Johnston A M. Mechanisms of inorganic-carbon acquisition in marine phytoplankton and their implications for the use of other resources. Limnology and Oceanography. 1991; 36(8):1701–1714.
View Article
Google Scholar

[139] View Article

[140] Google Scholar

[ref46] 46. Maberly SC. Exogenous sources of inorganic carbon for photosynthesis by marine macroalgae 1. J Phycol. 1999; 26(3): 439–449.
View Article
Google Scholar

[142] View Article

[143] Google Scholar

[ref47] 47. Johnston A M, Maberly S C, Raven J A. The acquisition of inorganic carbon by four red macroalgae. Oecologia.1992; 92(3): 317–326. pmid:28312597
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref48] 48. Rost B, Riebesell U, Burkhardt S, Sültemeyer D. Carbon Acquisition of Bloom-Forming Marine Phytoplankton. Limnol and Oceanogr. 2003; 48(1):55–67.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref49] 49. Mercado J M, Niell F X. Carbonic anhydrase activity and use of HCO₃^- in Bostrychia scorpioides (Ceramiales, Rhodophyceae). Eur J Phycol. 1999; 34:13–19
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref50] 50. Rost B, Richter K U, Riebesell U, Hansen P J. Inorganic carbon acquisition in red tide dinoflagellates. Plant Cell Environ. 2006; 29(5): 810–822. pmid:17087465
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref51] 51. Eberlein T, Van de Waal DB, Rost B. Differential effects of ocean acidification on carbon acquisition in two bloom-forming dinoflagellate species. Physiol plantarum. 2014;151(4): 468–479.
View Article
Google Scholar

[159] View Article

[160] Google Scholar

[ref52] 52. Drechsler Z, Sharkia R, Cabantchik Z I, Beer S. Bicarbonate uptake in the marine maxroalga Ulva sp. is inhibited by classical probes of anion exchange by red blood cells. Planta. 1993; 191:34–40
View Article
Google Scholar

[162] View Article

[163] Google Scholar

[ref53] 53. Larsson C, Axelsson L, Ryberg H, Beer S. Photosynthetic carbon utilization by Enteromorpha intestinalis (Chorophyta) form a Swedish rockpool. Eur J Phycol. 1997; 32:49–54
View Article
Google Scholar

[165] View Article

[166] Google Scholar

[ref54] 54. Trimborn S, Lundholm N, Thoms S, Richter K U, Krock B, Hansen P J, et al. Inorganic carbon acquisition in potentially toxic and non-toxic diatoms: the effect of pH-induced changes in seawater carbonate chemistry. Physiol Plantarum. 2008; 133(1):92–105.
View Article
Google Scholar

[168] View Article

[169] Google Scholar

[ref55] 55. Trimborn S, Wolf-Gladrow D, Richter K U, Rost B. The effect of pCO₂, on carbon acquisition and intracellular assimilation in four marine diatoms. J Exp Mar Biol Ecol. 2009; 376(1):26–36.
View Article
Google Scholar

[171] View Article

[172] Google Scholar

[ref56] 56. LOW-DÉCARIE E, Fussmann GF, Bell G. The effect of elevated CO₂ on growth and competition in experimental phytoplankton communities. Global Change Biology. 2011;17(8): 2525–2535.58.
View Article
Google Scholar

[174] View Article

[175] Google Scholar

[ref57] 57. Gordillo F J L, Niell F X, Figueroa F L. Non-photosyntheticenhancement of growth by high CO₂ level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta). Planta.2001;213:64–70 pmid:11523657
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

[ref58] 58. Chen X, Gao K. Effect of CO₂ concentrations on the activity of photosynthetic CO₂ fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum. Chinese Sci Bull. 2003;48(23): 2616–2620.
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref59] 59. Nielsen L T, Hallegraeff G M, Wright S W, Hansen P J. Effects of experimental seawater acidification on an estuarine plankton community. Aquat Microb Ecol. 2011; 65(3): 271–285.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref60] 60. Gao K, Ruan Z, Villafane V E, Gattuso J P, Helbling E W. Ocean acidification exacerbates the effect of UV radiation on the calcifying phytoplankter Emiliania huxleyi. Limnol Oceanogr. 2009; 54(6): 1855–1862.
View Article
Google Scholar

[187] View Article

[188] Google Scholar

[ref61] 61. Montechiaro F, Giordano M. Compositional homeostasis of the dinoflagellate Protoceratium reticulatum grown at three different pCO₂. J Plant Physiol. 2010; 167(2): 110–113. pmid:19740567
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref62] 62. Beardall J, Giordano M. Ecological implications of microalgal and cyanobacterial CO₂ concentrating mechanisms, and their regulation. Funct Plant Biol. 2002;29(3): 335–347.
View Article
Google Scholar

[194] View Article

[195] Google Scholar

[ref63] 63. Mercado J M, Javier F, Gordillo L, Niell F X, Figueroa F L. Effects of different levels of CO₂ on photosynthesis and cell components of the red alga Porphyra leucosticta. J Appl Phycol. 1999; 11(5):455–461.
View Article
Google Scholar

[197] View Article

[198] Google Scholar

[ref64] 64. Gordillo F J L, Niell F X, Figueroa F L. Non-photosynthetic enhancement of growth by high CO₂ level in the nitrophilic seaweed Ulva rigida C. Agardh (Chlorophyta). Planta. 2001; 213(1):64–70. pmid:11523657
View Article
PubMed/NCBI
Google Scholar

[200] View Article

[201] PubMed/NCBI

[202] Google Scholar

[ref65] 65. Hennon G M M, Quay P, Morales R, Swanson L M, Armbrust E V. Acclimation conditions modify physiological response of the diatom Thalassiosira pseudonana to elevated CO₂ concentrations in a nitrate-limited chemostat. J phycol. 2014; 50(2): 243–253. pmid:26988182
View Article
PubMed/NCBI
Google Scholar

[204] View Article

[205] PubMed/NCBI

[206] Google Scholar

[ref66] 66. Geider R, Osborne B A. Respiration and microalgal growth: a review of the quantitative relationship between dark respiration and growth. New Phytologist. 1989; 12(3): 327–341.
View Article
Google Scholar

[208] View Article

[209] Google Scholar

[ref67] 67. Amthor J S. Respiration in a future, higher CO₂ world. Plant Cell Environ. 1991; 14:13–20
View Article
Google Scholar

[211] View Article

[212] Google Scholar

[ref68] 68. Falkowski P G, Raven J A. Aquatic photosynthesis. Princeton University Press. 2013.

[ref69] 69. Li Q, Canvin DT. Energy Sources for HCO3− and CO₂ Transport in Air-Grown Cells of Synechococcus UTEX 625. Plant physiol. 1998;116(3): 1125–1132. pmid:9501145
View Article
PubMed/NCBI
Google Scholar

[215] View Article

[216] PubMed/NCBI

[217] Google Scholar

[ref70] 70. Tchernov D, Helman Y, Keren N, Luz B, Ohad I, Reinhold L, et al. Passive entry of CO₂ and its energy-dependent intracellular conversion to HCO3− in Cyanobacteria are driven by a photosystem I-generated ΔμH+. Biol Chem. 2001; 276(26): 23450–23455.
View Article
Google Scholar

[219] View Article

[220] Google Scholar

[ref71] 71. Gao K, Xu J, Gao G, et al. Rising CO₂ and increased light exposure synergistically reduce marine primary productivity. Nature Climate Change. 2012; 2(7):519–523.
View Article
Google Scholar

[222] View Article

[223] Google Scholar

Effect of CO₂-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi

Effect of CO₂-induced seawater acidification on growth, photosynthesis and inorganic carbon acquisition of the harmful bloom-forming marine microalga, Karenia mikimotoi

Figures

Abstract

Introduction

Materials and methods

Culture conditions and experimental design

Seawater carbonate chemistry

Growth and elemental analysis

Chlorophyll a

Photosynthetic oxygen evolution and respiration

Chlorophyll fluorescence measurements

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

pH drift experiment

Determination of carbonic anhydrase activity

Effect of inhibitors on chlorophyll fluorescence

Statistical analysis

Results

Seawater carbonate chemistry

Growth and elemental composition

Chlorophyll a

Photosynthetic oxygen evolution, dark respiration and Rubisco activities

Chlorophyll fluorescence

pH drift experiment and carbonic anhydrase activity

Effect of inhibitors on chlorophyll fluorescence

Discussion

Inorganic carbon acquisition

Growth, photosynthesis, respiration and photosynthetic electron transport

Elemental composition

Ecological and environmental implications

Acknowledgments

References