Strong shift from HCO₃⁻ to CO₂ uptake in Emiliania huxleyi with acidification: new approach unravels acclimation versus short-term pH effects

Abstract

Effects of ocean acidification on Emiliania huxleyi strain RCC 1216 (calcifying, diploid life-cycle stage) and RCC 1217 (non-calcifying, haploid life-cycle stage) were investigated by measuring growth, elemental composition, and production rates under different pCO₂ levels (380 and 950 μatm). In these differently acclimated cells, the photosynthetic carbon source was assessed by a ¹⁴C disequilibrium assay, conducted over a range of ecologically relevant pH values (7.9–8.7). In agreement with previous studies, we observed decreased calcification and stimulated biomass production in diploid cells under high pCO₂, but no CO₂-dependent changes in biomass production for haploid cells. In both life-cycle stages, the relative contributions of CO₂ and HCO₃ ⁻ uptake depended strongly on the assay pH. At pH values ≤ 8.1, cells preferentially used CO₂ (≥ 90 % CO₂), whereas at pH values ≥ 8.3, cells progressively increased the fraction of HCO₃ ⁻ uptake (~45 % CO₂ at pH 8.7 in diploid cells; ~55 % CO₂ at pH 8.5 in haploid cells). In contrast to the short-term effect of the assay pH, the pCO₂ acclimation history had no significant effect on the carbon uptake behavior. A numerical sensitivity study confirmed that the pH-modification in the ¹⁴C disequilibrium method yields reliable results, provided that model parameters (e.g., pH, temperature) are kept within typical measurement uncertainties. Our results demonstrate a high plasticity of E. huxleyi to rapidly adjust carbon acquisition to the external carbon supply and/or pH, and provide an explanation for the paradoxical observation of high CO₂ sensitivity despite the apparently high HCO₃ ⁻ usage seen in previous studies.

Electronic supplementary material

The online version of this article (doi:10.1007/s11120-014-9984-9) contains supplementary material, which is available to authorized users.

Introduction

Marine phytoplankton account for ~50 % of global primary production and are the main drivers of the marine "particulate organic carbon" (POC) pump (Falkowski et al. 1998; Field et al. 1998). Calcifying phytoplankton species also contribute to the "particulate inorganic carbon" (PIC) pump and thereby play a dual role in regulating marine biogeochemical cycling of carbon through their effects on surface ocean alkalinity (Broecker and Peng 1982; Zeebe and Wolf-Gladrow 2007). One key species of calcifying phytoplankton is the cosmopolitan and bloom-forming coccolithophore Emiliania huxleyi, which has been established as a model organism over the recent decades (Paasche 2002; Raven and Crawfurd 2012; Read et al. 2013; Westbroek et al. 1993). While the calcifying diploid life-cycle stage of this species has been intensively studied in field and laboratory experiments, the non-calcifying haploid stage has only recently gained attention due to its important ecological role. In blooms of diploid E. huxleyi, which are usually terminated by viruses, the haploid life-cycle stage functions as a virus-resistant backup population (Frada et al. 2012). Furthermore, the presence and absence of calcification in the differing life-cycle stages of E. huxleyi make them ideal candidates to investigate the cellular mechanisms of calcification and their interaction with photosynthesis under increasing oceanic CO₂ concentrations (Mackinder et al. 2010; Rokitta and Rost 2012).

Increasing pCO₂ in oceanic surface water directly affects carbonate chemistry by elevating the concentration of dissolved inorganic carbon (DIC) and shifting the carbon speciation toward higher CO₂ and H⁺ concentrations, a phenomenon often referred to as ocean acidification (OA; Caldeira and Wickett 2003; Wolf-Gladrow et al. 1999). Compared to preindustrial values, pH is expected to drop by 0.4–0.5 units until the end of this century. In several studies testing the effects of OA on E. huxleyi, diploid strains were found to exhibit strong, yet opposing responses in terms of biomass and calcite production. While biomass production was either unaffected or stimulated by increased pCO₂, calcification typically decreased and malformations of coccoliths increased (e.g., Hoppe et al. 2011; Langer et al. 2009; Riebesell et al. 2000). Bach et al. (2011) suggested that biomass production is stimulated by increasing CO₂ concentration at sub-saturating conditions, whereas calcification is specifically responsive to the associated decrease in pH. Such differential CO₂ and pH effects on biomass and calcite production are supported by the observation that OA distorts ion homeostasis and shifts the metabolism from oxidative to reductive pathways (Rokitta et al. 2012; Taylor et al. 2011). In a number of studies, the sensitivity of E. huxleyi toward OA has been attributed to its mode of inorganic carbon (C_i) acquisition, which is intrinsically responsive to changes in carbonate chemistry. Thus, for understanding the differential responses to OA, one needs to look at this crucial process of C_i assimilation.

Like most phytoplankton, E. huxleyi operates a CO₂ concentrating mechanism (CCM), which utilizes CO₂ and/or HCO₃ ⁻ uptake systems to accumulate CO₂ in the vicinity of RubisCO, and employs the enzyme carbonic anhydrase (CA) to accelerate the inter-conversion between these C_i species (see Reinfelder 2011 for review). For a long time, the CCM in E. huxleyi was assumed to rely on the CO₂ delivery by calcification (Anning et al. 1996; Sikes et al. 1980). More recently, however, studies have demonstrated that C_i fluxes for photosynthesis and calcification are independent (Herfort et al. 2004; Rost et al. 2002; Trimborn et al. 2007), and that these two processes may even compete for C_i substrates (Rokitta and Rost 2012). Most studies performed on the CCM of E. huxleyi to date yielded moderately high substrate affinities for C_i, which decreased slightly under OA scenarios (e.g., Rokitta and Rost 2012; Rost et al. 2003, Stojkovic et al. 2013). Moreover, low activity for extracellular CA and high contribution of HCO₃ ⁻ uptake for photosynthesis have been reported (e.g., Herfort et al. 2002; Rokitta and Rost 2012; Stojkovic et al. 2013; Trimborn et al. 2007). This high apparent HCO₃ ⁻ usage is puzzling, however, as it suggests biomass production to be rather insensitive to OA-related changes in CO₂ supply, which is in contrast to what studies usually have observed.

Most physiological methods characterizing the CCM and its functional elements are performed under standardized assay conditions, including a fixed pH value, and thus differing from treatment conditions. The pH and the concominant C_i speciation can, however, influence the cell’s physiology, in particular its C_i acquisition. When identifying the cause-effect relationship in OA responses, it is difficult to separate the effects of changes in C_i speciation from concomitant changes in H⁺ concentrations. Changes in external pH have been shown to directly drive changes in cytosolic pH in E. huxleyi, which, in turn, affected H⁺ gradients and membrane potentials (Suffrian et al. 2011; Taylor et al. 2011). This effect could indirectly impact secondary active transporters, e.g., the Cl⁻/HCO₃ ⁻ antiporter (Herfort et al. 2002; Rokitta et al. 2011). Moreover, the protonation of amino acid side chains can affect activity, specificity, and kinetics of enzymes and transporters involved in cellular processes (Badger 2003; Raven 2006). Hence, aside from altered concentrations of C_i species, pH itself could directly impact the mode of CCM (Raven 1990). These possible effects of the assay pH on C_i acquisition should be accounted for when performing experiments to characterize the CCM.

One common approach to determine the C_i source for photosynthesis is the application of the ¹⁴C disequilibrium method (Espie and Colman 1986), which has proven suitable for the study of marine phytoplankton in laboratory cultures (e.g., Elzenga et al. 2000; Rost et al. 2006a) and in natural field assemblages (e.g., Cassar et al. 2004; Martin and Tortell 2006; Tortell and Morel 2002; Tortell et al. 2008). The method makes use of the relatively slow chemical conversion between the CO₂ and HCO₃ ⁻ in the absence of CA (Johnson 1982), allowing for a differential labeling of these C_i species with ¹⁴C. This method is typically performed at pH of 8.5 ("assay pH"), deviating strongly from most natural in situ values and even more from the pH values applied in OA-experiments ("acclimation pH"). In this study, we aimed to disentangle the short-term effect of assay pH from the long-term effect of acclimation history on the photosynthetic C_i source of E. huxleyi. To this end, we grew haploid and diploid life-cycle stages at present-day (380 μatm) and elevated pCO₂ (950 μatm), and measured the responses in growth, elemental composition, and production rates. These low and high pCO₂-acclimated cells were then tested for their preferred C_i source by applying the ¹⁴C disequilibrium method, with assay conditions set to a range of ecologically relevant pH values (pH 7.9–8.7). The reliability of this new approach was tested by performing sensitivity studies.

Methods

pCO₂ acclimations

Haploid and diploid cells of E. huxleyi (strains RCC 1217 and RCC 1216, obtained from the Roscoff culture collection) were grown at 15 °C as dilute batch incubations. North Sea seawater medium (salinity 32.4) was sterile-filtered (0.2 μm) and enriched with vitamins and trace metals according to F/2 (Guillard and Ryther 1962), as well as phosphate and nitrate (100 and 6.25 μmol L⁻¹). Cells were exposed to a light:dark cycle (16:8 h) and saturating light (300 μmol photons m⁻² s⁻¹) provided by daylight lamps (FQ 54W/965HO, OSRAM, Munich, Germany). Light intensity was monitored with the LI-6252 datalogger (LI-COR, Lincoln, NE, USA) using a 4π-sensor (US-SQS/L, Walz, Effeltrich, Germany). Culturing was carried out in sterilized 2.4 L borosilicate bottles (Duran Group, Mainz, Germany) on roller tables to avoid sedimentation.

Prior to experiments, cells were acclimated to the respective pCO₂ and light conditions for at least 7 days (i.e., more than 10 generations). Prior to initiating cultures, medium was pre-aerated for at least 36 h with humidified, 0.2 μm-filtered air comprising pCO₂ values of 380 or 950 μatm (equivalent to 38.5 and 96.3 Pa, or ~15 and ~35 μmol kg⁻¹, respectively). Gas mixtures were created by a gas flow controller (CGM 2000 MCZ Umwelttechnik, Bad Nauheim, Germany) using pure CO₂ (Air Liquide Deutschland, Düsseldorf, Germany) and CO₂-free air (CO2RP280, Dominick Hunter, Willich, Germany). Sampling and measurements were done 4–8 h after the beginning of the light period (i.e., at midday) in exponential growth at densities of 40,000–60,000 cells mL⁻¹. Cultures showing a pH drift of > 0.05 were excluded from further analyses.

The carbonate system (Table 1) during the acclimations was assessed based on measurements of pH and total alkalinity (TA). The pH_NBS of the cultures was measured potentiometrically and corrected for temperature (pH-meter 3110; WTW, Weilheim, Germany). The electrode (A157, Schott Instruments, Mainz, Germany) was three-point calibrated with NBS certified standard buffers and the measurement uncertainty was 0.03 pH units. TA was determined by potentiometric titration (Dickson 1981; TitroLine alpha plus, Schott Instruments). Measurements were accuracy-corrected with certified reference materials (CRMs) supplied by A. Dickson (Scripps Institution of Oceanography, USA). Calculation of the carbonate system was performed using CO2sys (Pierrot et al. 2006). Input parameters were pH_NBS and TA, as well as temperature (15 °C), salinity (32.4), and pressure (1 dbar, according to 1 m depth; Hoppe et al. 2012). For all calculations, phosphate and silicate concentrations were assumed to be 7 and 17 μmol kg⁻¹, respectively, based on assessments of the media. Equilibrium constants for carbonic acid, K₁ and K₂ given by Mehrbach et al. (1973) and refit by Dickson and Millero (1987) were used. For the dissociation of sulfuric acid, the constants reported by Dickson (1990) were employed.

Table 1.

Carbonate chemistry of the pCO₂ acclimations at the time of harvesting and in cell-free media (reference); Attained pCO₂, DIC, HCO₃ ⁻, CO₃ ²⁻, and Ω_calcite are calculated based on measured pH_NBS and TA using CO2sys (Pierrot et al. 2006)

Strain, ploidy	Treatment pCO2 (μatm)	Attained pCO2 (μatm)	pHNBS	TA (μmol kg−1)	DIC (μmol kg−1)	CO2 (μmol kg−1)	HCO3 − (μmol kg−1)	CO3 2− (μmol kg−1)	Ωcalcite
RCC 1216, 2N	Low, 380	353 ± 8	8.19 ± 0.02	2,259 ± 19	2,023 ± 15	13 ± 0	1,857 ± 13	161 ± 3	3.9 ± 0.1
	High, 950	847 ± 55	7.86 ± 0.04	2,278 ± 20	2,156 ± 2	32 ± 2	2,060 ± 28	84 ± 4	2.0 ± 0.1
RCC 1217, 1N	Low, 380	345 ± 4	8.23 ± 0.00	2,317 ± 12	2,068 ± 10	13 ± 0	1,885 ± 10	170 ± 1	4.1 ± 0.0
	High, 950	837 ± 25	7.89 ± 0.01	2,317 ± 3	2,210 ± 5	32 ± 1	2,092 ± 5	86 ± 3	2.1 ± 0.1
Cell-free medium	Low, 380	405 ± 3	8.17 ± 0.00	2,304 ± 5	2,092 ± 5	15 ± 0	1,926 ± 5	151 ± 1	3.7 ± 0.0
	High, 950	997 ± 17	7.82 ± 0.01	2,305 ± 7	2,214 ± 12	38 ± 1	2,128 ± 11	75 ± 1	1.8 ± 0.0

Results are reported for 15 °C (n ≥ 3; ± SD)

Cell growth was assessed by daily cell counting with a Multisizer III hemocytometer (Beckman-Coulter, Fullerton, CA, USA) and the specific growth rates (μ) were calculated from daily increments (cf., Rokitta and Rost 2012). For the determination of total particulate carbon (TPC), POC and particulate organic nitrogen (PON), cell suspensions were vacuum-filtered (-200 mbar relative to atmosphere) onto pre-combusted (12 h, 500 °C) GF/F filters (1.2 μm; Whatman, Maidstone, UK), which were dried at 65 °C and analyzed with a EuroVector CHNS-O elemental analyzer (EuroEA, Milano, Italy). Before quantification of POC, filters were HCl-soaked (200 μL, 0.2 M) and dried to remove calcite. PIC was assessed as the difference between TPC and POC. By multiplying the POC and PIC cell quotas with μ, the respective production rates were derived (cf., Rokitta and Rost 2012). For Chl a measurements, cells were filtered onto cellulose nitrate filters (0.45 μm; Sartorius, Göttingen, Germany) and instantly frozen in liquid nitrogen. Chl a was extracted in 90 % acetone (v/v, Sigma, Munich, Germany) and determined fluorometrically (TD-700 fluorometer, Turner Designs, Sunnyvale, USA) following the protocol by Holm-Hansen and Riemann (1978). The calibration of the fluorometer was carried out with a commercially available Chl a standard (Anacystis nidulans, Sigma, Steinheim, Germany).

¹⁴C disequilibrium method

The C_i source for photosynthesis was determined by applying the ¹⁴C disequilibrium method (Elzenga et al. 2000; Espie and Colman 1986; Tortell and Morel 2002). In this method, a transient isotopic disequilibrium is induced by adding a small volume of a ¹⁴C_i "spike" solution with a relatively low pH (typically 7.0) into larger volume of buffered cell suspension with a relatively high pH (typically 8.5). The cell suspension contains dextran-bound sulfonamide (DBS) to eliminate possible external CA activity. Due to the pH-dependent speciation of DIC, the relative CO₂ concentration of the spike is high (~19 % of DIC at pH 7.0), compared to the cell suspension (~0.3 % of DIC at pH 8.5). When adding the spike to the cell suspension, the majority of the CO₂ added with the spike converts into HCO₃ ⁻ until equilibrium is achieved (Johnson 1982; Millero and Roy 1997). Consequently, the specific activity of CO₂ (${\text{SA}}_{{\text{CO}}_2}$, dpm (mol CO₂)⁻¹) is initially high and exponentially decays over time (Fig. 1). The slope of the ¹⁴C incorporation curve of a "CO₂ user" is, therefore, initially much steeper than during final linear ¹⁴C uptake, when isotopic equilibrium is achieved. In contrast, the slope of ¹⁴C incorporation for "HCO₃ ⁻ users" changes only marginally over time because ${\text{SA}}_{{\text{HCO}}_3^{-}}$ stays more or less constant during the assay.

Fig. 1.

Time-course of specific activities of CO₂ and HCO₃ ⁻ (medium and long dashed lines, respectively, here calculated for assay pH 8.5) in the isotopic disequilibrium method and examples for the ¹⁴C incorporation of the diploid life-cycle stage for predominant CO₂ usage ($f_{{\text{CO}}_2}=1.00$, squares) and considerable HCO₃ ⁻ usage ($f_{{\text{CO}}_2}=0.60$, triangles)

Quantification of the relative proportion of CO₂ or HCO₃ ⁻ usage was done by fitting data with the integral function of the ¹⁴C fixation rate (Elzenga et al. 2000; Espie and Colman 1986; Martin and Tortell 2006). The function includes terms representing the instantaneous fixation rate of DI¹⁴C, the fractional contribution of CO₂ $\left(f_{{\text{CO}}_2}\right)$ or HCO₃ ⁻ usage $\left(1-f_{{\text{CO}}_2}\right)$ to the overall C_i fixation and the specific activity (SA, dpm mol⁻¹) of these substrates at any given time (Eq. 1; Espie and Colman 1986; Elzenga et al. 2000; Tortell and Morel 2002). Strictly speaking, as HCO₃ ⁻ and CO₃ ²⁻ cannot be differentially labeled, $1-f_{{\text{CO}}_2}$ also comprises the potential fraction of CO₃ ²⁻ used.

$$\begin{array}{cc}\hfill \text{dpm}=& V_{\text{DI14C}}\left(f_{{\text{CO}}_2}\right)\left({\mathit{\alpha }}_{1}t+\left(\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}/{\text{SA}}_{\text{DIC}}\right)\left(1-e^{-{\mathit{\alpha }}_{1}t}\right)\right)/\phantom{\left({\mathit{\alpha }}_{1}t+\left(\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}/{\text{SA}}_{\text{DIC}}\right)\left(1-e^{-{\mathit{\alpha }}_{1}t}\right)\right){\mathit{\alpha }}_1}{\mathit{\alpha }}_1+V_{\text{DI14C}}\left(1-f_{{\text{CO}}_2}\right)\left({\mathit{\alpha }}_{2}t+\left(\mathrm{\Delta }{\text{SA}}_{{\text{HCO}}_3}/{\text{SA}}_{\text{DIC}}\right)\left(1-e^{-{\mathit{\alpha }}_{2}t}\right)\right)/\phantom{\left({\mathit{\alpha }}_{2}t+\left(\mathrm{\Delta }{\text{SA}}_{{\text{HCO}}_3}/{\text{SA}}_{\text{DIC}}\right)\left(1-e^{-{\mathit{\alpha }}_{2}t}\right)\right){\mathit{\alpha }}_2}{\mathit{\alpha }}_2\hfill \end{array}$$ 1

In this equation, V _DI14C is the total rate of ¹⁴C uptake; $f_{{\text{CO}}_2}$ is the fraction of uptake attributable to CO₂; α ₁ and α ₂ are the temperature-, salinity-, and pH-dependent first-order rate constants for CO₂ and HCO₃ ⁻ hydration and dehydration, respectively; t is the time (s); $\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}$ and $\mathrm{\Delta }{\text{SA}}_{{\text{HCO}}_3}$ are the differences between the initial and equilibrium values of the specific activities of CO₂ and HCO₃ ⁻, respectively; and SA_DIC is the specific activity of DIC. During steady-state photosynthesis, V_DI14C and $f_{{\text{CO}}_2}$ are assumed to be constant so that changes in the instantaneous ¹⁴C uptake rate reflect only changes in the specific activity of CO₂ and HCO₃ ⁻.

In the present study, the ¹⁴C disequilibrium method was modified to enable measurements over a range of ecologically relevant pH values (7.90–8.70). In order to maintain a suitably large initial isotopic disequilibrium $\left(\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}/{\text{SA}}_{\text{DIC}}\right)$, the pH of the ¹⁴C spike solutions needs to be adjusted in conjunction with the pH of the assay buffer. We, thus, used either MES or HEPES buffers to set the pH of spike solutions over the range of 5.75–7.30 (see Table 2 for exact pH values of assay and spike buffers). For the assays, 10–30 × 10⁶ cells were concentrated via gentle filtration over a polycarbonate filter (2 μm; Millipore, Billerica, MA, USA) to a final volume of 15 mL. During this filtration procedure, cells were kept in suspension, while the medium was gradually exchanged with buffered assay medium of the appropriate pH value. Assay media and spike buffers were prepared at least 1 day prior to the assay and stored in closed containers to avoid CO₂ exchange and pH drift. The pH value and temperatures of all buffers were measured immediately prior to assay runs. DIC concentration of the assay buffers was determined colorimetrically according to Stoll et al. (2001) using a TRAACS CS800 autoanalyzer (Seal Analytical, Norderstedt, Germany), and measurements were accuracy-corrected with CRMs supplied by A. Dickson (Scripps Institution of Oceanography, USA).

Table 2.

Chemical characteristics of ¹⁴C disequilibrium assay media and spike buffers, and the associated parameter values for model fits (Eq. 1)

Assay medium			Spike solution			Conditions for RCC 1216, 2N						Conditions for RCC 1217, 1N
pH	Buffer chemical	CO2 (%)	pH	Buffer chemical	CO2 (%)	DIC (μM)	CO2 (μM)	α 1	α 2	$\frac{\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}}{{\text{SA}}_{\text{DIC}}}$	$\frac{\mathrm{\Delta }{\text{SA}}_{{\text{HCO}}_3^{-}}}{{\text{SA}}_{\text{DIC}}}$	DIC (μM)	CO2 (μM)	α 1	α 2	$\frac{\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}}{{\text{SA}}_{\text{DIC}}}$	$\frac{\mathrm{\Delta }{\text{SA}}_{{\text{HCO}}_3^{-}}}{{\text{SA}}_{\text{DIC}}}$
7.90	BICINE	1.1	5.75	MES	80.4	2,210	23.4	0.0186	0.0197	29.09	−0.786	2,490	26.7	0.0176	0.0186	28.44	−0.786
8.10	BICINE	0.7	6.35	MES	50.7	2,250	14.6	0.0205	0.0225	30.08	−0.451	2,680	17.6	0.0194	0.0212	30.09	−0.454
8.30	BICINE	0.4	6.70	MES	31.5	2,290	8.9	0.0236	0.0272	30.46	−0.204	2,590	10.3	0.0223	0.0256	29.83	−0.206
8.50	BICINE	0.2	7.00	HEPES	18.7	2,380	5.4	0.0285	0.0355	31.37	−0.012	2,310	5.4	0.0270	0.0334	27.87	0.008
8.70	BICINE	0.1	7.30	HEPES	10.3	2,150	2.8	0.0364	0.0504	29.16	−0.237	–	–	–	–	–	–

Assays with the diploid cells (2N) were conducted at an assay temperature of 15.5 °C, a spike temperature of 23 °C, an added radioactivity of 315 kBq and a salinity of 32.4. Assays with the haploid cells (1N) were conducted at an assay temperature of 15.0 °C, a spike temperature of 23 °C, a spike radioactivity of 370 kBq and a salinity of 32.4

To initiate the assays, a volume of 4 mL buffered concentrated cell suspension was transferred into a temperature-controlled, illuminated glass cuvette (15 °C; 300 μmol photons m⁻² s⁻¹) to which 50 μM DBS was added (Ramidus, Lund, Sweden). Cells were continuously stirred in the light for at least 5 min prior to spike addition to reach steady-state photosynthesis. Spike solutions were prepared by adding NaH¹⁴CO₃ solution (1.88 GBq (mmol DIC)⁻¹; GE Healthcare, Amersham, UK) into a final volume of 200 μL of pH-buffered MilliQ water (various buffers at 20 mM; Table 2), yielding activities of ~370 kBq (10 μCi). Following the spike addition, 200 μL subsamples of the cell suspension were transferred into 2 mL HCl (6 M) at time points between 5 s and 12 min. Addition of these aliquots to the strong acid caused instant cell death and converted all DIC and PIC to CO₂. DI¹⁴C background was degassed in a custom-built desiccator for several days until samples were dry. Deionized water (1 mL) was then added to re-suspend samples prior to addition of 10 mL of scintillation cocktail (Ultima Gold AB, GMI, Ramsey, MN, USA), and the sample was vortexed thoroughly.

Acid-stable (i.e., organic) ¹⁴C activity in samples was counted with a Packard Tri-Carb Liquid Scintillation Counter (GMI). Blank samples, consisting of cell-free medium, were treated alongside the other samples. In the few cases where no blanks were available, time zero values were approximated by extrapolating the y-axis intercept from linear fitting of the first three data points of the ¹⁴C incorporation curves. Total radioactivity of the NaH¹⁴CO₃ stock solution was regularly quantified and compared to expected values to estimate loss of radioactivity or changes in counting efficiency. In all spike solutions, measured radioactivity ranged between 80 and 100 % of the theoretical values, and the actual radioactivity levels were used in the calculation of the specific activities. Blank-corrected data were fitted (Eq. 1), using a least-squares-fitting procedure. Applied fit parameters are given in Table 2. Furthermore, a detailed Excel spread sheet for calculating the fit parameters in dependence of the applied conditions (e.g., pH, temperature and DIC concentrations) is provided as Supplementary Material. Please note that in the calculation of initial and final specific activities, we accounted not only for changes in concentrations of ¹⁴C_i species but also for changes in concentrations of DI¹²C, ¹²CO₂, and H¹²CO₃ ⁻ upon spike addition. If these changes are neglected, $\mathrm{\Delta }{\text{SA}}_{{\text{CO}}_2}/{\text{SA}}_{\text{DIC}}$ will be significantly overestimated, leading to an underestimation of $f_{{\text{CO}}_2}$ (Eq. 1, Table 2, Supplementary material).

We used a numerical sensitivity study to examine how offsets in parameters such as pH, DIC concentrations, radioactivity, temperature, or blank values influence the derived estimates of $f_{{\text{CO}}_2}$. First, theoretical ¹⁴C incorporation curves for "HCO₃ ⁻ users" $\left(f_{{\text{CO}}_2}=0.25\right)$ and "CO₂ users" $\left(f_{{\text{CO}}_2}=0.80\right)$ were generated for two assay pH values (7.90 and 8.50) and used as a reference, assuming fixed values of DIC concentrations of 2,300 μmol kg⁻¹, assay temperature of 15 °C, spike solution temperature of 23 °C and spike radioactivity of 370 kBq. In a second step, model fits were obtained using slight offsets in these parameters (e.g., pH 7.95 and 7.85 instead of 7.90) to obtain the effect of parameter variability on $f_{{\text{CO}}_2}$ estimates. Sensitivity toward over- and underestimation of pH, temperature, DIC concentration, and radioactivity was tested. We further assessed the effects of blank values (±100 dpm) on $f_{{\text{CO}}_2}$ estimates as a function of different final ¹⁴C incorporation rates.

Statistics

All experiments were performed using at least biological triplicates (i.e., three independent, but equally treated cultures). When data were normally distributed (Shapiro-Wilk test) and showed equal variance (Equal-Variance Test), significance in difference between pCO₂ treatments was tested by performing student′s t-tests. When samples were not normally distributed or did not show equal variance, a rank sum test was performed instead. Null hypotheses were rejected when p ≤ 0.05, unless otherwise indicated.

Results

In diploid cells of E. huxleyi, the specific growth rate μ and PIC quotas did not change significantly in response to elevated pCO₂ (Table 3). While there was a small decrease in PIC production rates (−11 %), POC quotas and production rates increased strongly under elevated pCO₂ (+77 and +55 %, respectively). In conjunction with these changes, the quotas and production rates of TPC also increased (+28 and +23 %, respectively). The PIC:POC ratios of diploid cells decreased from 1.4 to 0.7 under elevated pCO₂, while the POC:PON ratios increased from 6.3 to 8.8. Chl a quotas were largely unaffected by the pCO₂ treatments, although Chl a:POC ratios decreased significantly from 0.022 to 0.012 pg pg⁻¹ under elevated pCO₂, owing to the change in POC quotas. In haploid cells, neither μ, elemental quotas or the respective production rates showed any significant response to elevated pCO₂ (Table 3). Similarly, Chl a quotas, Chl a:POC, and POC:PON ratios were all unaffected by the experimental CO₂ manipulations in the haploid strain.

Table 3.

Growth rates, elemental quotas and production rates, elemental ratios, as well as pigment composition of haploid (1N) and diploid (2N) cells of E. huxleyi, cultured at low (380 μatm) and elevated pCO₂ (950 μatm): μ (day⁻¹), POC quota (pg cell⁻¹), POC production (pg cell⁻¹ day⁻¹), PIC quota (pg cell⁻¹), PIC production (pg cell⁻¹ day⁻¹), TPC quota (pg cell⁻¹), TPC production (pg cell⁻¹ day⁻¹), PON quota (pg cell⁻¹), PON production (pg cell⁻¹ day⁻¹), PIC:POC ratio (mol:mol), POC:PON ratio (mol:mol), Chl a quotas (pg cell⁻¹), and Chl a:POC ratios (pg:pg)

Parameter	1N low pCO2	1 N high pCO2	p	2N low pCO2	2N high pCO2	p
μ	1.12 ± 0.04	1.08 ± 0.06	†	1.08 ± 0.05	1.04 ± 0.04	†
POC quota	10.76 ± 0.23	11.08 ± 1.19	†	8.35 ± 0.84	14.78 ± 1.91	**
POC production	12.09 ± 0.25	12.81 ± 0.44	†	9.02 ± 0.91	13.97 ± 0.63	*
PIC quota	0.48 ± 0.43	−0.18 ± 0.21	†	11.78 ± 0.78	10.90 ± 0.60	†
PIC production	–	–	†	12.71 ± 0.29	11.35 ± 0.90	**
TPC quota	11.23 ± 0.66	12.01 ± 1.27	†	20.13 ± 1.34	25.68 ± 2.00	*
TPC production	12.63 ± 0.70	12.51 ± 0.52	†	21.73 ± 1.05	26.77 ± 3.10	≤ 0.06
PON quota	1.39 ± 0.06	1.45 ± 0.09	†	1.54 ± 0.12	1.95 ± 0.22	*
PON production	1.56 ± 0.06	1.56 ± 0.08	†	1.66 ± 0.10	2.03 ± 0.30	†
PIC:POC	–	–	†	1.42 ± 0.14	0.75 ± 0.11	**
POC:PON	9.03 ± 0.19	8.90 ± 0.69	†	6.31 ± 0.30	8.83 ± 0.17	***
Chl a quota	0.10 ± 0.01	0.12 ± 0.01	†	0.18 ± 0.01	0.17 ± 0.01	†
Chl a :POC	0.009 ± 0.001	0.012 ± 0.001	†	0.022 ± 0.001	0.012 ± 0.001	***

For the haploid cells, PIC production and PIC:POC ratios were not calculated. Stars indicate statistical significance levels in differences between low and high pCO₂ treatments with * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. No significant difference (p > 0.05) is indicated by †

Under both pCO₂ acclimations, diploid cells were shown to be predominant "CO₂ users" under low assay pH ($f_{{\text{CO}}_2}$ ~ 1.0 at pH 7.9; Fig. 2a). With increasing assay pH, however, we observed a significant increase in relative HCO₃ ⁻ utilization. HCO₃ ⁻ uptake was induced at assay pH ≥ 8.3 (equivalent to CO₂ concentrations ≤ 9 μmol L⁻¹), reaching considerable contribution at high assay pH ($f_{{\text{CO}}_2}$ ~ 0.44 at pH 8.7). In contrast to the strong effect of the assay pH, the tested pCO₂ acclimations had no effect on the pH-dependent C_i uptake behavior (Fig. 2a). In other words, both low and high pCO₂-acclimated cells showed the same short-term response of $f_{{\text{CO}}_2}$ to assay pH. Like the diploid stage, haploid cells progressively changed from high CO₂ usage at low assay pH ($f_{{\text{CO}}_2}$ ~ 0.96 at pH 7.9) to substantial HCO₃ ⁻ contributions when assays were conducted in high pH assay buffers ($f_{{\text{CO}}_2}$ ~ 0.55 at pH 8.5; Fig. 2b). HCO₃ ⁻ uptake became relevant at pH ≥ 8.1 (equivalent to CO₂ concentrations ≤ 14 μmol L⁻¹), particularly in low pCO₂-acclimated cells. Except for haploid cells measured at pH 8.1, no significant differences in $f_{{\text{CO}}_2}$ were observed between the low and high pCO₂ acclimations (Fig. 2b).

Fig. 2.

Fraction of CO₂ usage $\left(f_{{\text{CO}}_2}\right)$ as a function of the assay pH in A the diploid E. huxleyi RCC 1216 and B the haploid RCC 1217 being acclimated to low pCO₂ (380 μatm, white triangles) and high pCO₂ (950 μatm, black circles)

The sensitivity analysis showed that an offset in the input pH of the buffered assay cell suspension (± 0.05 pH units) led to deviations in $f_{{\text{CO}}_2}$ of ≤ 0.09 (i.e., 9 percentage points) in "CO₂ users" and ≤ 0.02 in "HCO₃ ⁻ users" (Fig. 3a). An offset in the input temperature of the assay buffer (± 2 °C) led to a deviation in $f_{{\text{CO}}_2}$ of ≤ 0.09 in "CO₂ users" and ≤ 0.03 in "HCO₃ ⁻ users" (Fig. 3a). An offset in the input pH of the spike (± 0.05 pH units) changed the $f_{{\text{CO}}_2}$ estimates by ≤ 0.08 in "CO₂ users" and ≤ 0.03 in "HCO₃ ⁻ users" (Fig. 3a). Applying an offset in the input temperature of the spike (± 2 °C) caused a deviation in $f_{{\text{CO}}_2}$ by ≤ 0.06 in "CO₂ users" and had practically no effect on $f_{{\text{CO}}_2}$ in "HCO₃ ⁻ users" (≤ 0.01; Fig. 3a). An offset in the input DIC concentration of the buffer (± 100 μmol kg⁻¹) affected $f_{{\text{CO}}_2}$ by ≤ 0.08 in "CO₂ users" and ≤ 0.03 in "HCO₃ ⁻ users". Regarding the radioactivity of the spike (± 37 kBq), deviations in $f_{{\text{CO}}_2}$ were ≤ 0.12 in "CO₂ users" and ≤ 0.04 in "HCO₃ ⁻ users." Irrespective of CO₂ or HCO₃ ⁻ usage, offsets in blank estimations (± 100 dpm) led to deviating $f_{{\text{CO}}_2}$ by ≤ 0.27, but only when equilibrium ¹⁴C fixation rates were ≤ 1 dpm s⁻¹ (Fig. 3b). When steady-state ¹⁴C incorporation rates were ≥ 2 dpm s⁻¹ (i.e., average rate in diploid cells) and ≥ 4 dpm s⁻¹ (i.e., average rate in haploid cells), the deviations in $f_{{\text{CO}}_2}$ due to offsets in the blanks were ≤ 0.17 and ≤ 0.11, respectively.

Fig. 3.

Sensitivity in $f_{{\text{CO}}_2}$ estimates for "CO₂ users" ($f_{{\text{CO}}_2}=0.80$) and "HCO₃ ⁻ users" ($f_{{\text{CO}}_2}=0.25$) at low pH (7.9, in gray) and high pH (8.5, in white) A toward negative (inverted filled triangle) and positive (filled triangle) offsets in the pH, temperature, and DIC concentration of the assay buffer (pH_Assay, T _Assay, and [DIC]), as well as toward offsets pH, temperature, and radioactivity of the spike (pH_Spike, T _Spike, and RA), and B toward negative (inverted filled triangle) and positive (filled triangle) offsets in blank measurements (±100 dpm) in dependence of the final ¹⁴C incorporation rates. Sensitivity was assessed based on theoretical curves with constraints of a [DIC]_Assay = 2,300 μM, T _Assay = 15 °C, T _Spike = 23 °C, and RA_Spike = 37 kBq. Dashed lines indicate $f_{{\text{CO}}_2}$ values as expected for optimal experimental conditions

Discussion

Acclimation responses

This study corroborates previous findings on the general sensitivity of the diploid life-cycle stage of E. huxleyi toward OA (e.g., Feng et al. 2008; Langer et al. 2009; Riebesell et al. 2000). While growth rate was unaffected, OA reduced PIC production and stimulated POC production (Table 3). Consequently, the PIC:POC ratio was strongly decreased under OA, indicating a redirection of C_i fluxes between these two processes. Transcriptomics have previously attributed this redirection to an inhibition of calcification in response to impaired signal-transduction and ion-transport, as well as to stimulation in the production of glycoconjugates and lipids (Rokitta et al. 2012). In our study, also the TPC production increased significantly under OA (Table 3), indicating that not only C_i is allocated differently, but also the overall C_i uptake increases with the increasing pCO₂. Our data further suggest that less energy is required for the C_i acquisition under OA as more POC and TPC could be produced even though the Chl a quota remained unaffected by the pCO₂ treatment (Table 3). Improved energy-use efficiencies under OA have previously been proposed for the diploid life-cycle stage of E. huxleyi (Rokitta and Rost 2012).

In strong contrast to the diploid strain, the haploid life-cycle stage of E. huxleyi was insensitive toward OA with respect to growth rate and elemental composition (Table 3). The ability of the haploid cells to maintain homeostasis under OA has also been observed by Rokitta and Rost (2012). Even though the haploid cells appeared non-responsive toward OA on the phenomenological level (i.e., growth, elemental composition), transcriptomics have revealed significant changes at the subcellular level, such as an upregulation of catabolic pathways under OA (Rokitta et al. 2012). Based on the comparison of the life-cycle stages, Rokitta and co-workers concluded that the OA sensitivity in diploid cells originates from calcification, differences in C_i acquisition or both.

A number of studies have shown that E. huxleyi has moderately high C_i affinities and uses HCO₃ ⁻ as the primary C_i source (e.g., Herfort et al. 2002; Rokitta and Rost 2012; Rost et al. 2006b; Stojkovic et al. 2013), irrespective of the degree of calcification (Trimborn et al. 2007; Rokitta and Rost 2012). These characteristics would suggest E. huxleyi to be rather insensitive toward OA and the associated rise in CO₂ concentration, contrary to most results obtained for the diplont. As discussed below, this apparent discrepancy could originate from differences in conditions applied during short-term physiological measurements and those conditions cells experience in the long-term acclimation.

Modes of C_i acquisition

Our results demonstrate that the C_i source of both life-cycle stages of E. huxleyi is significantly influenced by the pH of the assay medium and the resulting carbonate chemistry (Fig. 2). With increasing pH in assay buffers, cells progressively changed from predominant CO₂ usage at lower pH values (≤ 8.1) to significant HCO₃ ⁻ contribution at higher pH (≥ 8.3). Surprisingly, this change occurred irrespectively of the pCO₂ conditions in the acclimation. To our knowledge, such a strong short-term pH-dependence in C_i acquisition has not been previously reported, which is most likely due to the fact that assays are typically performed under standardized pH values. Measuring physiological responses under one reference condition have the advantage that consequences of different acclimations can readily be compared in terms of altered capacities of certain processes, e.g., enzyme activities or transport rates. However, determination of the C_i source at one standard pH appears to impose a methodological bias, and our results, therefore, bear direct relevance to the interpretation of previous laboratory observations.

In view of the short-term pH effect on C_i acquisition, the contribution of HCO₃ ⁻ as a photosynthetic C_i source in E. huxleyi may have possibly been overestimated in previous studies. This overestimation is likely to be the most significant in those studies when ¹⁴C disequilibrium assays were conducted at pH 8.5 (e.g., Rokitta and Rost 2012; Rost et al. 2007). By looking at the C_i source determined at an assay pH mimicking the acclimation condition, we can now re-evaluate and in fact explain the responses of E. huxleyi toward elevated pCO₂. When assessing $f_{{\text{CO}}_2}$ using assay buffers of pH 7.9 and 8.1 (equivalent to the acclimation pH of high and low pCO₂ treatments), we observed predominant CO₂ uptake under both conditions (Fig. 2). Being "CO₂ user", cells were thus able to directly benefit from changes in the CO₂ concentrations in our acclimations (~15 μmol kg⁻¹ at 380 μatm and ~38 μmol kg⁻¹ at 950 μatm). For a "HCO₃ ⁻ user", however, it would be difficult to argue for a beneficial OA-effect as HCO₃ ⁻ concentrations do not differ much between treatments (~1,930 μmol kg⁻¹ at 380 μatm and ~2,130 μmol kg⁻¹ at 950 μatm). Our results thus suggest that biomass production in diploid cells not only profits from the declined calcification at high pCO₂, as suggested by Rokitta and Rost (2012) but also from the higher CO₂ supply under OA. As CO₂ usage is considered to be less costly than HCO₃ ⁻ uptake (Raven 1990), this could also explain the higher energy-use efficiency observed for E. huxleyi (Rokitta and Rost 2012).

Although the haploid life-cycle stage of E. huxleyi exhibited a pH-dependent C_i uptake behavior that was similar to the diploid (Fig. 2), the haploid cells did not show any CO₂-dependent stimulation in biomass production (Table 3). This could partly be related to the fact that the biomass production cannot profit from a down-scaling of calcification, simply because this process is absent in the haploid life-cycle stage. The lack of significantly stimulated biomass buildup under OA could also be attributed to the concomitant upregulation of catabolic pathways, such as higher lipid consumption, which is a specific feature of the haploid cells (Rokitta et al. 2012). After all, the similar C_i uptake behavior of both life-cycle stages confirms that photosynthetic HCO₃ ⁻ usage is not tied to calcification (Herfort et al. 2004; Trimborn et al. 2007; Bach et al. 2013) and that the preference for CO₂ or HCO₃ ⁻ is predominantly controlled by carbonate chemistry.

Our findings clearly demonstrate that the acclimation history, in both life-cycle stages, has little or no effect on the C_i usage of the cells (Fig. 2). In other words, the instantaneous effect of the assay conditions dominates over acclimation effects. We cannot preclude, however, that cells acclimated to higher pH values, where CO₂ supply becomes limiting, may increase their capacity for HCO₃ ⁻ uptake and acclimations effects would then be evident. Notwithstanding the potential for some acclimation effects, the extent to which short-term pH and/or CO₂ levels in the assay medium directly control cellular C_i usage is striking. This implies that even though E. huxleyi did not use significant amounts of HCO₃ ⁻ for photosynthesis, it must constitutively express a HCO₃ ⁻ transporter in all acclimations. Without the presence of a functional HCO₃ ⁻ transport system we could otherwise not explain the capacity for significant HCO₃ ⁻ uptake under short-term exposure to high pH (even in high pCO₂-acclimated cells).

In the diploid life-cycle stage, HCO₃ ⁻ transporter may be constitutively expressed to fuel calcification, as HCO₃ ⁻ was identified as the main C_i source for this process (Paasche 1964; Rost et al. 2002; Sikes et al. 1980). If CO₂ supply for photosynthesis becomes limiting, HCO₃ ⁻ transport could then also fuel photosynthesis. In the haploid cells, which do not calcify, we nonetheless observed the same capacity for HCO₃ ⁻ uptake, which suggests that HCO₃ ⁻ uptake capacity represents a fundamental component of the CCM of both life-cycle stages of E. huxleyi. Whether levels of protons or CO₂ concentrations are the main trigger for the shift between CO₂ and HCO₃ ⁻ uptake remains unclear, even though there is strong evidence that CO₂ supply is the main driver for the responses in photosynthesis (Bach et al. 2011).

Sensitivity analyses

In our sensitivity study, the applied offsets in pH (± 0.05 pH units), temperature (± 2 °C), DIC of the assay buffer (± 100 μM), and spike radioactivity (± 37 kBq) were larger than typical measurement errors to represent "worst-case scenarios". None of these offsets caused $f_{{\text{CO}}_2}$ estimates to deviate by more 0.12 in any of the pH treatments (Fig. 3a). When adequate efforts are taken to control these parameters (e.g., using reference buffers, thermostats), methodological uncertainties are thus negligible. DIC concentrations and radioactivity, however, are often not measured and in view of the potential drift over time, offsets can easily exceed typical measurement errors and lead to severe deviations in $f_{{\text{CO}}_2}$. For instance, ¹⁴CO₂ out-gassing causes the spike solution to progressively lose radioactivity. This loss of ¹⁴C can easily be > 20 % over the course of weeks or months, despite the high pH values of the stock solution and small headspace in the storage vial (Gattuso et al. 2010).

The average final ¹⁴C fixation rates, which depend on the biomass and radioactivity used, were 2.1 ± 0.8 dpm s⁻¹ in the runs with diploid and 6.6 ± 2.2 dpm s⁻¹ in those with haploid cells (Fig. 3b). In these ranges, offsets in blank values (± 100 dpm) can lead to biases in the estimated $f_{{\text{CO}}_2}$ by up to 0.20 (Fig. 3b). This strong sensitivity highlights the need to thoroughly determine blank values, but also to work with sufficiently high biomass and/or radioactivity to maximize ¹⁴C incorporation rates. When working with dense cell suspensions, however, self-shading or significant draw-down of DIC during the assay might bias results. Higher label addition generally increases the resolution of the assay and lowers the consequences of offsets in the blank value. It should be noted, however, that high concentrations of ¹⁴C in spike solutions can affect not only the isotopic but also the chemical conditions in the cuvette (e.g., pH and DIC).

Overall, our sensitivity study revealed that the ¹⁴C disequilibrium method is a straightforward and robust assay, which is very useful for resolving the C_i source of phytoplankton over a range of different pH values. It is important to realize, however, the pH of assay buffers has the potential to significantly affect the C_i uptake behavior of cells.

Conclusions

Our data clearly demonstrate that both life-cycle stages of E. huxleyi predominantly use CO₂ as C_i source for photosynthesis under typical present-day and future CO₂ levels, but constitutively express HCO₃ ⁻ transporters allowing them to directly use HCO₃ ⁻ when CO₂ becomes limiting. Under bloom conditions, where pH values can easily increase to 8.5 or higher, cells might, therefore, be able to maintain efficient C_i acquisition. Future research needs to investigate whether and how the assay pH governs the mode of C_i acquisition also in other coccolithophores species or phytoplankton taxa and how this may alter the energy budget of cells. Results from previous studies may need re-consideration in the light of our data showing strong short-term pH effects on C_i uptake of phytoplankton.

Electronic supplementary material

Below is the link to the electronic supplementary material.