Scholarly article on topic 'CO2-dependent carbon isotope fractionation in dinoflagellates relates to their inorganic carbon fluxes'

CO2-dependent carbon isotope fractionation in dinoflagellates relates to their inorganic carbon fluxes Academic research paper on "Environmental engineering"

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Abstract of research paper on Environmental engineering, author of scientific article — Mirja Hoins, Tim Eberlein, Dedmer B. Van de Waal, Appy Sluijs, Gert-Jan Reichart, et al.

Abstract Carbon isotope fractionation (εp) between the inorganic carbon source and organic matter has been proposed to be a function of pCO2. To understand the CO2-dependency of εp and species-specific differences therein, inorganic carbon fluxes in the four dinoflagellate species Alexandrium fundyense, Scrippsiella trochoidea, Gonyaulax spinifera and Protoceratium reticulatum have been measured by means of membrane-inlet mass spectrometry. In-vivo assays were carried out at different CO2 concentrations, representing a range of pCO2 from 180 to 1200μatm. The relative bicarbonate contribution (i.e. the ratio of bicarbonate uptake to total inorganic carbon uptake) and leakage (i.e. the ratio of CO2 efflux to total inorganic carbon uptake) varied from 0.2 to 0.5 and 0.4 to 0.7, respectively, and differed significantly between species. These ratios were fed into a single-compartment model, and εp values were calculated and compared to carbon isotope fractionation measured under the same conditions. For all investigated species, modeled and measured εp values were comparable (A. fundyense, S. trochoidea, P. reticulatum) and/or showed similar trends with pCO2 (A. fundyense, G. spinifera, P. reticulatum). Offsets are attributed to biases in inorganic flux measurements, an overestimated fractionation factor for the CO2-fixing enzyme RubisCO, or the fact that intracellular inorganic carbon fluxes were not taken into account in the model. This study demonstrates that CO2-dependency in εp can largely be explained by the inorganic carbon fluxes of the individual dinoflagellates.

Academic research paper on topic "CO2-dependent carbon isotope fractionation in dinoflagellates relates to their inorganic carbon fluxes"

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Journal of Experimental Marine Biology and Ecology

journal homepage: www.elsevier.com/locate/jembe

CO2-dependent carbon isotope fractionation in dinoflagellates relates to their inorganic carbon fluxes

Mirja Hoins aÄ*, Tim Eberlein b, Dedmer B. Van de Waalc, Appy Sluijs a, Gert-Jan Reicharta,d, Björn Rostb

a Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Budapestlaan 4,3584 CD Utrecht, The Netherlands

b Marine Biogeosciences, Alfred Wegener Institute, Helmholtz Centre for Polar- and Marine Research, Am Handelshafen 12,27570 Bremerhaven, Germany c Department of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10,6708 PB Wageningen, The Netherlands d Royal Netherlands Institute for Sea Research (NIOZ), Landsdiep 4, 1797 SZ 'tHorntje, Texel, The Netherlands

ARTICLE INFO ABSTRACT

Carbon isotope fractionation (ep) between the inorganic carbon source and organic matter has been proposed to be a function of pCO2. To understand the CO2-dependency of 8p and species-specific differences therein, inorganic carbon fluxes in the four dinoflagellate species Alexandriumfundyense, Scrippsiella trochoidea, Gonyaulax spinifera and Protoceratium reticulatum have been measured by means of membrane-inlet mass spectrometry. In-vivo assays were carried out at different CO2 concentrations, representing a range of pCO2 from 180 to 1200 ^iatm. The relative bicarbonate contribution (i.e. the ratio of bicarbonate uptake to total inorganic carbon uptake) and leakage (i.e. the ratio of CO2 efflux to total inorganic carbon uptake) varied from 0.2 to 0.5 and 0.4 to 0.7, respectively, and differed significantly between species. These ratios were fed into a single-compartment model, and 8p values were calculated and compared to carbon isotope fractionation measured under the same conditions. For all investigated species, modeled and measured 8p values were comparable (A. fundyense, S. trochoidea, P. reticulatum) and/or showed similar trends with pCO2 (A. fundyense, G. spinifera, P. reticulatum). Offsets are attributed to biases in inorganic flux measurements, an overestimated fractionation factor for the CO2-fixing enzyme RubisCO, or the fact that intracellular inorganic carbon fluxes were not taken into account in the model. This study demonstrates that CO2-dependency in 8p can largely be explained by the inorganic carbon fluxes of the individual dinoflagellates.

© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

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Article history:

Received 12 September 2015 Received in revised form 3 April 2016 Accepted 4 April 2016 Available online 30 April 2016

Keywords: CCM

CO2 uptake HCO- uptake Leakage

1. Introduction

During photosynthetic carbon fixation, the lighter carbon isotope 12C is preferred over the heavier carbon isotope 13C, thereby causing carbon isotope fractionation (sp) between the inorganic carbon (Q) source and the organic carbon. Values for sp of marine phytoplankton have been shown to be CO2-sensitive (e.g. Degens et al., 1968), and thus were discussed to serve as a proxy for past CO2 concentrations (Jasper and Hayes, 1990; Pagani, 2014; Van de Waal et al., 2013; Hoins et al., 2015). Large species-specific differences in sp have been

Abbreviations: Q, inorganic carbon; CCM, CO2-concentrating mechanism; Chl-a, Chlorophyll-a; ep, carbon isotope fractionation; £p-meas, measured carbon isotope fractionation; ep-mod, modeled carbon isotope fractionation; es, equilibrium fractionation between CO2 and HCO-; ef, kinetic fractionation associated with the CO2 fixation of RubisCO; LCO2, ratio of CO2 efflux relative to total Q uptake; DIC, dissolved inorganic carbon; HCO-, bicarbonate; Rhcc3, ratio of HCO- to total Q uptake; RubisCO, ribulose-1,5-bisphosphate Carboxylase/Oxygenase; CA, carbonic anhydrase; TA, total alkalinity.

* Corresponding author at: Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Budapestlaan 4,3584 CD Utrecht, The Netherlands.

E-mail addresses: mhoins@awi.de (M. Hoins), tim.eberlein@awi.de (T. Eberlein), D.vandeWaal@nioo.knaw.nl (D.B. Van de Waal), a.sluijs@uu.nl (A. Sluijs), g.j.reichart@uu.nl (G.-J. Reichart), bjoern.rost@awi.de (B. Rost).

described, which are yet poorly understood (e.g. Hinga et al., 1994; Burkhardt et al., 1999). Moreover, irrespective of the phytoplankton species investigated, most of these studies have solely described the relationship between sp and CO2, and only few have investigated the underlying physiological processes. Such mechanistic understanding is, however, needed to identify the reasons of the CO2-dependency of sp.

Carbon isotope fractionation of phytoplankton is primarily driven by the enzyme ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO), which is responsible for the fixation of CO2 into organic compounds. The intrinsic fractionation associated with RubisCO (sf) has been estimated to range between ~22 and 30%» (e.g. Roeske and O'Leary, 1984; Guy et al., 1993; Scott et al., 2007), even though a recent study obtained values as low as 11 % for the RubisCO of the coccolithophore Emiliania huxleyi (Boller et al., 2011). While RubisCO principally sets the upper limit of fractionation, other processes strongly determine the degree to which RubisCO can express its fractionation (Sharkey and Berry, 1985; Burkhardt et al., 1999; Rost et al., 2002). First, there is leakage, i.e. the amount of CO2 diffusing out of the cell in relation to Q uptake. With higher leakage, the intracellular Q pool is 'refreshed', thereby preventing accumulation of 13C and allowing RubisCO to approach its upper fractionation values. Second, the relative

http://dx.doi.org/10.1016/j.jembe.2016.04.001

0022-0981/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/40/).

contribution of bicarbonate (HCO3 ) to total Q uptake plays a role, as HCO- is enriched in 13C by -10%» relative to CO2 (Mook et al., 1974). An increasing HCO- contribution thus lowers sp. The enzyme carbonic anhydrase (CA), which accelerates the otherwise slow interconversion between CO2 and HCO-, can also influence sp under certain conditions, e.g. by influencing leakage as well as the relative HCO- contribution. All these processes play a role in the CO2-concentrating mechanisms (CCMs) of phytoplankton. Assessing the mode of CCMs may therefore help to understand the reasons for CO2-dependent changes in sp and species-specific differences therein.

Dinoflagellates are cosmopolitan unicellular algae that occur in many different environments, including eutrophic coastal regions and oligotrophic open oceans. In this study, we investigated whether the CO2-dependency of sp, which was found in the dinoflagellate species Alexandrium fundyense, Gonyaulax spinifera, Protoceratium reticulatum and Scrippsiella trochoidea (Burkhardt et al., 1999; Hoins et al., 2015), can be explained by changes in their Q fluxes. Characteristics of CCMs in the tested species, including their CA activities and Q fluxes, were measured by means of membrane-inlet mass spectrometry (MIMS). Results were fed into a single-compartment model that considers cellular leakage, the relative HCO- contribution as well as the carbon isotope fractionation of RubisCO (Sharkey and Berry, 1985; Burkhardt et al., 1999). The calculated carbon fractionation (sp_mod) was then compared to the measured carbon fractionation (sp_meas).

2. Material and methods

2.1. Incubations

Cultures of the dinoflagellate species A. fundyense (formerly Alexandrium tamarense strain Alex5; John et al., 2014), S. trochoidea (strain GeoB267; culture collection of the University of Bremen), G. spinifera (strain CCMP 409) and P. reticulatum (strain CCMP 1889) were grown in 0.2 |am filtered North Sea water (salinity 34), which was enriched with 100 |jmol L-1 nitrate and 6.25 |jmol L-1 phosphate. Metals and vitamins were added according to f/2 medium (Guillard and Ryther, 1962), except for FeCl3 (1.9 |jmol L-1), H2SeO3 (10 nmol L-1) and NiCl2 (6.3 nmol L-1) that were added according to K medium (Keller et al., 1987). Each of the strains was grown in 2.4 L air-tight borosilicate bottles at 15 °C and 250 ± 25 |amol photons m-2 s-1 at a 16:8 h light:dark cycle. Bottles were placed on roller tables in order to avoid sedimentation.

Dissolved CO2 concentrations ranged from ~5-50 |amol L-1 and were reached by pre-aerating culture medium with air containing 180,380, 800 and 1200 |jatm pCO2. The carbonate chemistry was calculated based on pH and total alkalinity (TA), using the program CO2sys (Pierrot et al., 2006). pH values were measured using a WTW 3110 pH meter equipped with a SenTix 41 Plus pH electrode (WTW, Weilheim, Germany), which was calibrated prior to measurements to the National Bureau of Standards (NBS) scale. An automated TitroLine burette system (SI Analytics, Mainz, Germany) was used to determine TA. Dissolved inorganic carbon (DIC) was determined colorimetrically using a QuAAtro autoanalyser (Seal Analytical, Mequon, USA). For more details on the carbonate chemistry in the acclimations, please refer to Eberlein et al. (2014) for A. fundyense and S. trochoidea and to Hoins et al. (2015) for G. spinifera and P. reticulatum.

To determine sp values, the isotopic composition of the organic material was measured using an Automated Nitrogen Carbon Analyser mass spectrometer (ANCA-SL 20-20, SerCon Ltd., Crewe, UK), and the isotopic composition of the DIC in growth medium was measured using a GasBench-11 coupled to a Thermo Delta-V advantage isotope ratio mass spectrometer (see Hoins et al., 2015 for details on isotope analysis). Prior to assays, cells were acclimated to the different CO2 concentrations for at least 7 generations (i.e. >21 days). To prevent changes in the carbonate chemistry, i.e.

keeping drawdown of DIC <3%, incubations were terminated at low cell densities (<400 cells mL-1).

2.2. MIMS assays

A custom-made membrane-inlet mass spectrometer (MIMS; Isoprime, GV Instruments, Manchester, UK; see Rost et al., 2007 for details) was used to determine CA activities and Q fluxes of A. fundyense and S. trochoidea acclimated to four different pCO2 (i.e. 180, 380, 800 and 1200 ^atm; Eberlein et al., 2014), and of G. spinifera and P. reticulatum acclimated to a low and high pCO2 (i.e. 180 and 800 |jatm). Assays were performed in an 8 mL temperature-controlled cuvette, equipped with a stirrer. Assay tests over ~1 h confirmed that conditions during the assay do not cause physiological stress (i.e. no decline in O2 production rates), and subsequent microscopic inspection did not reveal any visual effects on cell morphologies. Prior to the measurements, acclimated cells were concentrated using a 10 |am membrane filter (Millipore, Billerica, MA) by gentle vacuum filtration (<200 mbar) and stepwise transferred into Q-free medium buffered with a 4-(2-hydroxylethyl)-1-piperazine-ethanesulfonic acid (50 mmol-1 HEPES) solution at 15 ± 0.3 °C and a pH of 8.0 ± 0.1. Chlorophyll a (Chl-a) concentrations were determined fluoro-metrically by using a TD-700 Fluorometer (Turner Designs, Sunnyvale, CA, USA) and ranged between 0.15 and 1.70 |ag mL-1 during the assays.

To quantify activities of extracellular CA (eCA), the 18O depletion rate of doubly labeled 13C18O2 in seawater was determined by measuring the transient changes in 13C18O18O (m/z = 49), 13C18O16O (m/z = 47) and 13C16O16O (m/z = 45) in the dark, following the approach of Silvermann (1982). If cells possess eCA, exchange rates of 18O are accelerated relative to the spontaneous rate. To monitor the spontaneous rate, NaH13C18O3 label was injected to the cuvette, waiting until the m/z = 49 signal reached a steady-state decline. This rate was then compared to the steady-state decline after cells were added. Following Badger and Price (1989), eCA activity is expressed as percentage decrease in 18O-atom fraction upon the addition of cells, normalized to Chl-a. Consequently, 100 units (U) correspond to a doubling in the rate of interconversion between CO2 and HCO- per |ag Chl-a.

Photosynthetic O2 and Ci fluxes were determined following Badger et al. (1994). Making use of the chemical disequilibrium, this approach estimates CO2 and HCO- fluxes during steady-state photosynthesis. It is based on the simultaneous measurements of O2 and CO2 concentrations during consecutive light and dark intervals with increasing amounts of DIC. Oxygen fluxes in the dark and light are converted into Cj fluxes by applying a respiratory quotient of 1.0 and a photosynthetic quotient of 1.1 (Burkhardt et al., 2001; Rost et al., 2003). The light intensity in the cuvette was adjusted to the acclimation conditions (i.e. 250 ± 25 |jmol photons m-2 s-1). Net CO2 uptake was calculated from the steady-state decline in CO2 concentration at the end of the light period, corrected for the interconversion between CO2 and HCO-. The uptake of HCO- was calculated by subtracting net CO2 uptake from net Q uptake, and the CO2 efflux from the cells was estimated from the initial slope after turning off the light. Rate constants kj and k2 were determined based on temperature, salinity and pH (Zeebe and Wolf-Gladrow, 2001; Schulz et al., 2006), yielding mean values of 0.9241 (±0.0506) min-1 and 0.0085 (±0.0008) min-1, respectively. To eliminate any eCA activity, a prerequisite to apply the rate constants, we added dextran-bound sulfonamide (DBS; 50 ^mol L-1) to the cuvette. For more details on the calculations, please refer to Badger et al. (1994) and Schulz et al. (2007).

2.3. Single-compartment model

To calculate sp_mod, results for the relative HCO- contribution and leakage were fed into a single-compartment model after Sharkey and

Berry (1985) and Burkhardt et al. (1999):

sp- mod = RHCO3 x es + LCO2 x e f

where RHco3 represents the ratio of HCO— to total Q uptake, ss the equilibrium fractionation between CO2 and HCO— (— 10%»; Mook et al., 1974), LCO2 the ratio of CO2 efflux relative to total Q uptake, and sf the kinetic fractionation associated with the CO2 fixation of RubisCO, which was here assumed to be 28%» after Raven and Johnston (1991).

2.4. Statistical analysis

Shapiro-Wilk tests confirmed normality of the data. Significant differences between CO2 treatments were confirmed by a one-way ANOVA followed by post hoc comparison of the means using the Tukey HSD (a = 0.05; Table 1).

3. Results

3.1. CA activity

In A. fundyense and P. reticulatum, eCA activities were low with maximum activities of 156 U (|ag Chl-a)—1 and 44 U (|ag Chl-a) — 1, respectively. In S. trochoidea and G. spinifera, eCA activities were comparably high with up to 1600 U (^g Chl-a)—1 and 1100 U (^g Chl-a)—1, respectively. In neither of the species, eCA activities were responding to changes in pCO2. Please note that for G. spinifera and P. reticulatum no statistics could be applied due to the lack of replication.

3.2. HCO— contribution and leakage

Relative HCO— contribution was around 0.2 in A. fundyense and G. spinifera (Figs. 1A and 3A; Table 1), whereas S. trochoidea and P. reticulatum showed higher values of ~0.5 (Figs. 2A and 4A; Table 1). In other words, in A. fundyense and G. spinifera approximately 80% of the Ci taken up is in the form of CO2, whereas in S. trochoidea and P. reticulatum this was 50%. There was a significant decrease in HCO— contribution with increasing CO2 concentration in S. trochoidea, while

% 0.6 Q.

Π0.4 O t

i i ï Ï

i ] * i

■ HC037Total C, uptake

* Leakage

16 ■

Cl u 10

0.6 ST

&p-mod ep-meas

0 200 400 600 800 1000 1200 1400 pC02 (natm)

Fig. 1. Relative HCO— contribution, leakage and ep-mod and £p-meas in A. fundyense. Each data point represents the mean ± standard deviation (n = 3).

no such CO2-dependency was observed in any of the other tested species. Leakage differed significantly between the tested species, with values of up to 0.7 at 800 ^atm pCO2 in G. spinifera (Fig. 3A; Table 1) and lowest average values of ~0.5 in S. trochoidea and P. reticulatum,

Experimental conditions in dilute batch culture incubations (see also Eberlein et al., 2014; Hoins et al., 2015): average CO2 concentrations (|molL-1), total alkalinity (TA; |molL-1), dissolved inorganic carbon (DIC; |mol L-1) and pH (NBS scale). HCO- contribution, leakage, modeled carbon isotope fractionation (ep-mod) and measured carbon isotope fractionation (ep-meas) was derived under the same conditions.

pCO2 | atm

CO2 |mol L

TA | mol L-

DIC | mol L-

pH NBS

HCO3- contribution

Leakage

£p-mod A

A. fundyense 180 380 800 1200

5.9 ± 0.9a 11.5 ± 2.1b 25.9 ± 5.8c 36.5 ± 9.3d

2434 ± 3 2439 ± 1 2434 ± 2 2418 ± 1

1992 ± 10a 2117 ± 8b 2245 ± 8c 2283 ± 5d

8.50 ± 0.06a 8.27 ± 0.07b 7.97 ± 0.10c 7.83 ± 0.12d

0.22 ± 0.03 0.24 ± 0.04 0.24 ± 0.04 0.23 ± 0.08

0.44 ± 0.01a 0.46 ± 0.02a 0.53 ± 0.02b 0.63 ± 0.05c

10.1 ± 0.2a 10.6 ± 0.5a 12.6 ± 0.6b 15.3 ± 0.8c

9.0 ± 0.3a 10.2 ± 0.5b 12.7 ± 0.4c 12.1 ± 0.2c

S. trochoidea 180 380 800 1200

6.6 ± 0.2a 13.1 ± 0.5b 28.8 ± 2.0c 41.5 ± 3.6d

2386 ± 1 2388 ± 2

2385 ± 1

2386 ± 4

1872 ± 2a 2096 ± 3b 2223 ± 3c 2268 ± 9d

8.45 ± 0.01a 8.21 ± 0.02b 7.91 ± 0.03c 7.77 ± 0.04d

0.53 ± 0.03a b 0.55 ± 0.04a 0.48 ± 0.03b c 0.46 ± 0.04c

0.56 ± 0.06 0.53 ± 0.06 0.54 ± 0.01 0.48 ± 0.04

10.4 ±1.5 9.4 ± 1.5 10.3 ± 0.5 8.8 ± 1.1

6.0 ± 0.5a b

5.0 ± 0.1a

7.1 ± 0.7b 11.8 ± 0.7c

G. spinifera 180 380 800 1200

6.0 ± 1.1a 11.7 ± 2.5b 27.9 ± 7.4c 42.4 ± 7.9d

2447 ± 5 2461 ± 12 2475 ± 13 2459 ± 4

1962 ± 15a 2083 ± 1b 2224 ± 9c 2293 ± 5d

8.50 ± 0.05a 8.27 ± 0.07b 7.96 ± 0.10c 7.78 ± 0.06d

0.19 ±0.11 0.19 ±0.11

0.61 ± 0.01 0.71 ± 0.01

15.6 ± 0.9a 18.6 ± 1.7b

7.8 ± 1.0a 9.4 ± 0.4a 11.7 ± 0.7b 8.1 ± 0.5a

P. reticulatum 180 380 800 1200

7.1 ± 0.5a 13.9 ± 0.8b 31.0 ± 4.7c 45.2 ± 6.9d

2460 ± 8 2455 ± 2

2461 ± 12 2473 ± 19

2002 ± 2a 2121 ± 4b 2249 ± 23c 2288 ± 16d

8.43 ± 0.04a 8.21 ± 0.02b 7.88 ± 0.08c 7.75 ± 0.05d

0.44 ± 0.13 0.49 ± 0.19

0.50 ± 0.06 0.48 ± 0.09

9.58 ± 2.0 9.2 ± 1.9

8.4 ±1.8 8.4 ± 0.7 8.6 ± 2.0 9.9 ± 0.8

Values represent the mean of triplicate incubations (n = 3; ±SD). Superscript letters indicate significant differences between pCO2 treatments (P < 0.05).

ra 0.6 -

rc 0.4

8 0.2 i

i 1 * i

i I ' i

■ HC037Total C¡ uptake

* Leakage

CL :10 10 -

0.6 gf

—1-r

• Ei o E„.

0 200 400 600 800 1000 1200 1400 pC02 (natm)

Fig. 2. A) Relative HCO3 contribution, leakage and B) ep-mod and £p-meas in S. trochoidea. Each data point represents the mean ± standard deviation (n = 3).

Fig. 3. A) Relative HCO- contribution, leakage and B) ep-mod and £p-meas in G. spinifera. Each data point represents the mean ± standard deviation (n = 3).

respectively (Figs. 2A and 4A; Table 1). Only in A.fundyense, leakage was significantly CO2-dependent and increased from 0.44 to 0.63 (Fig. 1A; Table 1). For details on the kinetics of O2, CO2 and HCO- fluxes in A.fundyense and S. trochoidea, please refer to Eberlein et al. (2014).

33. Ci flux based sp calculations

Estimates for sp_mod are in the same range as sp_meas in A.fundyense and P. reticulatum (Figs. 1B and 4B; Table 1), while the model overestimated the fractionation by up to 5%o and 8%o in S. trochoidea and G. spinifera, respectively (Figs. 2B and 3B; Table 1). Except for S. trochoidea, sp_mod generally matches trends observed in sp_meas. In A.fundyense, for instance, sp_mod increased significantly from 10.1 to 15.3%», thereby closely matching sp_meas values (Fig. 1B; Table 1). Also in G. spinifera, sp_mod matches trends observed in sp_meas, if the highest pCO2 treatment of sp_meas is excluded. In this treatment, carbon isotope fractionation dropped significantly, most likely due to 2.5-fold increased cellular carbon contents (see discussion in Hoins et al., 2015). In S. trochoidea, neither the relative HCO- contribution nor leakage showed a CO2-dependency; hence sp_mod did not match the increase in sp_meas with increasing CO2 concentration (Fig. 2B; Table 1).

4. Discussion

4.1. CA activity plays a minor role in Ci fluxes

By expressing CA, many marine algae species accelerate the otherwise slow interconversion between CO2 and HCO-, thereby possibly facilitating the Cj uptake and internal Cj fluxes. In line with previous studies on dinoflagellates (Rost et al., 2006; Ratti et al., 2007), A. fundyense and P. reticulatum exhibit rather low eCA activities, even under low CO2 concentrations. In view of this, eCA is not expected to

significantly influence Q fluxes or sp in these species. In S. trochoidea and G. spinifera, however, eCA activities were high at all tested CO2 concentrations, comparable to values observed for temperate diatoms (Trimborn et al., 2009). Hence, the inhibition of eCA by the inhibitor DBS during the MIMS assay might have biased the Q fluxes, i.e. underestimated CO2 uptake (Rost et al., 2003), thereby potentially causing an underestimation of sp. As these were the species for which the model overestimated sp values, however, it can be concluded that eCA (and its inhibition by DBS during the C¡ flux measurements) did not influence the Ci fluxes much.

4.2. Species-specific differences in Ci fluxes

The HCO- contribution differed considerably between the tested species. While A. fundyense and G. spinifera showed a strong preference for CO2, S. trochoidea and P. reticulatum used CO2 and HCO- in equal proportions. The latter contradicts with the findings of an endpoint pH-drift experiment, suggesting that P. reticulatum is not able to efficiently use HCO- (Ratti et al., 2007). Testing other dinoflagellates with a modified pH-drift method, including the genus Protoceratium, demonstrated that the high pH itself can affect growth and thus interpretations about the used Q source based on pH-drift experiments must be considered with caution (Hansen et al., 2007). From an energetic point of view, high CO2 usage would be of advantage as CO2 can be taken up passively by diffusion, while HCO- is charged and thus has to be taken up by active uptake. And yet, the tested species covered a large part of their Cj demand by HCO-, as observed in S. trochoidea and P. reticulatum (Figs. 2Aand4A).

Similarly high HCO- contributions were observed for other dinofla-gellate species (Rost et al., 2006) and cyanobacteria (Price et al., 2008; Kranz et al., 2011). This preference for HCO- has been associated with the very low CO2-affinity of RubisCO type IB, which is expressed

Fig. 4. A) Relative HCO3 contribution, leakage and B) ep-mod and ep-meas in P. reticulatum. Each data point represents the mean ± standard deviation (n = 3).

in cyanobacteria, and RubisCO type II expressed in dinoflagellates (Badger et al., 1998; Whitney and Andrews, 1998). To compensate for this low affinity, high amounts of Q have to be accumulated, which in seawater can more easily be accomplished with the abundant HCO-ion rather than with CO2. In addition, HCO- is about 1000-fold less permeable to lipid membranes than CO2, making it the preferred Cj form to be accumulated within the cell (Price et al., 2008). In the case where species covered the majority of their Q demand by CO2, as observed in A.fundyense and G. spinifera (Figs. 1Aand 3A), one could therefore speculate about chloroplast-based Ci accumulation rather than pumping of HCO- at the cell wall (Badger et al., 1998). The observed differences in the preferred Q source and likely consequences for internal Q fluxes may also affect the overall leakage of cells.

Also leakage differed considerably among the species. A. fundyense showed a relatively low leakage of 0.44 at 180 ^atm pCO2, which increased to 0.63 at 1200 ^atm pCO2. Leakage in G. spinifera varied between 0.60 at 180 ^atm pCO2 and 0.70 at 800 ^atm pCO2. Leakage estimates in S. trochoidea and P. reticulatum were lower with ~0.50 and remained constant over the applied range of pCO2. These differences may be caused by different membrane permeabilities, which again potentially relate to the preferred Ci source. In fact, A. fundyense and G. spinifera both preferred CO2 over HCO- and likewise showed the highest degrees of leakage, thereby suggesting highly permeable membranes with respect to CO2. In these species, also CO2-related changes in the membrane permeability are indicated as they show significantly increased leakage under higher pCO2 (see also Eberlein et al., 2014 for A.fundyense, formerly A. tamarense).

4.3. Patterns in carbon isotope fractionation can be explained by Ci fluxes

Using results for HCO- contribution and leakage obtained in this study, carbon isotope fractionation was calculated and compared to

previous measurements (Figs. 1B-4B; see also Hoins et al., 2015). Generally, there is a good agreement as sp-mod and sp_meas values were in the same range (A. fundyense, S. trochoidea, P. reticulatum) and/or followed the same trend (A. fundyense, G. spinifera, P. reticulatum; Figs. 1B-4B). Despite the overall agreement between flux-based estimates and directly measured carbon isotope fractionation, sp_mod was overestimated in S. trochoidea and G. spinifera. Such offsets could principally be attributed to biases in the Ci flux measurements, i.e. uncertainties in the estimation of HCO- contribution and/or leakage. It has been argued, however, that the MIMS approach tends to overestimate the HCO- contribution (due to the constant pH of 8.0 during the assay, see Burkhardt et al., 2001), and rather underestimates cellular leakage (due to fact that CO2 fixation does not cease instantly upon darkening, see Badger et al., 1994). Hence, by correcting for these potential biases, i.e. assuming slightly lower HCO- contribution and higher leakage values, we would actually overestimate the fractionation even more for S. trochoidea and G. spinifera.

An alternative explanation for the overestimation by the model may be attributed to the fractionation factor of RubisCO, which we assumed to be 28%o (Raven and Johnston, 1991). Recent studies have found lower values, even as low as 11%» as in the case of the coccolithophore Emiliania huxleyi (Boller et al., 2011). Even though there are no indications for such low fractionation values in the highly conserved type II RubisCO, a lower fractionation would bring modeled and measured sp values closer in S. trochoidea and G. spinifera. In A. fundyense and P. reticulatum, however, it would lead to underestimated sp_mod. Hence, we would refrain from assuming much lower fractionation values for RubisCO type II in dinoflagellates in our calculations. Lastly, the fact that internal Ci fluxes were not taken into account might have also contributed to the offsets between sp_mod and sp_meas. Models that incorporate internal Ci cycling have, however, caused even higher sp-mod, as these processes work against the 13C accumulation within the chloroplasts (Cassar et al., 2006; Schulz et al., 2007) or the carboxysome (Eichner et al., 2015). Therefore, although the values and trends in carbon isotope fractionation are relatively well understood based on our physiological experiments, differences between theory and measurements are at present not fully resolved.

5. Conclusions

Our study demonstrates that carbon isotope fractionation in dinoflagellates can, to a large degree, be explained by considering their Cj fluxes. Relative HCO- contribution and/or leakage were CO2-dependent in A. fundyense, S. trochoidea and G. spinifera, which in turn can explain the CO2-dependency of their sp observed in previous studies (Hoins et al., 2015). To further advance our understanding of the sp patterns in dinoflagellates, Ci fluxes measurements should be performed at in situ pH (Kottmeier et al., 2014, 2016) and ideally differentiate between 13C and 12C fluxes (McNevin et al., 2006).

Acknowledgments

This research was funded through the Darwin Centre for Biogeosci-ences Grant 3021, awarded to GJR and AS, and the European Research Council under the Seventh Framework Program of the European Community through ERC Starting Grants #259627 to AS and #205150 to BR. DBvdW and BR thank BIOACID, financed by the German Ministry of Education and Research. This work was carried out under the program of the Netherlands Earth System Science Centre (NESSC). We thank Urban Tillmann (Alfred Wegener Institute) and Karin Zonneveld (Marum, Bremen University) for providing the dinoflagellate species Alexandrium fundyense Alex5 and Scrippsiella trochoidea GeoB267, respectively, and Ulrike Richter, Laura Wischnewski, Jana Hölscher (Alfred Wegener Institute) and Arnold van Dijk (Utrecht University) for technical support. [SS]

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