# Constraints on coccolithophores under ocean acidification obtained from boron and carbon geochemical approaches

Yi-Wei Liu, Sebastian Rokitta, Björn Rost, Robert A. Eagle

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Yi-Wei Liu, Sebastian Rokitta, Björn Rost, Robert A. Eagle. Constraints on coccolithophores under ocean acidification obtained from boron and carbon geochemical approaches. Geochimica et Cosmochimica Acta, 2021, 315, pp.317-332. 10.1016/j.gca.2021.09.025. hal-03452046

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Constraints on coccolithophores under ocean acidification obtained from boron and carbon geochemical approaches

Yi-Wei Liu<sup>a,b,*</sup>, Sebastian D Rokitta<sup>c</sup>, Björn Rost<sup>c,d</sup>, Robert A. Eagle<sup>b,e,f,*</sup>

Received 21 September 2020; accepted in revised form 20 September 2021; Available online 25 September 2021

Abstract

Ocean acidification (OA) appears to have diverse impacts on calcareous coccolithophores, but the cellular processes under-
lying these responses are not well understood. Here we use stable boron and carbon isotopes, B/Ca ratios, as well as inorganic
and organic carbon production rates to investigate the carbon utilization and the internal pH regulation at the site of calci-
fication in *Emiliania huxleyi*, *Calcidiscus leptoporus* and *Pleurochrysis carterae* cultured over a wide *pCO*₂ range (180–1000
µatm). Despite large variability, the geochemistry data indicate species-specific modes of pH control and differences in the
utilization of inorganic carbon. Boron isotope data suggest that all three species generally upregulate the pH of the calcifica-
tion fluid (pH<sub>CF</sub>) compared to surrounding seawater, which coincides with relatively constant growth rates and cellular ratios
of inorganic to organic carbon. Furthermore, species exhibit different strategies in regulating their pH<sub>CF</sub>, i.e., two species
maintain homeostasis (pH<sub>CF</sub> = ~8.7), while one species shows a constant offset to the surrounding seawater (ΔpH = ~0.6
units) over the entire tested *pCO*₂ range. In addition to these different strategies, carbon isotope data suggests that high plas-
ticity in the utilization of dissolved inorganic carbon might be an explanation for species-specific differences in coccol-
ithophore responses to OA.

© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Keywords: Ocean acidification; Coccolithophores; Carbon isotopes; Boron isotopes

# 1. INTRODUCTION

Coccolithophores are calcifying microalgae and are
among the most important primary producers in the ocean
(Eikrem et al., 2016). They utilize dissolved inorganic car-
bon (DIC) for both photosynthesis and calcification, and
play an important role in regulating carbonate chemistry

<footnote><sup>a</sup> Institute of Earth Sciences, Academia Sinica, 128, Sec. 2, Academia Road, Nangang, Taipei 11529, Taiwan</footnote>

<footnote><sup>b</sup> Université de Brest, UBO, CNRS, IRD, Ifremer, Institut Universitaire Européen de la Mer, LEMAR, Rue Dumont d'Urville, 29280 Plouzané, France</footnote>

<footnote><sup>c</sup> Alfred Wegener Institute - Helmholtz-Centre for Polar- and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany</footnote>

<footnote><sup>d</sup> FB Biology/Chemistry, University of Bremen, Leobener Strasse, 28359 Bremen, Germany</footnote>

<footnote><sup>e</sup> Institute of the Environment and Sustainability, University of California – Los Angeles, La Kretz Hall, 619 Charles E. Young Dr. E #300, Los Angeles CA90024, USA</footnote>

<footnote><sup>f</sup> Atmospheric and Oceanic Sciences Department, University of California –Los Angeles, Math Sciences Building, 520 Portola Plaza, Los Angeles, CA 90095, USA</footnote>

<footnote>* Corresponding authors at: Institute of Earth Sciences, Academia Sinica, 128, Sec. 2, Academia Road, Nangang, Taipei 11529, Taiwan (Y.-W. Liu). Institute of the Environment and Sustainability, University of California – Los Angeles, La Kretz Hall, 619 Charles E. Young Dr. E #300, Los Angeles CA90024, USA (R.A. Eagle).</footnote>

<footnote>E-mail addresses: liuyiwei@earth.sinica.edu.tw (Y.-W. Liu),
robeagle@g.ucla.edu (R.A. Eagle).</footnote>
as well as mediating the flux of carbon dioxide ($CO_2$) from the atmosphere into the deeper ocean through the biological carbon pump (Rost and Riebesell, 2004). Experiments have shown that the particulate inorganic and organic carbon (PIC, POC) production of coccolithophores, as well as their responses to ocean acidification (OA), vary among species, and even among strains within a species (Langer et al., 2006; Langer et al., 2009; Meyer and Riebesell, 2015).

*Emiliania huxleyi* is the most abundant coccolithophore in surface waters, and culture studies show that also *E. huxleyi* has highly variable and strain-specific responses in experimentally manipulated OA scenarios (Hoppe et al., 2011; Iglesias-Rodriguez et al., 2008; Langer et al., 2009; Riebesell et al., 2000). Recent studies suggest that this species will shift the relative uptake of inorganic carbon from $HCO_3$ towards $CO_2$ under low pH conditions, and that seawater $[H^+]$ is the primary driver for these OA responses (Kottmeier et al., 2014; Kottmeier et al., 2016b). However, how this species controls the pH and carbonate chemistry in the calcification fluid ($pH_{CF}$; $DIC_{CF}$) is still largely unknown.

*Calcidiscus leptoporus* is a cosmopolitan species thriving in tropical to subtropical oligotrophic warm-water masses. Typical coccolith and coccospHERE sizes of *C. leptoporus* range from 3 to 11 µm and 5 to 20 µm, respectively (Geisen et al., 2002; Knappertsbusch et al., 1997), which is significantly larger than *E. huxleyi*. Its coccoliths can be dominant in sediments due to their comparably high dissolution resistance (Boeckel and Baumann, 2004; Dittert et al., 1999). The fossil records of *C. leptoporus* can be traced back to 23 Ma ago (Geisen et al., 2004; Knappertsbusch, 2000). In contrast to *E. huxleyi*, recent culture studies suggest that the calcification process of *C. leptoporus* is not negatively affected by OA (Fiorini et al., 2011; Langer et al., 2006).

*Pleurochrysis* (also known as *Chrysotila*) *carterae* is a motile coccolithophore species that is widely distributed along the temperate coasts of Europe (van den Hoek et al., 1995). Different from the well-studied coccolithophore species *E. huxleyi*, the considerably larger *P. carterae* has a relatively high content of particulate organic carbon (POC) compared to the calcite, with typical PIC: POC ratios lower than 0.5 (e.g., Casareto et al. (2009); White et al. (2018)). In addition, special polysaccharides form organic nucleation matrices and assist in calcification, which has been recognized as a distinct biomineralization pathway in this species (Marsh, 2003). The organic matrix is occluded in the calcite during the precipitation process, causing a high organic content of the calcite composite material formed by *P. carterae* (Hood et al., 2016; Moheimani and Borowitzka, 2011). These differences suggest that *P. carterae* allocates DIC to the calcification vesicle in a different way than the aforementioned species, and also point towards differences in its internal carbonate chemistry regulation.

The chemical composition of coccolith calcite can be used as a tracer for physiological processes in the organisms and for environmental conditions during calcification. Previous studies have utilized $^{13}$C in both, organic components and coccolith calcite to infer carbon utilization in the

organism (Hermoso, 2015; Hermoso et al., 2016; McClelland et al., 2017; Rickaby et al., 2010; Rost et al., 2002). Similar to other phytoplankton, the extent to which stable carbon is fractionated can be attributed to fluxes of DIC into the cell, leakage of carbon from the chloroplast or the cell, the location and activity of the enzyme carbonic anhydrase (CA) and/or the rate of calcification (Eichner et al., 2015; Hoins et al., 2016; Hoins et al., 2015; McClelland et al., 2017). Although CA may not always play a role with regard to fractionation (Hoins et al., 2016; McClelland et al., 2017), Eichner et al. (2015) has shown that CA-like activities have the potential to modify isotopic values of carbon species and thus overall fractionation. As proposed models describe the observed fractionation only adequately for certain species or $pCO_2$ ranges (e.g., Hoins et al. (2016); Hoins et al. (2015); McClelland et al. (2017)), we apparently still lack sufficient understanding of the processes controlling the isotopic fractionation, which is especially true for coccolithophore species of different origin and ecology. Thus, more laboratory and field data are needed to better constrain the underlying processes and to improve interpretations of the carbon fractionation data from experiments or geological records.

In addition, boron geochemistry can be used to constrain the $pH_{CF}$ and other carbonate system parameters. This approach has been widely used in corals and other calcifiers (Liu et al., 2020; McCulloch et al., 2018), but is less developed in coccolithophores, partly due to the difficulty of measuring the low boron contents of coccoliths. The few studies available on coccolithophores have shown that the B/Ca ratio and its pH-dependency can vary among species and strains, but it was postulated that a combination of B/Ca and boron isotope measurements could be used to disentangle how coccolithophores regulate their internal carbonate chemistry (Stoll et al., 2012). A follow-up study suggested, however, that coccolith $\delta^{11}B$ of *E. huxleyi* is insensitive to seawater pH changes and might be affected by the nutrient concentration in the culture media (Henehan, 2015). More recently, Liu et al. (2018) showed that the widespread coastal coccolithophore *Ochrosphaera neapolitana* maintains a homeostatic pH at the calcification site. Whether the pH homeostasis and the level of pH regulation is a species-specific characteristic thus remains unresolved.

To address these open questions, we here use a combined $\delta^{13}C$ and boron geochemistry approach to study carbon utilization and pH regulation in three coccolithophore species (*E. huxleyi*, *C. leptoporus* and *P. carterae*) that are common members of phytoplankton assemblages but originate from different lineages with contrasting ecologies. These species were acclimated to a range of $pCO_2$/pH reflecting not only oceanic carbon chemistry from glacial to interglacial times, but also the conditions projected for the year 2100 (IPCC, 2013). We assess their (1) photosynthetic and calcification responses to $CO_2$-induced OA using their PIC/POC ratio, (2) utilization of carbon species in calcification and photosynthesis using stable carbon isotopes, and (3) the ability to regulate the carbonate chemistry of the calcifying fluid using boron isotopes and B/Ca ratios as a pH proxy.
## 2. MATERIALS AND METHODS

### 2.1. Culture experiment

Three strains of different coccolithophore species *Emilia-huxleyi* (RCC 1216, a heavily calcified 'R' morphotype), *Calcidiscus leptoporus* (RCC 1129) and *Pleurochrysis carterae* (PCC156) were grown as dilute-batch cultures at 17 ± 1 °C in 0.2 µm (PES) filtered North-Sea seawater medium (Salinity ~ 33 PSU) that was enriched with nutrients (100 µM NO₃⁻; 6 µM PO₄³⁻) as well as vitamins and trace metals according to the F/2 recipe by Guillard and Ryther (1962). The three species were selected because of their generally different cell sizes, calcification levels and responses to OA (Meyer and Riebesell, 2015; Moheimani and Borowitzka, 2011). The algal cultures were grown under a 16:8h light:dark cycle with an irradiance of 150 ± 15 µmol photons m⁻² s⁻¹ (OSRAM daylight lamps Biolux 965) that was measured with a 4π-sensor (US-SQS/L, ULM 500; Walz, Effeltrich, Germany). Cultivation vessels were 2.5 L polycarbonate bottles (Nalgene; New York, USA) on automated roller tables to maintain suspension of cells. Before inoculating cells into the experiments, cells were pre-acclimated to experimental conditions and the respective *pCO₂* for at least 10 division cycles. Throughout the experiment, we refrained from using glass-made material to avoid a bleeding of boron compounds into the water that would interfere with analytical procedures. To adjust *pCO₂*, culture media were pre-aerated with humidified, 0.2 µm filtered air with adjusted *pCO₂* (180, 380, 750, 1000 µatm) that were created using a gas-flow controller (CGM2000; MCZ Umwelttechnik, Bad Nauheim, Germany), which mixed pure *CO₂* (AirLiquide, Düsseldorf, Germany) into *CO₂*-free air (*CO₂*-stripper; Domnick-Hunter, Willich, Germany). Target *pCO₂* values were monitored regularly with a non-dispersive infrared analyzer (LI6252, Li-Cor) and via the measured wet-chemical parameters.

Algae samplings were done 6–10 hours after the beginning of the light period, i.e., at midday with mean algal densities of 52000 mL⁻¹ (*E. huxleyi*), 4300 mL⁻¹ (*C. leptoporus*) and 4900 mL⁻¹ (*P. carterae*). The cell densities were kept intentionally low to prevent large drifts in carbonate chemistry while obtaining sufficient materials for elemental and isotopic analyses. Cell densities differed slightly between biological replicates at the time of sampling because of small deviations in the inoculation and/or growth rates. In few instances, cell densities exceeded target values leading to larger shifts in carbonate chemistry. The carbonate systems in the cultures were determined in the beginning and the end of the cultivation: The seawater pH (*pH*<sub>sw</sub>; in NBS scale) was assessed daily or every other day with a regularly calibrated handheld device (826 pH mobile device with an Aquatrode Plus; Metrohm, Filderstadt, Germany) and corrected for daily offsets by including measurements of a TRIS reference buffer (Dickson et al., 2007). The instrumental uncertainty given by the manufacturer is ± 0.001 pH unit. The pH reproducibility of each measurement was typically smaller than 0.003 pH unit (n = 3). The variation between the three biological

replicates was typically smaller than 0.03 pH units. The *pH*<sub>sw</sub> value reported in the figures and supplemental information is the average of the initial and the end pH measurements, and the uncertainty of the pH reported (1 SD) denotes the variation of the pH in the cultivation vessel during the experiment instead of the uncertainty of the pH measurements. DIC was assessed from sterile-filtered media with the colorimetric method developed by Stoll et al. (2001), using a QuAAtro autoanalyzer (Seal Analytical, Norderstedt, Germany). For completeness, also TA was assessed from linear Gran titration plots (Dickson, 1981), which were produced using an autosampler-coupled burette system (TitroLine alpha plus, Schott, Mainz, Germany). Both, DIC and TA were corrected for daily offsets by including certified reference materials (CRM) obtained from A. Dickson (Scripps Institution of Oceanography). The complete carbonate systems were calculated based on *pH*<sub>sw</sub> and dissolved inorganic carbon (DIC) using the CO2SYS package (Pierrot et al., 2006), as well as temperature, salinity and pressure (Hoppe et al., 2012). For all calculations, the concentrations of phosphate and silicate were assumed to be 6 and 1 µM, respectively. Equilibrium constants for carbonic acid were adapted from Dickson and Millero (1987), who refitted the data obtained by Mehrbach et al. (1973). For sulfuric acid, the dissociation constants described by Dickson, 1990 were used and total boron concentration reported by Lee et al. (2010) was used.

Based on our DIC data, we have shown that DIC drawdown was <152 µmol kg⁻¹, which translate into a theoretical nitrate and phosphate drawdown of 24 and 1.5 µM, respectively. Thus, the supplied nutrient levels of 100 µM nitrate and 6.6 µM phosphate must have remained sufficiently high, ruling out nutrient limitation.

### 2.2. Growth, elemental and isotopic analysis

Algal culture growth was monitored by daily cell counts with a particle counter (Multi-Sizer III, Beckman-Coulter, Fullerton, CA, USA), and specific growth rates (µ) were assessed from daily increments using the equation $\mu = (\ln(c_1) - \ln(c_0)) \Delta t^{-1}$, where c₀ and c₁ are the cell concentrations at two time points and Δt is the time interval in days.

For the determination of total particulate carbon (TPC), particulate organic carbon (POC), as well as the carbon isotopic signatures (δ¹³C), cultures were gently vacuum-filtered (–200 mbar relative to the atmosphere) onto pre-combusted (3 h at 500 °C) GF/F filters (Whatman, Maidstone, UK), which were dried at 50 °C and analyzed on an ANCA SL 20–20 stable isotope mass spectrometer (SerCon; Crewe, UK). Before the quantification of POC, the respective filters were soaked with 0.2 M HCl and dried again, to remove calcite. Particulate inorganic carbon (PIC) was assessed as the replicate-specific difference between TPC and POC. By multiplying elemental quotas with specific growth rates, the respective production rates could be assessed (Rokitta and Rost, 2012).

For the assessment of the boron isotopic signatures, trace elemental ratios and carbon isotopic signatures in coccolith calcite, cell cultures were concentrated by gentle filtration on polycarbonate membrane filters (47 mm
diameter, 1 µm pore size; Whatman® Nuclepore™, GE Healthcare, UK) and concentrated algal suspension was centrifuged (~10 min at 13.000g) to pellet cells. The supernatant was then discarded and the cell pellet was dried at 50 °C in an oven. Dry samples were packed and transferred to a clean lab for further cleaning and chemical treatments.

Before chemical analysis, the dry samples were first rinsed with Milli-Q water (MQ; 18 MΩ·cm) three times to remove remaining salt precipitates on the cells. About 100 µg of sample split was then prepared for stable isotope analysis. The δ¹³C of coccolith calcite was analyzed on a Finnigan MAT Kiel IV preparation device coupled directly to the inlet of a Finnigan MAT 253 triple collector isotope ratio mass spectrometer in the stable isotope lab in the Global Change Research Center, National Taiwan University, Taiwan. The reproducibility of the reference standard NBS-19 and an in-house carbonate standard MAB were better than ±0.09‰ (2 SD, n = 4) and ±0.15‰ (2 SD, n = 16), respectively.

Stable carbon isotopes of dissolved inorganic carbon (δ¹³C_DIC) were measured via a Finnigan MAT Delta Plus XL mass spectrometer in continuous flow mode connected to a Gas Bench with a CombiPAL autosampler in the Stable Isotope Lab in the Department of Geological and Atmospheric Sciences, Iowa State University, USA. Reference standards (NBS-18, NBS-19) were used for isotopic corrections, and to convert data to be relative to the Vienna PeeDee Belemnite (VPDB) value. At least one reference standard was used for every five samples. The combined uncertainty of analytical reproducibility and average correction factor for δ¹³C_DIC is ± 0.09‰ (VPDB).

Boron isotopic composition in coccolith samples was measured following the method from Liu et al. (2013, 2015). Micro-sublimation and total evaporation negative ion thermal ionization mass spectrometry (TE-NTIMS) techniques were applied. The methods were used due to limited amount of boron available and to avoid extra contamination introduced during purification. In brief, less than 50 µL of standard or sample solution with [B] = ~750 ppb was loaded onto a cap of conical bottom Savillex PFA vial for 15 hours of sublimation. After purification, 2 µL of 30% ultrapure H₂O₂ was added to remove additional organic materials and eliminate potential interferences of CN when running isotope analysis. 1 µL of standard or sample was then loaded onto a refined Re-filament after 1 µL of boron free seawater (BFSW) was loaded. Boron isotopes were analyzed using a Thermo Fisher Triton multicollector thermal ionization mass spectrometer operating in negative ion mode (NTIMS) at the Institute of Earth Sciences, Academia Sinica, Taiwan. The same amount of carbonate standard JCp-1 was processed using the same protocols as the coccolith samples to monitor the robustness of the method. Boron isotopic composition in seawater and coccolith samples were reported as δ¹¹B, where

$$ \delta^{11}\text{B} = \left[ \frac{(^{11}\text{B}/^{10}\text{B})_{\text{sample}}}{(^{11}\text{B}/^{10}\text{B})_{\text{SRM 951a}}} - 1 \right] \times 1000 \ (\%) \quad (1) $$

with ¹¹B/¹⁰B for boric acid standard SRM 951a was 4.034 5 ± 0.0084‰ (2 SD, n = 24).

Boron isotopic composition in seawater medium was analyzed on a Finnigan Neptune multicollector-inductively coupled plasma mass spectrometer (MC-ICPMS) at IFREMER, France. The reproducibility of δ¹¹B determinations in seawater analyzed on MC-ICPMS was ±0.58‰ (2 SD, n = 12) and the reproducibility of δ¹¹B determinations in international coral standard JCp-1 analyzed on NTIMS were ±1.9‰ (2 SD, n = 23) and 1.66‰ (2 SD, n = 38), before and after replacing an ion source, respectively.

There are two dominant dissolved boron species in seawater, boric acid and the borate ion. The relative proportion of these two species is pH-dependent and the reaction can be described by the following reaction equation:

$$ B(OH)_3 + H_2O \rightleftharpoons B(OH)_4^- + H^+ \quad (2) $$

The isotope exchange between two species follows the reaction below:

$$ ^{10}\text{B}(\text{OH})_3 + ^{11}\text{B}(\text{OH})_4^- \rightleftharpoons ^{11}\text{B}(\text{OH})_3 + ^{10}\text{B}(\text{OH})_4^- \quad (3) $$

where ¹¹B is enriched in boric acid compared to the borate ion. The boron isotopic signature can therefore be determined after combining the reactions above, with a fractionation factor (α) between the two species of 1.0272 (Klochko et al., 2006). Although more recent studies have derived a range of fractionation factors based on theoretical calculations (1.026–1.028 (Nir et al., 2015; Rustad et al., 2010)), the uncertainties of these are larger than the value obtained by Klochko et al. (2006). Therefore, α = 1.0272 is widely used by the community and also applied in the present study.

It has been hypothesized and also observed that marine carbonates primarily incorporate B(OH)₄⁻ into the carbonate structure during their shell growth, and therefore seawater pH not only dictates the amount of B(OH)₄⁻ in seawater but also the isotopic composition of boron in marine carbonates. In this case, the seawater pH can be derived based on the equation:

$$ \text{pH} = \text{pK}_b - \log \left( - \frac{\delta^{11}\text{B}_{\text{sw}} - \delta^{11}\text{B}_{\text{carbonate}}}{\delta^{11}\text{B}_{\text{sw}} - \alpha\delta^{11}\text{B}_{\text{carbonate}} - 1000(\alpha - 1)} \right) \quad (4) $$

Marine calcifiers precipitate their calcium carbonate skeletons/shells from an enclosed or semi-enclosed compartment, where the pH can be regulated and thus potentially differ from the ambient bulk seawater. Instead of directly reflecting ambient seawater pH, the boron isotopic signature is therefore dictated by the pH of the calcification site fluid pH, and can be calculated based on the adjusted equation:

$$ \text{pH}_{\text{CF}} = \text{pK}_{\text{b}} - \log \left( - \frac{\delta^{11}\text{B}_{\text{CF}} - \delta^{11}\text{B}_{\text{carbonate}}}{\delta^{11}\text{B}_{\text{CF}} - \alpha\delta^{11}\text{B}_{\text{carbonate}} - 1000(\alpha - 1)} \right) \quad (5) $$

It has been proposed that boric acid is the only dissolved boron species that permeates membranes (Stoll et al., 2012), based on findings of nearly identical boron concentrations in external and internal medium of plant cells (Brown and
Hu, 1993; Seresinhe and Oertli, 1991), suggesting a passive transport of boron. Alternatively, although energetically costly, it has been demonstrated that active transport of borate ion via transporters BOR or NIP is possible during boron deficiency (Takano et al., 2005; Takano et al., 2002; Tanaka and Fujiwara, 2008). Because boron concentration in seawater is ~4-fold higher than that in soil, Stoll et al. (2012) therefore suggest passive diffusion of non-charged boric acid as the principle boron uptake mechanism in coccolithophores. Because of the high permeability of lipid bilayers to boric acid, the boron concentration is thought to reach close-to-equilibrium levels across cell membranes (Dordas and Brown, 2000). However, boron isotopic composition of the coastal coccolithophore species *Ochrosphaera neapolitana* shows lower values than predicted based on the model that assumes that boric acid exclusively enters the coccolith vesicle (Liu et al., 2018). Whether boric acid is the only species that can be transported across coccolithophore cell membrane and vesicle is still under investigation. Therefore, here we test both scenarios, in which either the boron isotopic signature is the same in the calcifying fluid and the seawater ($\delta^{11}\text{B}_{\text{CF}} = \delta^{11}\text{B}_{\text{SW}}$), or the boron isotopic signature of the calcifying fluid carries the isotopic signal of boric acid in the seawater ($\delta^{11}\text{B}_{\text{CF}} = \delta^{11}\text{B}_{\text{seawater boric acid}}$).

The coccolith B/Ca analysis were conducted on a high resolution ICP-MS ELEMENT XR at the Institute of Earth Sciences, Academia Sinica. The reproducibility of B/Ca is generally better than ±2% (2 RSD, relative standard deviation) for B/Ca values greater than 200 µmol/mol, and still ±12% (2 RSD) for B/Ca values smaller than 25 µmol/mol. To avoid interference of B from the organic matrix, an improved oxidation and cleaning protocol has been applied. The validation of the stepwise cleaning protocol is detailed in the supplementary materials (S1). In brief, samples were treated overnight with 15% H₂O₂ (pH adjusted to >8 by adding NaOH) before dissolving the coccolith carbonate. Although it has been suggested that a combined reductive and oxidation cleaning protocols are more efficient to remove organic matrix that bonded with coccoliths (Blanco-Ameijeiras et al., 2012; Henehan, 2015), it is also known that reductive cleaning protocols can result in substantial loss of sample materials. As complex cleaning procedures could risk introducing higher blanks, we applied a cleaning protocol using a single but stronger oxidation reaction. Based on tests with different concentrations of H₂O₂ (Fig. S1), 15% solutions were applied to our samples. The calcium running concentration of the standards and solution was 2 mM.

To further evaluate the potential interferences of organic remnants, we also monitored other trace elemental ratios. Particularly, Fe/Ca and P/Ca ratios of coccoliths have been used to assess the effectiveness of cleaning coccolith samples (Blanco-Ameijeiras et al., 2012). Although we do not have P/Ca results available, we do have Fe/Ca ratios monitored for both, samples of the original experiment as well as from the additional cleaning test, allowing us to further assess the cleanness of the sample.

Our results suggest that high Fe/Ca ratios do not significantly bias B/Ca ratios if the ratios are lower than 1000 µmol/mol (df = 8, *P* = 0.08, CI = 95%; Fig. S3). This was because the B/Ca values were nearly identical based on the current analytical resolution, which is 12% for 25 µmol/mol of B/Ca. This Fe/Ca level is also below the range of coccolithophore Fe/Ca after applying the optimal cleaning protocol on the cultured *E. huxleyi* and *C. leptoporus* reported in Blanco-Ameijeiras et al. (2012) (Fig. S2(d)). We therefore set 1000 µmol/mol of Fe/Ca as additional filter threshold to re-assess the data set of our pH control experiment. Based on our described screening procedure, we removed one data point from *C. leptoporus* and two data points from *P. carterae*, respectively (Fig. S4). We find that our cleaning methods give B/Ca values that are comparable to those reported for *E. huxleyi* by Diez Fernández et al. (2015) and Henehan (2015) (Fig. S2). In addition, the vast majority of Fe/Ca data fall within the range reported in Blanco-Ameijeiras et al. (2012) for the combined reductive and oxidative cleaning. Thus, we overall conclude that our B/Ca data is comparable to that reported in previous publications (Blanco-Ameijeiras et al., 2012; Diez Fernández et al., 2015; Henehan, 2015), and thereby we have confidence in our boron isotope data.

In addition to the aforementioned analysis, about 1 mL of culture was filtered onto 25 mm polycarbonate filters (Whatman) and sputtered for scanning electron microscopy (SEM) analysis (Figs. S5–S7). The overall sample processing protocols are illustrated in Fig. S8.

## 2.3. Statistical evaluation

All linear regressions were calculated with least squares method to check for statistically significant trends with respect to seawater pH treatments. Trends were deemed significant when *P* values were ≤0.05. Propagated errors of boron-inferred pH<sub>CF</sub> were calculated via Monte Carlo simulations.

# 3. RESULTS

## 3.1. Growth rates and PIC/POC variations

Coccolithophore specific growth rates, PIC/POC ratios, POC and PIC quotas as well as POC and PIC productions are plotted against culture seawater pH in Fig. 1. There is no statistically significant trend in specific growth rates, PIC/POC ratios, or carbon production rates in any of the tested species. Average cell growth rates of *E. huxleyi*, *C. leptoporus*, and *P. carterae* are 0.83, 0.36, and 0.83 d⁻¹, respectively (Fig. 1(a)). The specific growth rates of *P. carterae* increased during the last two days before harvesting, resulting in large standard deviations for the growth rate estimates in this species. Nevertheless, a positive correlation is found between the PIC quota of *E. huxleyi* and seawater pH (R² = 0.40, *P* < 0.03), albeit weak. The PIC/POC ratios of the three coccolithophore species do not show consistent responses to pH<sub>SW</sub> (Fig. 1(b)), but correlate with diverse
<figure><img src="image_2.png" /><figcaption>Fig. 1. The growth, PIC/POC, POC quota, POC production, PIC quota, and PIC production as a function of pH<sub>SW</sub>. The (a) specific growth (μ), (b) PIC/POC ratios, (c) POC quota (pg cell<sup>-1</sup>), (d) POC production (pg cell<sup>-1</sup> day<sup>-1</sup>), (e) PIC quota (pg cell<sup>-1</sup>), and (f) PIC production (pg cell<sup>-1</sup> day<sup>-1</sup>) of coccolithophore species *E. huxleyi*, *C. leptoporus*, and *P. carterae* are shown in red circles, blue squares, and green diamonds, respectively. The error bars denote one standard deviation (SD) of the analysis or propagated uncertainty of the calculation.</figcaption></figure>

biological parameters. In *E. huxleyi*, PIC/POC shows a lar-
ger variation in the low pH treatments. The PIC/POC ratio
in *C. leptoporus* is negatively correlated to POC quota
(R² = 0.42, P = 0.02), but is positively correlated to the
PIC production rate (R² = 0.68, P < 0.01). The PIC/POC
ratio in *P. carterae* is positively correlated to the PIC quota
(R² = 0.72, P < 0.01).

The PIC/POC ratios of the three species show distinc-
tively different values, with the values of *E. huxleyi* being
the highest, followed by *C. leptoporus*, and the PIC/POC
ratios of *P. carterae* being the lowest. Note that the PIC/
POC ratios of *C. leptoporus*, which is thought to be heavily
calcified, are lower than expected based on literature values
(Diner et al., 2015; Fiorini et al., 2011; Hermoso et al.,
2014; Langer et al., 2006). By checking the SEM images
(see Supplementary material S2, and Figs. S5–S7), we
found a major presence of holococcoliths instead of the typ-
ical heterococcoliths of *C. leptoporus* that are more heavily
calcified, which explains the relatively low PIC/POC values
in our study. Although variations exist within the growth
rates and PIC/POC ratios in each treatment of the experi-
ment, the results demonstrate that the selected species cover
different ranges of growth rates and PIC/POC ratios. More-
over, PIC/POC ratios do not systematically follow a certain

response pattern that holds true across these taxonomically
different coccolithophores.

**3.2. Carbon and boron isotope results**

The isotopic composition of the POC and PIC were nor-
malized to the carbon isotopic composition of the culture
seawater DIC in order to remove signals derived from
differences in the inorganic carbon source (e.g., from fossil
CO2 introduced by bubbling). Therefore, the carbon
isotope results are reported with a Δ notation. Here, we
define that Δ¹³Cₚool = δ¹³Cₚool - δ¹³Cₔₗc, and Δ¹³Cₚic =
δ¹³Cₚic - δ¹³Cₔₗc. The Δ¹³Cₚool and Δ¹³Cₚic values of the
three species range between -30‰ and -10‰, and between
-2.5‰ and 2‰, respectively (Fig. 2). Within each species,
we found similar pH-dependencies between the Δ¹³Cₚool
and Δ¹³Cₚic. In *E. huxleyi*, there is no trend between seawa-
ter pH and carbon isotopes in either Δ¹³Cₚool and Δ¹³Cₚic.
In *C. leptoporus*, Δ¹³Cₚool and Δ¹³Cₚic are threshold nega-
tively and negatively correlated to the seawater pH respec-
tively (R² = 0.67, P < 0.01 and R² = 0.98, P < 0.01). In *P.
carterae*, Δ¹³Cₚool and Δ¹³Cₚic values show positive corre-
lation with the seawater pH (R² = 0.74, P < 0.01 and
R² = 0.33, P = 0.06, respectively).
<figure><img src="image_3.png" /><figcaption>Fig. 2. Stable carbon and boron isotope results as a function of seawater pH (a) and (b) show the carbon isotopic composition of particulate organic and inorganic carbon, respectively. (c) and (d) are boron isotopic compositions and the pH offsets between calcification site fluid and the ambient seawater, respectively. *E. huxleyi*, *C. leptoporus*, and *P. carterae* are shown in red circles, blue squares, and green diamonds, respectively. The error bars of carbon and boron isotopes and calculated pH offsets represent 2 SD of the results, while the horizontal error bars show the 1 SD of the pH values. Note that the 2 SD of boron isotopes here were obtained from the long-term reproducibility of international coral standard JCp-1 because the coccolith material were limited for replications for most of the sample. Gray lines are theoretical borate fractionation curves (α = 1.0272; Klochko et al., 2006). Solid red, blue and green lines denote statistically significant linear/nonlinear trends of the data set (Tables S1–S5). Dotted lines indicate weaker significance in linear trends (0.05 < *p* < 0.1).</figcaption></figure>

Boron isotopic compositions range between 15‰ and 35‰ (Fig. 2(c)). The δ¹¹B values of *E. huxleyi* show little change across the different pH treatments, while the δ¹¹B values of *C. leptoporus* generally display larger variability even within the same treatments. For *P. carterae*, we found that δ¹¹B values were positively correlated to seawater pH (R² = 0.64, *P* < 0.01). These boron isotopic data can be translated to a pH~CF~ range between 7.8 and 9.5 (Fig. 2(c), and Fig. 3(a)), based on the assumption that δ¹¹B~CF~ = δ¹¹B~SW~ and that only borate ions are incorporated into the calcite structure. To further evaluate the abilities of coccolithophores to regulate the pH~CF~, we calculate the ΔpH values and plot them against pH~SW~ (Fig. 2(d)). By doing so, it becomes clear that *E. huxleyi* and *C. leptoporus* elevate their pH~CF~ by about 1.0 pH unit at pH~SW~ 7.8, whereas they only elevated their pH~CF~ by less than 0.5 pH units at pH~SW~ > 8.3. In contrast, *P. carterae* shows no trend in ΔpH, i.e., they elevate the pH~CF~ by about 0.6 units independent of the external pH~SW~. Note that the assumption of the canonical boron systematics applied here might not hold true for all marine organisms. If the alternative hypothesis is used, i.e., that only boric acid is permeable to cell membranes (Stoll et al., 2012), calculations yield slightly lower pH~CF~ values (Fig. 3(b)), resulting in even more pronounced species-specific differences in the relationship between ΔpH and pH~SW~ (Fig. 3(c)). Importantly, both models for interpreting coccolith δ¹¹B

indicate that pH~CF~ levels are all at least about 0.3 pH unit above the ambient pH~SW~.

### 3.3. B/Ca

A suite of trace elements was measured, allowing us to report ratios between these trace elements and specifically calcium with the intent to use these ratios as further indicators of biological processes. The complete data can be found in the **supplementary materials** while here we only focus our analyses on the B/Ca ratio. The cleaning and analytical protocols were optimized on *E. huxleyi* (Fig. S1, 2, and **supplementary material S1**) and then applied to all species. To further evaluate the cleanness of the sample, we applied an additional quality filter to the dataset, and exclude the results if Fe/Ca ratios are higher than 1000 µmol/mol which we judged to indicate organic and potentially iron oxide contamination (see SI for in depth justification). Generally, B/Ca ratios are different among species. In *E. huxleyi*, the B/Ca ratios are constantly below 25 µmol/mol, in *C. leptoporus*, ratios are close to 100 µmol/mol, but in *P. carterae*, determined ratios are between 14 and 150 µmol/mol (Fig. 4(a)). Regarding the latter, the B/Ca ratios exhibit high variability of about 50 µmol/mol within a treatment, and over 50 µmol/mol between treatments. Overall, there is no significant trend in either of the species against culture seawater pH.
<figure><img src="image_4.png" /><figcaption>Fig. 3. The δ¹¹B-inferred pH~CF~ values of the three coccolithophore species and their pH offsets to the culture seawater pH. (a) and (b) show the pH~CF~ values that calculated under two different boron transport scenarios. (a) shows the results assuming that both boric acid and the borate ion can be transported through the coccolithophore cell membrane, and the δ¹¹B value in the calcification site is in isotope equilibrium with the culture seawater δ¹¹B value. (b) shows the results assuming only boric acid is permeable to the coccolithophore cell membrane, so the total boron concentration and the δ¹¹B value of the calcification fluid varies depending on the boric acid concentration and its δ¹¹B value of the culture medium under the control pH conditions. The solid lines show statistically significant linear correlations of the data in the panels.</figcaption></figure>

# 4. DISCUSSION

## 4.1. Growth, elemental quotas and production rates

In *E. huxleyi*, the applied pH treatments did not significantly affect growth rates or quotas of POC and PIC (Fig. 1(a, c, e)). Consequently, derived PIC/POC ratios did not show significant trends in response to OA (Fig. 1(b)). However, the quotas of PIC and POC slightly decreased in more acidic treatments in *E. huxleyi*. This is in line with previous observations for this strain of *E. huxleyi*, especially for the here applied growth-saturating light levels (Kottmeier et al., 2016b; Rokitta and Rost, 2012). Reported PIC production rates showed decreasing or no trend with pH, depending on different culture approaches and conditions (e.g., light regimes) that significantly affect experimental outcomes.

In *C. leptopus* and *P. carterae*, growth rates were slightly decreased in the lowest pH scenarios, but when con-

sidering the whole range of applied pH and the inherent variability of the data, no significant trends in growth rates as well as in quotas and production rates of POC and PIC could be recognized by the applied tests. In contrast to the slightly declining trend in PIC and POC quota observed in *E. huxleyi*, the PIC and POC quota of *C. leptopus* and *P. carterae* are more resistant to low pH or are even slightly increased under the most acidic condition. The above results suggest that although the PIC/POC ratios of all three species show no clear response to OA, this strain of *E. huxleyi* might still be negatively impacted as both, inorganic and organic production decrease under low seawater pH.

## 4.2. Carbon isotopic composition and implications for inorganic carbon sources

*Δ¹³C~POC~* and *Δ¹³C~PIC~* behave similarly, but show species-specific trends depending on seawater pH (Fig. 2
<figure><img src="image_5.png" /><figcaption>Fig. 4. The B/Ca compositions of coccolithophores as a function of seawater pH (a) The B/Ca of *E. huxleyi*, *C. leptoporus*, and *P. carterae* are shown in red circles, blue squares, and green diamonds, respectively.</figcaption></figure>

(a, b)), being either invariant (as in *E. huxleyi*), decreasing (as in *C. leptoporus*) or increasing (as in *P. carterae*). These differences in trends among species have been proposed to originate mainly from the interplay of species-specific controls on influx of CO₂ vs. HCO₃⁻, CO₂ leakage from chloroplasts or the cytosol, as well as Rayleigh fractionation resulting from the interplay of RubisCO fractionation and leakage (McClelland et al., 2017). Disequilibria between CO₂ and HCO₃⁻ due to changes of vesicle pH or unidirectional “CA-like” processes (Eichner et al., 2015; Wilkes and Pearson, 2019) can also potentially determine how much the intrinsic fractionation of RubisCO can manifest in the isotopic value of POC, and in case of coccolithophores also of PIC (Bolton and Stoll, 2013; Holtz et al., 2017). In the following, we discuss the potential explanations for each species.

Most intuitive is the interpretation of the composition of P. carterae: Δ¹³CₚOC values increase with increasing pH (Fig. 2(a, b)), suggesting higher CO₂ usage for photosynthesis under lower pH, which is in line with the current understanding of algal CO₂ concentrating mechanisms (Giordano et al., 2005; Kottmeier et al., 2016b; Reinfelder, 2011). The Δ¹³CₚIC values are close to zero and display only an insignificant and numerically small trend with seawater pH (R² = 0.33, P = 0.06). Even though the observed deviation from zero could result from vital effects, e.g., an imprint from RubisCO on the internal isotope signal (McClelland et al., 2017), this value is yet perfectly in line with the notion that calcification does not fractionate strongly against its primary inorganic carbon source HCO₃⁻ (Rost et al., 2002; Sikes et al., 1980). The here observed trends towards higher Δ¹³CₚIC and Δ¹³CₚOC values with increasing seawater pH have also been reported for another coastal coccolithophore, *O. neapolitana* (Liu et al., 2018), although those values were ~12‰ heavier (Fig. 5). Despite this offset, which cannot be explained with changes in carbon sources, the similar shifts of Δ¹³CₚIC and Δ¹³CₚOC with seawater pH suggest that both coastal coccolithophore species increasingly utilize more CO₂ under more acidic conditions.

For *E. huxleyi*, the Δ¹³CₚOC and Δ¹³CₚIC suggest major utilization of HCO₃⁻ for both, biomass buildup and calcification, which has been observed before (e.g., Kottmeier et al. (2016a, 2016b); Rokitta and Rost (2012)). The lack of pH-dependent trends, i.e., the comparably stable Δ¹³CₚOC and Δ¹³CₚIC values indicate that changing carbon sources and changing leakage cancel each other out in *E. huxleyi* under the here applied growth conditions. There are fundamental uncertainties, however, regarding the fractionation behavior of the prime carbon fixing enzyme RubisCO in *E. huxleyi*, which hinder a more detailed discussion of the data (Boller et al., 2011).

Based on the Δ¹³CₚOC, *C. leptoporus* seems to primarily use HCO₃⁻ in all pH scenarios, similar to what has been observed in *E. huxleyi*. Remarkably, both Δ¹³CₚOC and Δ¹³CₚIC decrease when seawater pH increases, suggesting an apparently ceasing contribution of HCO₃⁻ to photosynthesis and calcification. This, however, contradicts our understanding of inorganic carbon transport and its pH/CO₂-dependent regulation (Reinfelder, 2011). Such patterns can thus only be explained by a significantly higher leakage over the plasmalemma under high pH that overcompensates the signal originating from the likely heavier carbon source under these conditions. Such a higher leakage can result from the steeper outward CO₂ gradient under high pH (i.e., low [CO₂]) and higher HCO₃⁻ pumping that result from the alleviation of H⁺ inhibition (Kottmeier et al., 2016a).

### 4.3. Boron isotopic composition and its implication for calcification fluid pH

The boron isotope composition of the investigated coccolithophores show large variations in the experiments, spanning from 17‰ to 36‰ (Fig. 2(c)). *E. huxleyi* shows relatively constant values at about 25‰, which is in line
<figure><img src="image_6.png" /><figcaption>Fig. 5. Comparisons of stable carbon isotopes of the coccolithophore species examined in this study to other coccolithophore results from literature. (a) $\Delta^{13}C_{POC}$ and (b) $\Delta^{13}C_{PIC}$ values vs. atmospheric $pCO_2$ are plotted.</figcaption></figure>

with reported $\delta^{11}B$ value ranging from 18‰ to 25‰ (Henehan, 2015). *C. leptoporus* exhibited $\delta^{11}B$ values between 19‰ to 35‰. For both species, no significant trend with decreasing seawater pH could be determined, which is consistent to previous findings in other coccolithophore species (Liu et al., 2018). *P. carterae* showed values from 17 to 36‰ that exhibit a significant correlation with seawater pH across the treatments. Absolute $\delta^{11}B$ values are different from those observed in *O. neapolitana* (Liu et al., 2018) due to the differences in the culture seawater $\delta^{11}B$ values. However, the range of $\delta^{11}B$ values derived in this study is comparable with these previous works, showing fractionation of about 15‰ to 22‰ with regard to the culture seawater $\delta^{11}B$ values (Henehan, 2015; Liu et al., 2018). Some of the $\delta^{11}B$ values of *C. leptoporus* and *P. carterae* determined here are about 5‰ to 10‰ higher than in those of *E. huxleyi* and *O. neapolitana*. These observed differences among species and deviations between studies have to be attributed to species-specific regulation of vesicle pH or, as an alternative hypothesis, they represent differences in uptake and transport of boron to the coccolith vesicle (see following sections).

Using the canonical systematics of the $\delta^{11}B$ proxy applications (Hemming and Hanson, 1992; Zeebe and Wolf-

Gladrow, 2001) yields $\delta^{11}B$ inferred $pH_{CF}$ values ranging between 7.8 and 9.5 for the investigated coccolithophores (Fig. 2(c)). These findings reflect that all three species increase $pH_{CF}$ by at least 0.5 pH units, potentially up to ~1.5 pH units, above the surrounding $pH_{SW}$ in almost all culture scenarios (Fig. 2(d)) to facilitate calcite precipitation. This underlines that those species are principally able to control their internal chemistry and overcome external pH levels to a considerable extent.

*E. huxleyi* and *C. leptoporus* experience little effect of $pH_{SW}$ on their $\delta^{11}B$ signal (Fig. 2(c)). Apparently, they sustain relatively constant $pH_{CF}$ of about 8.6 to 9.0 (Fig. 3(a)) to facilitate calcite precipitation (Fig. 1(e)) in all applied pH treatments, which is in good agreement with previous studies on *E. huxleyi* and *O. neapolitana* (Henehan, 2015; Liu et al., 2018). Hence, depending on external $pH_{SW}$, regulation of $pH_{CF}$ is required to different extents, i.e., less effort under high pH. In contrast, *P. carterae* reveals a significant positive trend of increasing $\delta^{11}B$ with $pH_{SW}$ (Fig. 2(c)), which implies that $pH_{CF}$ (Fig. 3(a)) is kept at a constant pH offset of ~0.6 pH units with respect to the surrounding seawater (Fig. 2(d)). A possible explanation is that *P. carterae*'s calcification process is based on efficient vesicular $Ca^{2+}$ (and potentially dissolved boron) delivery and is
strongly supported by the prominent coccolithosomes, which contain an organic polysaccharide matrix used for controlling calcite crystal growth (Holtz et al., 2013; Marsh and Dickinson, 1997). This molecular and subcellular control over coccolith formation may lower the need for strong pH elevation to achieve calcite precipitation conditions.

Additionally, we can evaluate the potential DIC<sub>CF</sub> levels of the three coccolithophore species using both δ<sup>11</sup>B and B/Ca for the calculation. Assuming a fixed minimum boron partition coefficient (K<sub>D</sub>) of 0.0004 (Sanyal et al., 2000; Stoll et al., 2012), we derived that the DIC<sub>CF</sub> in *E. huxleyi* is about 2–3 times higher than the DIC<sub>CF</sub> in *C. leptoporus* and *P. carterae* (Fig. S9(b), Eq. (S1)). The calculated DIC<sub>CF</sub> differences will be larger if we assume that the borate ion primarily competes with CO<sub>3</sub><sup>2-</sup> instead of HCO<sub>3</sub><sup>-</sup> when forming coccolith calcite (Fig. S9(c); see more detailed discussions on the potential control on the coccolith B/Ca in *supplementary materials S2*). Irrespective of how DIC<sub>CF</sub> is calculated, for *E. huxleyi* and *C. leptoporus*, data suggest an interesting anticorrelation between achieved DIC<sub>CF</sub> and pH<sub>CF</sub>: *E. huxleyi*, a small cell, elevates DIC<sub>CF</sub> 2–3 times higher than *C. leptoporus*, but achieves a lower pH<sub>CF</sub>. The larger *C. leptoporus*, in turn, achieves a comparably low DIC<sub>CF</sub>, but the highest pH<sub>CF</sub> in our dataset. This way, the two species apparently create calcite-precipitating conditions in different ways. Given that *P. carterae*'s mode of calcification appears fundamentally different from the two aforementioned species, our data underline the high diversity in species-specific calcification mechanisms that were (re-)invented among the coccolithophores multiple times along their natural history (De Vargas et al., 2007).

### 4.4. Evaluation of assumptions on the δ<sup>11</sup>B proxy

The application of the canonical δ<sup>11</sup>B proxy for pH<sub>CF</sub> in coccolithophores was based on several assumptions, most importantly, that only borate ions are incorporated into the coccolith calcite. Although recent NMR studies have revealed the complexity of boron incorporation into biogenic carbonates (Klochko et al., 2009; Noireaux et al., 2015; Rollion-Bard et al., 2011), the isotope signatures of studied carbonates suggest the variable proportions of trigonal B in the carbonates might be due to re-coordination of the tetrahedral B(OH)<sub>4</sub><sup>-</sup>. An independent study with Near-Edge X-ray Absorption Fine Structure further provides some evidence that the trigonally coordinated BO<sub>3</sub><sup>-</sup> exists in calcite structure as a result of re-coordination and therefore strengthens the use of B as a pH proxy (Branson et al., 2015). However, it has also been suggested that both boric acid and borate ion incorporate into calcite (Cusack et al., 2015; Uchikawa et al., 2015). This can, at least for coralline red algae (Liu et al., 2020; Sutton et al., 2018), much better explain measured δ<sup>11</sup>B values that are otherwise too high in some cases (>40‰) to be translated to a corresponding pH<sub>CF</sub>. In contrast, in our study the lowest measured δ<sup>11</sup>B value of ~17.5‰ from coccoliths is quite close to the lower limit of the theoretical seawater borate δ<sup>11</sup>B curve calculated for the culture conditions. This would correspond to

a maximum of ~10% of total boron being recruited from the boric acid pool for the process of coccolith formation.

Alternatively, we have calculated the pH<sub>CF</sub>, as classically done for marine calcifiers, i.e., by assuming that the total boron isotopic composition of the calcification vesicle is in isotope equilibrium to the ambient seawater (δ<sup>11</sup>B<sub>CF</sub> = δ<sup>11</sup>B<sub>SW</sub>), and that only borate ion will be incorporated into coccolith calcite. We found that the pH<sub>CF</sub> can vary across the pH treatments by as much as ~1.5 pH units (Fig. 3 (a)), sometimes reaching as high as ~9.5 in two of the species. It has been suggested that photosynthetic activities of organisms or the symbionts, as well as active proton regulation, can potentially raise the pH of the calcifying fluid to above 8.6 or even up to pH 9.3 in some organisms such as corals and coralline red alga (Ries, 2011; Short et al., 2015). Still, the absolute pH<sub>CF</sub> values determined when applying this assumption are generally higher than reported observations of cytosolic or vesicle pH in *E. huxleyi* based on fluorescent dyes (Anning et al., 1996; Suffrian et al., 2011).

Based on observations in terrestrial plants and some animals (Dordas and Brown, 2000; Tanaka and Fujiwara, 2008), it has alternatively been proposed that boric acid is the primary dissolved boron source that crosses the cell membrane of coccolithophores by passive diffusion (Stoll et al., 2012). Taking this as an alternative hypothesis, the calculated pH<sub>CF</sub> values are shifted down to mostly below pH 9.0 across the different treatments (Fig. 3(b)). Moreover, the range of the calculated pH<sub>CF</sub> values would be about 0.2 units narrower and result in an increased sensitivity of pH<sub>CF</sub> across the pH treatments for the three species (Fig. 3(c)). In this case, more of the determined pH<sub>CF</sub> values would overlap with the independent determinations using pH sensitive dyes (Anning et al., 1996).

The partition coefficient of B to inorganic calcite has been estimated to be about 0.001 in an experiment with seeded crystal growth (Sanyal et al., 2000), but the solution chemistry was not fully documented and may therefore bear uncertainties. A lower estimation of K<sub>D</sub> (0.0004–0.0005) has been proposed with the assumption that the seeded crystals were re-equilibrated with the experimental solution (Stoll et al., 2012). This lower estimation is similar to what has been found for foraminifera grown in seawater (Rollion-Bard and Erez, 2010). Thus, Stoll et al. (2012) assumed a fixed K<sub>D</sub> = 0.0005 for coccolithophores, and modeled the expected B/Ca and δ<sup>11</sup>B values of coccolith calcite under fixed or various pH<sub>CF</sub> and vesicle DIC conditions (revised in Fig. 6). Here, we also compared our results to their model and found that only B/Ca ratios of *E. huxleyi* might fit in their “constant pH<sub>CF</sub>” scenario (Fig. 6(a)). However, note that in the model, the δ<sup>11</sup>B value of the calcification fluid will still change under different pH<sub>SW</sub> due to the changes of δ<sup>11</sup>B of boric acid transported to the cell.

In contrast to the model mentioned above, the δ<sup>11</sup>B values we measured for *E. huxleyi* suggest a near identical δ<sup>11</sup>B value of the calcification fluid, and thus contradict the assumption of diffusive boric acid uptake (Fig. 6(d, e)). Additionally, the higher B/Ca ratios measured for *C. leptoporus* and *P. carterae* (Fig. 4) suggest that K<sub>D</sub> must be higher and/or variable to correctly describe calcite produc-
<figure><img src="image_7.png" /><figcaption>Fig. 6. Comparisons between the observed coccolithophore B/Ca and δ¹¹B and the model proposed by Stoll et al. (2012). Lines in (a) and (d) show predictions of coccolith B/Ca and δ¹¹B values for different pH~CF~ assuming pH is fixed in the coccolithophore calcification vesicle despite changing pH~SW~. Lines in (b) and (e) show predictions of coccolith B/Ca and δ¹¹B values when vesicle pH is variable but DIC is fixed. Lines in (c) depict coccolith B/Ca and δ¹¹B values when both vesicle pH and DIC are variable. The shaded areas show the calculated B/Ca range of the pH interval. Data of present study are shown in solid symbols, with 1 RSD, 2SD, and 1 SD error bars in the B/Ca, δ¹¹B, and pH~SW~ values, respectively. Note that the pH~SW~ values are all presented in total scale in this figure.</figcaption></figure>

tion in these coccolithophore species. Although the δ¹¹B-inferred pH~CF~ values in the second scenario (involving diffusive boric acid uptake) are lower and closer to what has been estimated from independent techniques (Anning et al., 1996), the above evaluations suggest that the assumptions of this scenario do not adequately describe the obtained data.

Although the actual boron uptake mechanism of coccolithophores is still unresolved, our boron isotope data pro-

vide more evidence that boric acid might not be the sole boron species that can pass through the cell membrane. Furthermore, our results reflect a general pH upregulation strategy among coccolithophore species under OA conditions. In addition, the experimental setting helps to evaluate the potential use of coccolith δ¹¹B as a paleo pH proxy. Based on our results, in only one of the four species examined, the coccolith δ¹¹B is sensitive to the changes of environmental seawater pH (Liu et al., 2018; this study)
suggesting coccolith calcite may not be an attractive target for paleo-ocean pH reconstruction. The systematic decline of δ¹¹B in *P. carterae* may indicate that this species could function as a potential archive for the use of paleo seawater pH reconstruction. However, that being said, the large variation within a treatment, the little amount of calcite as well as limited preservation in sediments likely hinders the applicability. Furthermore, an improvement of analytical precision for low boron concentration samples is required to better calibrate this potential proxy tool in the future.

## 5. CONCLUSION

Our results show highly variable signatures of incorporated boron and carbon isotopes, and B/Ca among three coccolithophore species across a wide range of seawater pH. The different isotopic compositions of organic and inorganic particulate carbon indicate different carbon utilization strategies among these species. Species-specific strategies are also evident from the boron isotopes and B/Ca data measured in the calcite. Our results highlight variability in the ability to regulate calcification fluid pH and to control carbonate chemistry in response to ocean acidification. Furthermore, our data demonstrates that the investigated species follow different mechanisms of calcite production and the associated carbon utilization. These findings are independent of underlying assumptions, and shed light on limitations of current models that aim to describe coccolithophores’ boron uptake mechanisms, and the partition of boron species during coccolithophore calcification. Experiments like ours will add to our understanding of ion transport processes and how they may differ between species (Brownlee et al., 2020).

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## ACKNOWLEDGEMENTS

We acknowledge Dr. Ina Benner and Dr. Chuan Ku for their helps in preparing additional coccolith materials for validation of coccolith cleaning protocols. This work was initially supported by an award to RAE under the the "Laboratoire d'Excellence" LabexMER (ANR-10-LABX-19), co-funded by a grant from the French government under the program "Investissements d'Avenir" and latterly a postdoctoral fellowship to YWL at Academia Sinica. This work was also supported by the Ministry of Science and Technology, Taiwan (MOST) to YWL. RAE also acknowledges support from National Science Foundation grant OCE-1437166 and the Pritzker Endowment to UCLA IoES.

## AUTHOR CONTRIBUTIONS

RAE and YWL designed the research with input from SR and BR. YWL and SR performed the culturing. YWL

performed the geochemical analyses and calculations. YWL drafted the manuscript with input from RAE, all authors contributed to the final manuscript.

## DATA AVAILABILITY

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

## APPENDIX A. SUPPLEMENTARY MATERIAL

Supplementary data to this article can be found online at https://doi.org/10.1016/j.gca.2021.09.025.

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Associate editor: Heather Stoll