Role of extracellular carbonic anhydrase in the polar diatom Fragilariopsis cylindrus

Summary

• Diatoms are major primary producers in productive polar oceans, where cold temperatures and high CO₂ solubility raise questions as to whether they rely on the CO₂‐concentrating mechanism (CCM) to saturate photosynthesis. Knowledge of CCM function will help better predict the sensitivity of diatom primary production to warming and acidifying polar oceans.

• Here, we characterized the CCM in the polar diatom, Fragilariopsis cylindrus, using membrane inlet mass spectrometry (MIMS) and a numerical model. Quantification of the role of external carbonic anhydrase (eCA) was enabled through the addition of a surface boundary layer to the numerical model.

• We found that F. cylindrus displayed an active CCM across a wide range of inorganic carbon availability, but that the CCM was not required under air‐equilibrated CO₂ concentrations. eCA was an essential component of the CCM, facilitating HCO₃ ⁻ uptake. When eCA was inhibited, CO₂ was the primary carbon source. The eCA role in supporting HCO₃ ⁻ uptake was independent of temperature changes.

• eCA‐supported HCO₃ ⁻ transport is likely a mechanism for F. cylindrus to cope with fluctuating concentrations of inorganic carbon over a dynamic seasonal cycle in polar oceans. Our work challenges the hypothesis that CCMs are not required at cold temperatures.

Short abstract

See also the Commentary on this article by Wheeler, 251: 913–915.

Introduction

CO₂‐concentrating mechanisms (CCMs) in marine phytoplankton encompass a wide range of mechanisms by which CO₂ is concentrated around RuBisCO within a cell to nearly saturate photosynthesis (Reinfelder, 2011). CCMs enable phytoplankton to access the large reservoir of HCO₃ ⁻ in seawater, circumventing any CO₂ limitation of photosynthesis. While the presence of a CCM suggests that phytoplankton CO₂ fixation would be less sensitive to the direct effects of ocean acidification (increased CO₂ and lowered pH), studies have found mixed responses that seem species‐ and environment‐dependent (Gao et al., 2014; Dutkiewicz et al., 2015; Schulz et al., 2017). It has been hypothesized that CCMs can be energetically costly (Raven et al., 2014), and at higher CO₂ availability, a decreased reliance on CCMs could enhance algal growth (Clement et al., 2016). However, the complexity of CCMs and their diversity across taxa, coupled with spatial and temporal variation in the marine environment, has raised questions as to whether marine carbon fixation is sensitive to rising CO₂ and lowering pH (Dutkiewicz et al., 2015).

Whether CCMs play an important role in cold waters is uncertain. Polar oceans cover 20% of the global ocean and polar primary production supports ecologically unique ecosystems, drives a strong biological carbon pump, and impacts many other large‐scale biogeochemical processes (Ducklow et al., 2007). Due to extreme environments and remote locations, studying primary production in these environments is difficult. Ecosystems in these regions are highly sensitive to the effects of rising anthropogenic CO₂, and observations have already recorded ecosystem regime changes in some polar regions (Schofield et al., 2010; Intergovernmental Panel on Climate Change (IPCC), 2022). However, empirical studies have found that rates of primary production in polar oceans appear to be largely insensitive to the direct effects of ocean acidification, which has been attributed to the already high concentration of CO₂ that is close to saturating photosynthesis (e.g. 25 μM CO₂ in sea water vs RuBisCO K_CO2 c. 17 μM at 0°C, Young et al., 2015a), thus reducing the need for a CCM (Eberlein et al., 2017; Hoppe et al., 2018; Torstensson et al., 2021).

Diatoms are the major primary producers in polar oceans and all to date are known to possess CCMs (Tortell et al., 2008a,b, 2010; Trimborn et al., 2013; Kranz et al., 2015). Studies examining the effect of ocean acidification on polar diatoms have found mixed results, from negative (e.g. Chaetoceros flexuosus) to neutral or positive (e.g. Thalassiosira hyalina), depending on species and community interactions (Tortell et al., 2008a,b; Feng et al., 2010; Trimborn et al., 2013, 2017; Young et al., 2015b; Eberlein et al., 2017; Hancock et al., 2018; Hoppe et al., 2018; Torstensson et al., 2021; Andrew et al., 2022). It appears that while polar diatoms rely heavily on CO₂ (Trimborn et al., 2013; Kranz et al., 2015), their CCM is functional and regulated in response to carbonate chemistry (Young et al., 2015b).

A number of studies have described the mechanisms of CCM function in diatoms (Delille et al., 2007; Hopkinson et al., 2011, 2013, 2016; Hopkinson, 2014; Shen et al., 2017; Tsuji et al., 2017; Li & Young, 2023; Keys et al., 2024). There appears to be two main mechanisms, one by which an external carbonic anhydrase (eCA) plays a critical role in inorganic carbon uptake (e.g. Thalassiosira pseudonana) and the other by which eCA is absent and inorganic carbon uptake is dependent on HCO₃ ⁻ transporters (e.g. Phaeodactylum tricornutum). To date, there has not been a careful culture study of the CCM in polar diatoms, though field studies have measured high relative rates of eCA (Martin & Tortell, 2006; Kranz et al., 2015). A recent study by Li & Young (2023) on the effect of temperature on the CCM of P. tricornutum demonstrated that the reduced ATP requirement of the CCM at lower temperatures was balanced by an equivalent reduction in CO₂ fixation rates (Li & Young, 2023). In permanently cold polar waters, low temperatures can affect the CCM by increasing CO₂ solubility, slowing diffusion and chemical reaction rates, lowering the half‐saturation constants of enzymes, and altering membrane permeability. In particular, the uncatalyzed equilibration between CO₂ and HCO₃ ⁻ is prohibitively slow at cold temperatures and thus polar diatoms may have an increased dependence on CAs.

Here, we investigated the importance of eCA across a range of CO₂ and constant pH by changing total dissolved inorganic carbon (DIC) for the model polar diatom Fragilariopsis cylindrus. We characterized the CCM using membrane inlet mass spectrometry (MIMS) and an adapted diatom CCM model to elucidate the role of eCA. The results highlight the constitutive dependence on eCA for F. cylindrus when taking up HCO₃ ⁻ from the environment, despite temperature changes.

Materials and Methods

Cell culture conditions

Fragilariopsis cylindrus CCMP1102 (a Southern Ocean isolate) cells were cultured in Instant Ocean solution buffered with 25 mM HEPES (pH 8.10) and supplemented with f/2 nutrients (Guillard, 1975) with 0.1 mM silicate. The pH was tuned in accordance with growth temperature and calibrated using thymol blue as described previously (Zhang & Byrne, 1996). Cultures were maintained at experimental temperatures of either 3°C, −2°C, or 9°C, salinity 35‰ (or 40‰ for −2°C and 3°C comparison), and light intensity was set at c. 120 μmol m⁻² s⁻¹ from white LED light with a 16 h : 8 h light : dark cycle. Experimental cells were acclimated for more than a month before subculturing. Cells were sampled during exponential growth at a density between 5 × 10⁵ ml⁻¹ and 1 × 10⁶ ml⁻¹. Cell numbers and sizes were measured using the Beckman Coulter Z1 cell counter.

Membrane inlet mass spectrometry and gas concentration calibrations

Fluxes of isotopically labeled CO₂ and O₂ were measured using membrane inlet mass spectrometry (MIMS) using a setup similar to Hopkinson et al. (2011, 2013). The cell chamber was cooled to the experimental temperature (3°C, −2°C, or 9°C) using the water jacket and circulating water bath with c. 15% (v/v) propylene glycol. Each day before cell measurements, the MIMS was calibrated as described by Cousins et al. (2010). Calibration for CO₂ concentration was done by adding known amounts of ¹³C‐labeled bicarbonate to 1.2 ml 0.1 N HCl (plus 1 M NaCl at −2°C) at a given temperature. ¹³C‐CO₂ mass readings (m/q 45 current) and concentrations (0–327 μM) fit well as a linear relationship (Supporting Information Fig. S1). Factoring the ¹³C/¹²C ratio in the enriched ¹³C‐HCO₃ ⁻ sample, slopes of linear fits were used for calibrating CO₂ concentrations later in data processing.

The minimum backgrounds of mass 44 (¹²CO₂) and 45 (¹³CO₂) were taken after CO₂ calibration in HCl by adding 25 μl 10 M NaOH. However, we observed that O₂ concentrations had a slight impact on the signal from these masses, so a further calibration of ¹⁶O‐CO₂ (mass 44 and mass 45) was done to correct for the impact from O₂ (mass 32). The O₂ correction was done by adding Na₂S₂O₄ to remove O₂ in the NaOH solution and assuming a linear relationship (Fig. S1). We calibrated O₂ concentrations at the experimental temperature by bubbling air through water or seawater for at least 30 min, using the equations from Mortimer (1981) and Garcia & Gordon (1992). The minimum background signal of O₂ was acquired by adding Na₂S₂O₄ (Fig. S1). A linear relationship between O₂ concentration and signal current was assumed. The O₂ signal was normalized to Ar (mass 40) to correct for air consumption by the MIMS during experimental runs. Conversion of signal current to gas concentrations was calculated using a customized Python script, ‘MIMS_CO2_calib.py’ (available at GitHub: https://github.com/limengwsu/Fcyl_CCM).

Measuring CO₂ and O₂ signals with F. cylindrus cells

Cells were harvested by centrifugation (5000  g , 2 min) and washed once, followed by pelleting at 10 000  g for 1 min, and then resuspended in 25 mM HEPES‐buffered (pH 8.10) DIC‐free artificial seawater (ASW). The cells were dark‐adapted (> 20 min) before added (20 μl stock) to the MIMS reaction chamber and added at a final cell density c. 8.6 ± 2.6 × 10⁶ ml⁻¹ (mean ± SD) in 1.2 ml total volume. Cells were added to a pre‐equilibrated mixture of CO₂ and ¹⁸O, ¹³C‐labeled NaHCO₃ (final concentration c. 2 mM), in the presence or absence of 100 μM acetazolamide (AZ). Cell suspensions were gently mixed via a stir bar (300 rpm). After a quasi‐steady state was reached in the dark (> 10 min), a light was turned on (c. 250 μmol m⁻² s⁻¹, AmScope 150 W halogen bulb) for 20–30 min followed by a light off period. A second light on process was repeated with the same cells (Fig. 1). Usually, two experiments, with AZ vs without AZ, were carried out on the same day, with samples taken from the same cell culture bottle. For each treatment, at least three biological replicates were carried out involving different cells on different days.

Fig. 1.

Example of trace of isotopic fluxes during a Fragilariopsis cylindrus CO₂‐concentrating mechanism (CCM) experiment using MIMS. (a, b) Present O₂ (Ar corrected) and CO₂ signal traces of a typical F. cylindrus CCM experiment at 3°C with and without external carbonic anhydrase (eCA) inhibitor acetazolamide (AZ) (100 μM), respectively. Note that ¹²C¹⁶O₂ is not shown but included in the total CO₂ for clarity. The time points t0 through t5 mark the sequential events, t₀: adding NaH¹³C¹⁸O₃, t₁: addition of F. cylindrus cells, t₂: first light on, t₃: first light off, t₄: second light on, and t₅: second light off. Shaded areas indicate dark periods in the membrane inlet mass spectrometry (MIMS) reaction chamber. (c, d) A zoomed‐in view around timepoint t1 with curve fitting (dashed line) displaying the accelerated ¹⁸O‐exchange between ¹³C‐CO₂ and H₂O after cell addition, with and without inhibitor, respectively.

Fluxes of O₂ (Ar corrected) in the light and dark measured rates of photosynthesis and dark respiration, respectively. From dark to light transition, diatom cells need to activate photosynthetic machineries to achieve optimum steady state rate (Hopkinson et al., 2011). To avoid confounding impacts of dark adaptation, two ‘light on’ periods (Fig. 1) were measured for steady state photosynthetic rate and CO₂ signals. The two ‘light on’ periods do not differ in their net O₂ evolutionary rates (Fig. S2a). Similarly, salinity (35 vs 40‰) does little to net O₂ evolutionary rates (Fig. S2b). In the ‘Results’ section, photosynthetic rates and CCM (carbon usage) were derived from the second ‘light on’ period. Data from 3°C were pooled and presented in the main text, while temperature‐dependent differences were shown in Fig. S2.

For low DIC experiments, cells were illuminated without additional DIC except for residual DIC and that from respiration. After the first light period, external ¹³C‐labeled NaHCO₃ was added in sequentially increasing concentrations. For experiments testing a sudden shift in pH, cells were cultured at 8.10 and then transferred to pH 7.6 or pH 8.6 for the MIMS measurements.

Data analysis, modeling, and statistical analysis

Changes in gas concentrations ([CO₂] and [O₂]) over time were processed using customized Python scripts. The principles of calculating physicochemical parameters, photosynthetic rates, and CO₂/HCO₃ ⁻ uptake have been described by earlier studies (Badger et al., 1994; Hopkinson et al., 2011, 2013; Kranz et al., 2015) and are detailed in Methods S1 Data Analysis for determining F. cylindrus physiochemical parameters, CA activities, and carbon usage.

We adapted the ‘Chloroplast pump’ and ‘Antarctica diatom’ models of Hopkinson et al. (2011); Hopkinson (2014); Kranz et al. (2015) to model the carbon fluxes of DIC in F. cylindrus cells by incorporating respiration and updated parameters measured in this study, appropriate for a cold temperature environment (see Table S1 for details). Using CO₂ drawdown and O₂ signal traces measured from MIMS, the model used curve fitting estimates for CO₂/HCO₃ ⁻ permeability and CA activity within the pyrenoid, and the HCO₃ ⁻ uptake activity of the chloroplast (Fig. S3). We updated the above‐cited models by estimating CO₂ concentration at the cell‐surface layer and CO₂/HCO₃ ⁻ fluxes across the cytoplasmic membrane when eCA is active (Methods S1 Data Analysis). Sample data and code for analysis are available on GitHub (under Fcyl_CCM repository, fit_CO2_Fc.py). The signal processing, modeling, statistical analyses, and curve fitting are largely dependent on the Scipy package in Python (Virtanen et al., 2020), and some data plots were generated using the seaborn package (Waskom, 2021). CCM modeling was done as described in Kranz et al. (2015), with the translation of the MATLAB code to a Python script with updated description and parameters (see Methods S1 CCM Modeling for details).

Results

Inhibition of F. cylindrus eCA lowered its affinity to inorganic carbon

Net O₂ evolution as a function of DIC and CO₂ was measured in the polar diatom F. cylindrus at 3°C (Fig. 2). With our experimental setup, the minimum DIC (c. 400 μM) did not reach lower than the K_0.5 for cells with active eCA. For comparison, the curve fitting results showed that the presence of eCA inhibitor acetazolamide (AZ) resulted in higher half saturation constants (K_0.5) of c. 700 μM for DIC or 4.2 ± 1.2 μM for CO₂ compared to 111 ± 88 μM DIC and 1.0 ± 0.7 μM CO₂ for the control (Table 1). The inhibited K_0.5 values were similar to a K_0.5CO2 of c. 4 μM found for eCA‐inhibited polar diatoms measured in coastal waters along the western Antarctic Peninsula (Kranz et al., 2015). While this demonstrates an active role of eCA at low CO₂ concentrations, at air‐equilibrated seawater concentrations of inorganic carbon (c. 2 mM DIC or c. 20 μM CO₂) there was no difference in O₂ evolution between control and AZ‐treated cells, indicating that eCA was not required to saturate photosynthesis under these conditions.

Fig. 2.

Low dissolved inorganic carbon (DIC)/CO₂ limits Fragilariopsis cylindrus photosynthesis in the presence of external carbonic anhydrase (eCA) inhibitor acetazolamide (AZ). (a, b) Photosynthetic rate (net O₂ production) in untreated and AZ‐treated cells across a range of steady state extracellular DIC and CO₂ concentrations at 3°C, respectively. Michaelis‐Menton curves (dashed lines) and 95% confidence interval of the best fit (shading) are shown for untreated (green) and AZ‐treated (orange) cells. Vertical lines mark the calculated values of K_0.5.

Table 1.

Michaelis–Menten constants for photosynthesis vs inorganic carbon curves for Fragilariopsis cylindrus.

Treatment	K0.5CO2(μM)	K0.5HCO3‐(μM)	V max (10−18 mol O2 per cell s−1)	n
	Mean ± SD	Mean ± SD	Mean ± SD
With AZ	4.2 ± 1.2	723 ± 229	7.7 ± 0.8	5
No AZ	1.0 ± 0.7	111 ± 88	6.3 ± 0.5	5

MIMS measurements to quantify the F. cylindrus CCM

To further understand the role of eCA in the CCM, we modeled the F. cylindrus CCM, using MIMS‐measured O₂ and isotopic CO₂ signal traces, coupled with known physiochemical parameters to estimate inorganic carbon (Ci) fluxes across different cell compartments (See Methods S1 for details) This approach was an extension of previous studies (Hopkinson et al., 2011, 2013; Kranz et al., 2015), with the adjustment of parameters for polar ocean temperatures (e.g. slower diffusion and reaction rates, greater solubility) and the smaller cell size of F. cylindrus. In addition, we included a surface boundary layer to quantify the activity of eCA by following Hopkinson et al. (2013), and estimated uptake of CO₂ and HCO₃ ⁻ during photosynthesis with an active eCA.

Passive fluxes as measured by MIMS

The ¹⁸O‐labeled CO₂ traces of F. cylindrus cells in the dark were used to quantify the physicochemical parameters of passive carbon fluxes, that is, mass transfer coefficients of CO₂ and HCO₃ ⁻ (f_c and f_b), extracellular CA (eCA) activity, and intracellular/cytosolic CA (iCA) activity (See Methods S1 for full details). Photosynthesis (O₂ production) and respiration (rate of O₂ consumption) were also measured in the light and dark, respectively.

An example trace of isotopic fluxes during a MIMS experiment is shown in Fig. 1(a,b). The addition of NaH¹³C¹⁸O₃ (c. 2 mM final concentration) into DIC‐free buffer at time zero (t₀) led to a rise of total CO₂ and ¹³C‐CO₂ signals (including ¹³C¹⁸O¹⁸O, ¹³C¹⁸O¹⁶O, and ¹³C¹⁶O¹⁶O through ¹⁸O‐exchange between H₂O and HCO₃ ⁻) until the equilibrium between total CO₂ and DIC is reached. Curves fitted during this slowly equilibrating step (between t₀ and t₁) were used to estimate the first‐order kinetic rate constants of uncatalyzed CO₂ and HCO₃ ⁻ interconversion (k₁ and k₂, Table S2). At time 1 (t₁) F. cylindrus were added in the dark, which resulted in a drawdown of O₂ and release of total CO₂ (respiration). Subsequent illumination/dark periods resulted in fluxes in O₂ and total CO₂ due to photosynthesis and respiration, respectively. The addition of F. cylindrus at t₁ also resulted in a rapid decrease in ¹³C¹⁸O¹⁸O in the bulk medium as it was taken up by the cells. The presence of CA (both eCA and iCA) accelerated the exchange of ¹⁸O between H₂O and HCO₃ ⁻, an increase in ¹³C¹⁸O¹⁶O and ¹³C¹⁶O¹⁶O. The initial rapid drop of ¹³C¹⁸O¹⁸O and increase in ¹³C¹⁶O¹⁶O, mainly driven by the influx of ¹³C¹⁸O¹⁸O and accelerated ¹⁸O exchange by iCA (Fig. S4), is more pronounced in eCA‐inhibited cells compared to control (see Fig. 1c vs 1D for a zoomed‐in view, and Fig. S4 for ¹⁸O‐depletion comparisons). This occurs because inhibiting eCA slows the production of ¹³C¹⁸O₂ from H¹³C¹⁸O₃ ⁻ dehydration in the bulk media, while the diffusion of ¹³C¹⁸O₂ into the cells and its rapid conversion to ¹³C¹⁶O₂ remain unaffected. When the process reaches a quasi‐steady state, ¹³C‐labeled CO₂ fluxes between cells and bulk media slowed down (after 2–3 min after adding cells), the rates of ¹⁸O‐depletion of (not the concentrations of ¹⁸O‐depleted) CO₂ and DIC are faster in reactions with active eCA (Fig. 1c vs 1d, Fig. S4). Curve fits when eCA was inhibited (Fig. 1c) were used to calculate the mass transfer coefficients of CO₂ and HCO₃ ⁻ (f_c and f_b), as described in previous studies using the one‐compartment model (Hopkinson et al., 2011). In agreement with earlier studies on diatoms (Hopkinson et al., 2011, 2013; Kranz et al., 2015), the F. cylindrus cells were more permeable to CO₂ than HCO₃ ⁻, with the magnitude of f_c, f_b estimated being 2.30 ± 0.64 × 10⁻⁹ and 1.90 ± 1.06 × 10⁻¹² cm³ per cell s⁻¹ (n = 12) respectively (Table S1). In the presence of eCA inhibition, first order rate constant for iCA (k_cf) was also calculated being 99 ± 36 s⁻¹ (n = 12; Table S1).

Measuring eCA activity and inorganic carbon uptake in the presence of eCA

Characterization of the CCM requires measurement of the uptake of CO₂ and HCO₃ ⁻ into the cell in the presence of an active CA. However, it is commonly believed that the rapid interconversion of CO₂ and HCO₃ ⁻ in the presence of eCA, which has one of the fastest catalytic rates (k_cat/K_0.5 up to 10⁸ s⁻¹ M⁻¹; West et al., 2012), would make separating cellular uptake of CO₂ and HCO₃ ⁻ impossible (Badger et al., 1994). Here, we demonstrated that use of high cell numbers with sufficient enzyme quantity would allow us to quantify the catalyzed rate by eCA using MIMS and measure mass fluxes of CO₂ and HCO₃ ⁻ in the presence of eCA.

First, eCA activity was measured using the MIMS previously and the method of (Hopkinson et al., 2013). If we assume that eCA activity functioned uniformly throughout the bulk environment, the catalyzed rate can be quantified as the accelerated CO₂ hydration rate, k_eCA1 (unconfined eCA model; Fig. 3a; Martin & Tortell, 2006; Tortell et al., 2010). In another approach, we assumed eCA was contained within a surface boundary layer between the cytoplasmic membrane and the frustule to calculate the eCA‐driven CO₂ hydration rate, k_sf (confined eCA model; Fig. 3b; Hopkinson et al., 2013). First‐order rate constants for eCA‐catalyzed CO₂ hydration (k_sf or k_eCA1) in the unconfined and confined models were normalized to uncatalyzed CO₂ hydration rates (k₁) and are shown in Fig. 3c. Estimated eCA activity in the unconfined eCA model (k_eCA1) was 2–4 fold faster than the uncatalyzed rate. However, constraining eCA to a surface boundary layer in the confined model resulted in eCA activity (k_sf) being c. 10 000‐fold faster than the uncatalyzed rate due to the smaller volume of the surface boundary layer compared to the bulk media (Fig. 3c).

Fig. 3.

Estimated external carbonic anhydrase (eCA) activity using bulk (unconfined) and surface boundary layer (confined) models, and the impact on calculated CO₂ uptake. (a, b) Unconfined and confined eCA conceptual models, respectively, were used for estimating eCA activity and CO₂ uptake. The pyrenoid structure was omitted for simplicity. Unconfined model: uptake of CO₂ and HCO₃ ⁻ (U_CO2 and U_HCO3‐), rate of CO₂ hydration/HCO₃ ⁻ dehydration in bulk (k_eCA1, k_eCA2) and cytoplasm (k_cf, k_cr). Confined model: uptake of CO₂ and HCO₃ ⁻ from bulk to surface (U_CO2bulk and U_HCO3‐bulk) and from surface to cell (U_CO2M and U_HCO3‐M), rate of CO₂ hydration/HCO₃ ⁻ dehydration in the bulk (k₁, k₂) and the surface layer (k_sf, k_sr). Boxplots showing (c) estimated eCA activities normalized to uncatalyzed CO₂ hydration rate and (d) estimated CO₂ uptake as a fraction of inorganic carbon supply during photosynthesis (${\mathrm{f}}_{{\mathrm{CO}}_2}$) in the unconfined model (${\mathrm{U}}_{{\mathrm{CO}}_2}$) or confined model (${\mathrm{U}}_{{\mathrm{CO}}_2\text{bulk}}$ and ${\mathrm{U}}_{{\mathrm{CO}}_2\mathrm{M}}$). CO₂ uptake rates were normalized to O₂ evolution rate (mol CO₂ : mol O₂).

Inhibition of eCA switches carbon uptake from HCO₃ ⁻ to CO₂

With quantified rates of eCA, CO₂ uptake (U_CO2) and HCO₃ ⁻ uptake (U_HCO3 ⁻) could be calculated in the presence of eCA using the approach of (Badger et al., 1994; Hopkinson et al., 2011) by replacing uncatalyzed rate constants (k₁, k₂) with eCA activities (Eq.  S14). Fig. 3 shows CO₂ uptake rates normalized to O₂ evolution and expressed as a fraction of inorganic uptake (f_CO2) using the unconfined model (U_CO2) and the confined model, which separates uptake into the surface layer, U_CO2bulk (using Eq. S12) and across the cytoplasmic membrane, U_CO2M (using Methods S1 Eq. S19–S24, and pers. comm. Badger). Positive f_CO2 indicated uptake into the cell, while negative values indicated CO₂ was leaked. Across 14 MIMS runs with saturating DIC, both the unconfined and confined models had f_CO2 values near zero or negative, an indication that HCO₃ ⁻, not CO₂, was the main inorganic carbon source during photosynthesis.

By contrast, when eCA was inhibited, we found that CO₂ was primarily used as a carbon source across all DIC concentrations tested (Fig. 4), which was in agreement with Kranz et al. (2015). We observed this same trend regardless of temperatures tested (i.e. at −2°C and 9°C HCO₃ ⁻ was the main carbon source with eCA active and CO₂ was preferentially taken up with eCA inactive; Fig. S2C). These results suggest that an active eCA was required for efficient HCO₃ ⁻ uptake across the cytoplasmic membrane.

Fig. 4.

Dissolved inorganic carbon (DIC) usage of Fragilariopsis cylindrus in the presence and absence of external carbonic anhydrase (eCA) inhibitor. CO₂ uptake rate (a) and HCO₃ ⁻ uptake rate (b) plotted vs corresponding carbon species concentration.

While our data suggested that eCA played a minimal role in supplying CO₂ from the bulk environment, it may be important for recycling leaked CO₂ (Trimborn et al., 2008) from the cytoplasm or to supply CO₂ by accelerating the interconversion of HCO₃ ⁻ and CO₂ at the surface boundary layer. To determine whether eCA played a role in recycling CO₂, net fluxes of CO₂ formation catalyzed by eCA (F_CO2eCA, Eq. S25) were calculated and compared to carbon uptake across the cytoplasmic membrane in the confined model (U_CO2M and U_HCO3‐M; Fig. 5a). When eCA supplies CO₂, F_CO2eCA will be positive. If eCA plays a role in recycling CO₂ then F_CO2eCA will be negative; that is, the net effect of eCA is converting CO₂ to HCO₃ ⁻ at the surface boundary layer. We found that when there was net CO₂ leakage across the cytoplasmic membrane, the net flux of eCA activity was for CO₂ hydration (negative F_CO2eCA), suggesting a recycling role. On the few occasions where CO₂ was taken into the cell when eCA was active, net eCA activity was for HCO₃ ⁻ dehydration (positive F_CO2eCA), facilitating the supply of CO₂. When compared to HCO₃ ⁻ uptake across the cytoplasmic membrane (U_HCO3‐M), the CO₂ recycle/supply role of eCA is a minor one, no > 20% in our observations (Fig. 5a). Nevertheless, the fraction of CO₂ supplied or recycled by eCA to total CO₂ flux across the cytoplasmic membrane was found to be positively correlated with eCA activity (Fig. 5b).

Fig. 5.

Net CO₂ flux catalyzed by external carbonic anhydrase (eCA) (${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}$) at the surface boundary layer. (a) ${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}$ calculated using Eq. S25 was normalized to CO₂ and HCO₃ ⁻ fluxes across the cytoplasmic membrane (${\mathrm{U}}_{{\mathrm{CO}}_2\mathrm{M}}$ and ${\mathrm{U}}_{{\mathrm{HCO}}_3‐\mathrm{M}}$). A negative ${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}$ corresponded to net CO₂ hydration and indicates eCA was recycling CO₂ leaked from cytoplasm (negative ${\mathrm{U}}_{{\mathrm{CO}}_2\mathrm{M}}$). The normalized value of ${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}$ to ${\mathrm{U}}_{{\mathrm{CO}}_2\mathrm{M}}$ indicates the fraction of recycled CO₂ or supplied CO₂ by eCA. Note that ${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}$ is relatively small when compared to HCO₃ ⁻ uptake across cytoplasmic membrane (${\mathrm{U}}_{{\mathrm{HCO}}_3‐\mathrm{M}}$). (b) Correlation between eCA activity and fraction of CO₂ recycle or supply by eCA (${\mathrm{F}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{eCA}}/{\mathrm{U}}_{\mathrm{C}{\mathrm{O}}_2\mathrm{M}}$).

Modeling F. cylindrus CCM

To model the F. cylindrus CCM at 3°C, we extended the ‘chloroplast pump’ CCM model (Hopkinson et al., 2011; Li & Young, 2023), using the confined model that restricted eCA activity to within the surface boundary layer (Hopkinson et al., 2013). The model was constrained by our MIMS measurements of temperature‐dependent physiochemical parameters and rates of photosynthesis and respiration. Physiochemical parameters include f_c (Table S2) and the half‐saturation constant for cytoplasmic membrane HCO₃ ⁻ transporter, Km_Bc, which was calculated from fitting data shown in Figs 4(b) and S5. Constants in the model were adjusted for the measurement temperature of 3°C (see Table S1 for details).

Fig. 6 shows model output of steady state net fluxes and intracellular concentrations for CO₂ and HCO₃ ⁻ in F. cylindrus with eCA active and inactive in a media of 2 mM DIC, pH 8.1 (Fig. 6a,b, respectively). In both scenarios, the driving force of the CCM in F. cylindrus was active pumping of HCO₃ ⁻ from the cytosol into the chloroplast. With eCA active (Fig. 6a), the resulting low cytosolic [HCO₃ ⁻] was replenished by active uptake of HCO₃ ⁻ across the cell membrane. However, when eCA is inactive (Fig. 6b), there is no HCO₃ ⁻ transport across the cytosolic membrane, and net diffusion of CO₂ into the cytosol is the primary source of carbon into the cell. Bulk media concentrations of CO₂ differ between 6A and 6B, due to the closed volume of the MIMS chamber and the absence of a CA in the bulk media. The differences in the net diffusion of CO₂ into the surface layer from the bulk resulted in steady state bulk [CO₂] being lower and not in equilibrium with [HCO₃ ⁻]. This is consistent with the observation of a faster decline of measured total CO₂ signals when eCA is inhibited by AZ (Fig. 1a vs 1b). In both cases, leaked CO₂ from the chloroplast and mitochondria was recycled via iCA in the cytosol (Fig. 6).

Fig. 6.

CO₂‐concentrating mechanism (CCM) model of inorganic carbon fluxes in Fragilariopsis cylindrus with or without external carbonic anhydrase (eCA) at pH 8.1. The role of eCA in supporting HCO₃ ⁻ uptake from surface to cytoplasm (${\mathrm{U}}_{{\mathrm{HCO}}_3‐\mathrm{M}}$) was modeled. Inorganic carbon net fluxes (units of 10⁻¹⁸ mol per cell s⁻¹) were labeled by the side of arrows, with arrow line width representing relative flux rate. The gray and blue arrows represent the diffusive and active transport fluxes of inorganic carbon, respectively, while the black, green, and magenta arrows denote fluxes associated with interspecies carbon conversion, photosynthetic fixation, and respiration. Only net fluxes equal to or > 10⁻²⁰ mol per cell s⁻¹ are shown. CO₂ and HCO₃ ⁻ concentrations in different compartments are labeled with concentration units μM and mM, respectively. AZ, acetazolamide.

We next modeled a scenario of low [CO₂] (2 mM DIC, pH 8.6). This scenario can occur during a diatom bloom, where pH can reach as high as 8.6 (Kranz et al., 2015). F. cylindrus with eCA active transported HCO₃ ⁻ into the cytosol and was able to sustain a high photosynthetic rate (Fig. S6A). By contrast, without eCA, a favorable [CO₂] gradient for CO₂ diffusion into the cell required cytosolic [CO₂] to be extremely low (c. 3 μM); consequently, this also resulted in low cytosolic [HCO₃ ⁻] (Fig. S6B). Low cytosolic [HCO₃ ⁻] limited the rate of HCO₃ ⁻ transport into the chloroplast, ultimately slowing the rate of carbon fixation. The dependence of HCO₃ ⁻ uptake on eCA activity was consistent with the observed higher cellular K_0.5 for inorganic carbon when eCA was inhibited (see Fig. 2). Experimental validations of modeled results were performed by exposing F. cylindrus cells cultured at pH 8.1 to pH 7.6 and pH 8.6 environmental shocks, and O₂ evolution rates were measured with varying DIC concentrations (Fig. S7). We observed a decline in the rate of O₂ evolution at pH 8.6. This decline was enhanced with eCA inhibition (Fig. S7a). By contrast, at pH 7.6 (Fig. S7b), the presence or absence of eCA inhibitor did not affect DIC response curves of F. cylindrus O₂ evolution.

Discussion

The diatom F. cylindrus is used as a model species for polar diatoms due to its abundance in both polar seawater and sea ice, and is found at both poles (Kang & Fryxell, 1992; Mock & Hoch, 2005; Mock et al., 2017). Previous studies have speculated that polar diatoms do not require a CCM to be productive and that F. cylindrus primarily uses CO₂ for its inorganic carbon source (Kranz et al., 2015). However, contrasting evidence suggested that many polar diatoms, including F. cylindrus, use a CCM that takes up HCO₃ ⁻ as the major inorganic carbon source (Tortell et al., 2008a,b), which is regulated in response to the carbon environment (Young et al., 2015b). Here we quantified CCM function in F. cylindrus and demonstrated that eCA activity was not required under air‐equilibrated polar seawater concentrations of DIC (2 mM, pH 8.1, 3°C), though eCA was essential at low CO₂, and at high pH (e.g. during phytoplankton blooms, 2 mM DIC, pH 8.6, 3°C) to maintain photosynthetic rates. In addition, we found that the role of eCA was primarily to facilitate uptake of HCO₃ ⁻, not CO₂, into the cell, though the exact mechanism is yet to be elucidated.

At cold temperatures, eCA plays an important role by overcoming the prohibitively slow uncatalyzed interconversion between CO₂ and HCO₃ ⁻. The expression of diatom eCA (both as transcripts and as activity normalized to uncatalyzed rates) is generally enhanced in polar oceans (Tortell et al., 2008a; Kranz et al., 2015; Pierella Karlusich et al., 2021). Our finding that eCA in polar diatoms was only essential to maintain photosynthetic rates under low CO₂ concentrations has been observed for temperate diatom species, for example, Skeletonema costatum, Thalassiosira weissflogii, Thalassiosira pseudanana, and Cylindrotheca fusiformis (Burkhardt et al., 2001; Rost et al., 2003; Hopkinson et al., 2013; Tsuji et al., 2017). These studies on temperate diatoms and others on eukaryotic algae proposed that the role of eCA was to facilitate the supply of CO₂ across the cytoplasmic membrane (Moroney et al., 1985; Elzenga et al., 2000; Hopkinson et al., 2013; Kranz et al., 2015) and/or facilitate the recycling of CO₂ under high pH and low CO₂ conditions (Trimborn et al., 2008). Our approach, regardless of whether an unconfined or confined model of eCA activity was used (Fig. 3), was able to tease apart the inorganic carbon usage of F. cylindrus when eCA is active. Our data suggested that when eCA is active, F. cylindrus uses primarily HCO₃ ⁻; by contrast, when eCA was inhibited, F. cylindrus mostly depends on CO₂ diffusion for photosynthesis.

We argue that eCA was required to facilitate the transport of HCO₃ ⁻ across the cytosolic membrane in F. cylindrus. To test this argument, we tested the alternative hypothesis that eCA was supplying CO₂ in F. cylindrus by modeling a CCM that relies on CO₂ diffusion with an active eCA, but no cytoplasmic HCO₃ ⁻ uptake, and simulated a CO₂ signal (Fig. S8, dashed line). This alternative CCM model, CO₂ uptake and supplying CO₂ by eCA, was unable to replicate our observations using a MIMS of a minimal change in CO₂ concentrations during photosynthesis (Fig. 1b t2‐t3 and t4‐t5). We hypothesized that F. cylindrus used an eCA‐dependent HCO₃ ⁻ transport system. An eCA‐dependent HCO₃ ⁻ transport system has been previously demonstrated in the human AE1/CAII system (Sterling et al., 2001) but this is the first time, to our knowledge, it has been described in marine CCMs. This quantitative interpretation of the role of eCA still allows the role of eCA to supply or recycle CO₂ at the surface boundary layer (Trimborn et al., 2008; Hopkinson et al., 2013), with rate and net direction of supply/recycle depending on eCA activity and the cytosolic CO₂ concentration, respectively. Molecular biology approaches have demonstrated the diversity of diatom CCM structure and function (Shen et al., 2017; Tsuji et al., 2017), including the presence/absence of numerous genes of different classes of CAs and SLC‐type HCO₃ ⁻ transporters. However, localization is still uncertain, and there continues to be novel forms identified (e.g. iota class of CA; Jensen et al., 2019). According to the available F. cylindrus genome data (Mock et al., 2017), there are six annotated SLC‐type HCO₃ ⁻ transporters, providing possible candidates responsible for the CA‐supported HCO₃ ⁻ transport. Yet it still leaves a puzzle as to why F. cylindrus and other diatoms dependent on eCA do not directly use the most abundant and freely available HCO₃ ⁻ through transporters like ptSLC4‐2 (Nakajima et al., 2013). Diatoms may have diverse systems for DIC uptake. While we do not rule out the possibility, without molecular evidence, an AZ‐sensitive HCO₃ ⁻ transporter independent of eCA is less likely.

The relative utilization of CO₂ vs HCO₃ ⁻ in relation to eCA by diatoms has long been debated. A variety of approaches have been used to distinguish between these pathways, including the isotopic disequilibrium technique, MIMS, modeling, and estimates based on ¹³C carbon isotope fractionation. Diatom Ci uptake and possession of an eCA vary across species and under differing conditions (Elzenga et al., 2000; Hopkinson et al., 2013). In a Bering Sea phytoplankton field study, Martin & Tortell (2006) applied the isotopic disequilibrium technique and found that inhibition of eCA reduced both HCO₃ ⁻ utilization and photosynthetic activity, leading them to suggest that AZ treatment may directly affect, or inhibit, the HCO₃ ⁻ transport system. Similar responses were measured across 17 cultured diatom species, leading Martin & Tortell (2008) to propose a role for eCA in HCO₃ ⁻ transport. However, Trimborn et al. (2008) combined MIMS measurements with ¹³C carbon isotope fractionation analyses for three large diatoms and concluded that eCA functions to recycle CO₂ at the diatom cell surface. Through modeling and curve fitting, Hopkinson et al. (2013) further refined the MIMS approach to quantify eCA activity in Thalassiosira pseudonana (CCMP1335) and concluded that the ‘most logical’ role of eCA is to enhance CO₂ supply. This MIMS‐based quantification of eCA activity has since been applied in several studies (Kranz et al., 2015; Chrachri et al., 2018; Keys et al., 2024), although eCA activity was generally not incorporated into Ci uptake estimates in these analyses. Results from recent use of microelectrodes to monitor pH and CO₃ ²⁻ dynamics at the cell surface in different diatom species (Chrachri et al., 2018; Keys et al., 2024, 2026) were also interpreted as eCA having a role in CO₂ supply. Our results with F. cylindrus are not in disagreement with measurements of Chrachri et al. (2018), with the interpretation that eCA facilitates HCO₃ ⁻ uptake and indirectly modulates cell surface carbonate chemistry. However, there are clearly species‐specific differences, as photosynthetic rates by F. cylindrus were not impacted by eCA inhibition at high DIC (2 mM). The different interpretations for the role of eCA for Ci supply across different methodologies, species, and conditions highlight the challenges of interpreting indirect measurements. It is likely that there are species‐ and condition‐specific responses, with different diatom species exhibiting distinct Ci uptake strategies.

This study highlights that caution should be taken when interpreting physiological results in the presence of inhibitors alone. In the presence of the eCA inhibitor AZ, our results agreed with previous studies that showed CO₂ as the primary source of entry to the diatom cells (Burkhardt et al., 2001; Rost et al., 2003; Kranz et al., 2015). In field research, inorganic carbon usage in the marine phytoplankton community is often measured via the disequilibrium assay, where eCA inhibitors are used to eliminate the fast interconversion between ¹⁴C‐labeled HCO₃ ⁻ and CO₂ (Martin & Tortell, 2006; Rost et al., 2007; Tortell et al., 2008a, 2010; Kranz et al., 2015). If eCA facilitated HCO₃ ⁻ transport is an important function of CCMs across the phytoplankton community, the results obtained from experiments with eCA inhibitors may underestimate the usage of HCO₃ ⁻ (Martin & Tortell, 2006; Tortell et al., 2010). For future studies, it may be helpful to combine and compare the techniques, including MIMS, disequilibrium assay, microelectrode measurements, isotope fractionation, and quantitative modeling for different diatoms with eCA activity. The role of eCA can be either supplying or recycling CO₂, but its role in facilitating HCO₃ ⁻ transport awaits more studies, especially those that provide molecular evidence.

A question remains as to why eCA is highly expressed and regulated in polar diatoms? In polar oceans, CO₂ concentrations are dynamic, not often at equilibrium with the atmosphere. While deep upwelling of CO₂, accumulation of respired CO₂ under ice over winter (Tortell et al., 2014) and high CO₂ solubility can result in CO₂ concentrations well above that needed to saturate RuBisCO (Young et al., 2015a), phytoplankton blooms in seawater and sea ice brines during spring can result in a pH > 8.6 and rapid drawdown of CO₂ (Gleitz et al., 1995; Delille et al., 2007). The eCA‐supported HCO₃ ⁻ transport may be a mechanism for F. cylindrus to cope with fluctuating CO₂ conditions in polar ocean and sea ice habitats. Changes in pH due to ocean acidification are likely to be small compared to the wide variation experienced seasonally, suggesting that carbon fixation by polar diatoms will be robust in the face of ocean acidification.

Competing interests

None declared.

Author contributions

JY designed the experiments, ML carried out and optimized the experiments, and performed data analysis. JY and ML wrote the paper.

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Supporting information

Fig. S1 MIMS CO₂ concentration calibration and rate constant estimations.

Fig. S2 Photosynthetic rates and inorganic carbon usage by Fragilariopsis cylindrus during photosynthesis.

Fig. S3 Curve fitting results to constrain bicarbonate uptake activity into cytoplasm and chloroplast for modeling.

Fig. S4 The impact of eCA on the depletion rate of ¹⁸O‐labeled CO₂ in the dark.

Fig. S5 The impact of [HCO₃ ⁻] on membrane bicarbonate uptake rate at 3°C.

Fig. S6 CCM model of inorganic carbon fluxes in Fragilariopsis cylindrus at 3°C, pH 8.6.

Fig. S7 The impact of eCA inhibitor on photosynthetic rate with varying DIC concentrations at pH 8.6 and pH 7.6.

Fig. S8 The impact of eCA activity on MIMS CO₂ signal.

Methods S1 Additional text for methods of data analysis and CCM modeling.

Table S1 Fcyl CCM model parameters at 3°C.

Table S2 Physiochemical parameters calculated from passive fluxes of CO₂ in Fragilariopsis cylindrus.

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Data availability

The Python scripts for data analyses and CCM modeling are available on GitHub: https://github.com/limengwsu/Fcyl_CCM.