The constant increase in greenhouse gases resulting from anthropogenic activities, which significantly contribute to global warming, represents one of the main challenges of this century, requiring close collaboration between researchers and industrial stakeholders to develop effective solutions. Over the years, several strategies have been proposed for capturing and removing carbon dioxide (CO₂), such as chemical or physical absorption, membrane separation, and CO₂ bio-fixation (e.g., Daneshvar et al., 2022; Moheimani, 2005). To date, amine-based alkaline solvent absorption remains the dominant CO₂ capture method because it is technologically more mature and less expensive than most alternative technologies, although several disadvantages persist, including high energy consumption for solvent regeneration, low CO₂ absorption capacity, poor thermal stability, and high corrosivity (Liang et al., 2016). For this reason, current CO₂ removal efforts are increasingly shifting toward nature-based solutions, such as CO₂ bio-fixation through microalgae cultivation, which could offer a greener and more sustainable alternative to conventional methodologies.

Indeed, photosynthetic unicellular microorganisms are expected to have 10–50 times higher CO₂ bio-fixation rate than terrestrial plants, owing to their much faster growth rates, up to 400 times higher (Falkowski, 2012; Ighalo et al., 2022). Combined with the advantages of yielding high-value products, requiring limited land area, and enabling efficient biomass production and harvesting in photobioreactors, microalgae cultivation represents a strategic alternative to traditional CO₂ mitigation approaches (Cheng et al., 2025; Lam et al., 2012).

Since the 1990s, microalgae such as green and red algae or diatoms have been extensively studied for Carbon Capture Usage and Storage (CCUS) applications (e.g., Cheng et al., 2025; Hoque et al., 2025). However, even though coccolithophores are well studied in fundamental marine carbon-cycle research, they remain comparatively less explored in applied CCUS cultivation systems. These golden-brown marine microalgae possess the distinctive ability to cover their cells with calcite scales–called coccoliths–ranging from 1 to 20 µm in size, which together form an exoskeleton known as the coccosphere (Brownlee et al., 2021; Young, 1997; Monteiro et al., 2016). Thus, for coccolithophores, the total mass comprises both the organic fraction and the inorganic CaCO₃. Since CaCO₃ makes up the main component of the coccolithophore exoskeleton, based on the stoichiometric composition of CaCO₃, the carbon contained in the mineral phase corresponds to ∼12% of its mass. Therefore, these organisms have the potential to sequester more carbon than other microalgae lacking this life strategy (Villiot et al., 2021). In most microalgal systems, the CO₂ captured during growth is eventually released back to the atmosphere when the biomass or derived bioproducts are degraded or consumed. By contrast, in coccolithophores a substantial fraction of the carbon precipitates as CaCO₃ in coccoliths, and it is stored over long timescales in the deep-sea sediments, providing a unique form of carbon sequestration among microalgae (Brownlee et al., 2021; Moore et al., 2021). In nature, coccolithophores account for approximately 20% of oceanic primary productivity and contribute up to 50% of the ∼1.6 Pg y^-1 of calcite produced in the pelagic zone (Brownlee et al., 2021; Monteiro et al., 2016; Taylor et al., 2017). They are thus key players in global carbon biogeochemical cycles (e.g., Rost and Riebesell, 2004; Ziveri et al., 2007). Their natural ability to permanently remove inorganic carbon from seawater and the atmosphere and to store it within their CaCO₃ exoskeleton has led to their identification as promising candidates for new technologies in CCUS (Brownlee et al., 2021; Moore et al., 2021).

Coccolithophore carbon production varies greatly among species. For instance, Gephyrocapsa huxleyi produces less than 6 pg C cell^-1, whereas larger species like Scyphosphaera apsteinii contain up to 200 times more carbon. The PIC:POC ratio also varies significantly between and within species. For example, Chrysotila carterae exhibits ratios ranging from 0.17 (Gafar et al., 2019) to 0.8 (Zou et al., 2017), whereas Calcidiscus quadriperforatus reaches 2.08 (Gafar et al., 2019). The ecological preference of coccolithophores for oligotrophic regions (Winter et al., 1994; Young et al., 2005) may have limited research on their response to nutrient-rich conditions and their potential applications in bioremediation or biotechnology. However, some opportunistic taxa–such as Chrysotila roscoffensis and G. huxleyi–are well adapted to turbulent, eutrophic environments (Dimiza et al., 2020; Reifel et al., 2001) and are known to seasonally form large oceanic blooms (Brownlee et al., 2021; Reifel et al., 2001).

This theoretically high potential has been explored in only a few studies so far, particularly from a biotechnological perspective. Only a limited number of papers have investigated the intensive cultivation of coccolithophores in photobioreactors (Jakob et al., 2018; Moheimani et al., 2011; Moheimani and Borowitzka, 2011), and just one has examined cultivation in raceway ponds (Moheimani and Borowitzka, 2006). These studies reported that C. carterae exhibited the highest overall performance in plate photobioreactors, reaching dry weight (DW), lipid, and CaCO₃ productivities of 0.54, 0.12, and 0.06 g L^-1 d^-1, respectively. Regardless of reactor configuration, C. carterae achieved the highest productivity, followed by G. huxleyi and Gephyrocapsa oceanica. Moreover, C. carterae contained the greatest proportions of both lipids (20%–25% of DW) and CaCO₃ (11%–12%). When cultured in outdoor raceway ponds, C. carterae maintained a total productivity of approximately 0.19 g L^-1 d^-1, with CaCO₃ contributing up to 10% of DW. This corresponds to an annual areal productivity of about 60 t ha^-1 y^-1 of total mass and roughly 5.5 t ha^-1 y^-1 of CaCO₃ (Moheimani and Borowitzka, 2006). Despite these promising results, no further research has been conducted on this topic.

A crucial aspect of evaluating coccolithophores for CCUS applications is strain selection. Key traits to consider include: i) the amount of CaCO₃ produced relative to the organic fraction, ii) the capacity to reach high cellular densities, iii) growth rate, iv) response to medium composition, v) tolerance to stressors such as culture mixing, and vi) specific coccolith morphology for potential product reuse. We selected three strains: one of G. huxleyi and two belonging to the species C. roscoffensis. G. huxleyi is a non-motile species (4–10 µm) carrying 10–50 coccoliths per coccosphere, each 2–5 µm in size (Young et al., 2022) and displaying a highly variable PIC:POC ratio (0.24–1.38; Gafar et al., 2019). Chrysotila spp. are flagellated, motile species (12–15 µm) bearing smaller coccoliths (∼1–2 µm) (Young et al., 2022) and showing lower PIC:POC ratios (∼0.17; Gafar et al., 2019) than G. huxleyi. The three coccolithophore strains were chosen based on (i) published evidence of their successful cultivation in bioreactors and raceway ponds and their potential for biotechnological exploitation (e.g., C. carterae; Moheimani and Borowitzka, 2006), (ii) reports of intense natural blooms in high-nutrient environments (e.g., C. roscoffensis; Reifel et al., 2001), and (iii) the high calcification capacity and bloom-forming behaviour of G. huxleyi, which is known to produce multiple layers of coccoliths. Despite these individual studies, a targeted comparison of their performance under the high-nutrient regimes required to sustain calcified biomass production for CO₂ capture is still lacking; the present screening was specifically designed to address this gap.

Since nitrogen (N) and phosphorus (P) are fundamental building blocks for the organic fraction and for the efficient utilization of supplied CO₂ (Daneshvar et al., 2022; Jakob et al., 2018; Zuccaro et al., 2020), and since coccolithophores typically thrive in oligotrophic oceanic waters (Han et al., 2025; Winter et al., 1994), it is essential to investigate their tolerance to higher nutrient concentrations as a preliminary step toward maximizing yield. The amount of carbon supplied is also a key factor sustaining biomass production, especially for coccolithophores, which require CO₂ for photosynthesis as well as bicarbonate ions (HCO₃ ⁻) for calcification processes (Brownlee et al., 2015; Takano et al., 1995). Therefore, we tested the response of the above-mentioned strains under increasing macronutrient levels–namely, N, P, and carbon in the form of nitrates (NO₃ ⁻), phosphates (PO₄ ^3-), and sodium bicarbonate (NaHCO₃), respectively–evaluating also their potential for CO₂ sequestration within both the organic fraction and CaCO₃ exoskeleton. For the C. roscoffensis strains, the effects of elevated N and P concentrations relative to the standard maintenance medium, along with additional carbon supply, were examined here for the first time.

The ultimate aim of this study is to identify the strain with the highest yield under non-limiting conditions, enhancing both organic and inorganic production, and to provide a preliminary evaluation of the feasibility of large-scale CCUS applications for coccolithophores.

To date, the scientific community studying coccolithophores has focused primarily on their physiology and ecology, with only a few articles exploring strategies to increase monospecific coccolithophore culture densities and enhance yields. Only in a few studies, C. carterae has been successfully cultivated in both closed photobioreactors and raceway ponds, achieving promising areal productivity using f/2 medium; thus at the same macronutrient levels of our mod-f/2 experiments (i.e., ∼75 mg/L NaNO₃ and 5 mg/L NaH₂PO₄·H₂O). Only three previous studies have modified macronutrient and carbon supply (mainly in G. huxleyi) compared to the standard medium composition with the objective of improving coccolithophore production for biotechnological applications (Takano et al., 1995; Jakob et al., 2018; Moreira et al., 2023). In our calculations, we used a typical microalgal biomass composition derived from the Redfield molar ratio (C:N:P = 106:16:1). When converted to a mass basis, the ratio is usually rounded to 50:10:1, i.e., ∼50 wt% C, 10 wt% N and 1 wt% P, a commonly used approximation for microalgal biomass in mass-balance calculations (Zuccaro et al., 2020). Under this assumption, if N accounts for ∼10 wt% of m_bio, a supply of 12 mg/L N (total N in the mod-f/2; total P: 1.43 mg/L) can support at most ∼120 mg/L m_bio. Therefore, we designed experiments to test tolerance to six-fold higher nutrients (total N: 70 mg/L; total P: 8.11 mg/L, hereafter N70) and to evaluate whether additional inorganic carbon (daily NaHCO₃ additions; N70 + C) could further increase growth.

For all the three strains screened, nutrient enrichment up to 70 mg/L N and 8 mg/L P (N70 condition) did not lead to an increase in m_tot. Therefore, the main differences are observed with carbon addition compared to the other two conditions, showing a general increase in OD and DW (Figure 1; Table 1). This experimental outcome confirms that carbon availability remained a pivotal limiting factor for further increase in m_tot.

Based on a qualitative comparison of the OD trend in the growth curves, G. huxleyi enters the stationary phase faster than CS1 and CS2 under all tested conditions, stabilizing at around an OD of ∼0.5 in mod-f/2 and ∼0.4 in N70 at t7, corresponding to a DW of around 0.17 g/L and 0.13 g/L, respectively (Figure 1; Supplementary Figures S2, S3). In G. huxleyi, the addition of carbon leads to more than a 5-fold increase in OD at t7 (0.59 g/L), which can be related to increased coccolith production since the recorded average PIC:POC ratio equals 2.1 ± 1.1 (Table 1). This pronounced rise in PIC:POC under N70 + C is unusually high compared with values reported in literature for G. huxleyi (the highest previous value reported is 1.38; Gafar et al., 2019), and might reflect species-specific calcification responses to elevated HCO₃ ⁻. However, the intrinsic variability among biological replicates must be considered when making this assumption. Considering this PIC:POC ratio, the calculated accumulation of m_bio for G. huxleyi is 0.06 ± 0.02 g/L, which corresponds to about 10% of m_tot. The same amount of m_bio is obtained under the N70 condition, where it accounts for 55% of DW since the PIC:POC ratio decreases to 0.2 ± 0.05 (Figures 2B,C). Thus, although the OD increases markedly with NaHCO₃ additions, m_bio is not scaling proportionally because a large fraction of the signal and m_tot increase derives from coccolith production. The low m_bio production in G. huxleyi is also confirmed by the nutrient consumption data reported in Table 2. The N consumed at t7 is the lowest measured among the studied strains, reaching up to 7.4 ± 2.2 mg/L in N70 + C compared to 32.4 ± 0.5 and 47.4 ± 1.7 mg/L for CS1 and CS2, respectively, under the same treatment. The same trend is recorded for P, with values of 1.9 ± 0.01 mg/L (N70) and 3.3 ± 0.08 mg/L (N70 + C), representing the lowest P consumption among the selected strains (Table 2). These uptake patterns reinforce the interpretation that increased OD in G. huxleyi under high HCO₃ ⁻ primarily reflects enhanced coccolith production rather than increased m_bio.

Similarly to G. huxleyi, the strains of the species C. roscoffensis show similar OD trends in both mod-f/2 and N70 conditions. As described above for G. huxleyi, the addition of carbon in N70 + C increases OD up to 1.08 (DW 0.48 g/L) in CS1 and even higher for CS2, which reaches an OD of 2.46 in the stationary phase, corresponding to a DW of 1.2 g/L–the highest measured DW among the strains and tested conditions (Figure 1; Table 1). For C. roscoffensis, the PIC:POC ratio remains mostly stable, especially for CS1 with an average value of 0.3 in N70 and 0.2 ± 0.04 in N70 + C; only CS2 shows a slight increase in the PIC:POC ratio, up to 0.3 ± 0.01, under the carbon-replete condition (Table 1; Figure 2C). Consequently, for C. roscoffensis strains, OD increases reflect substantial gains in m_bio as well as m_carb.

For the C. roscoffensis strains, macronutrient consumption is usually higher than for G. huxleyi, reaching N up to 32.4 ± 0.5 mg/L and 47.4 ± 1.7 mg/L for CS1 and CS2, respectively, in N70 + C (Table 2; Figure 2A). Even though the percentage of N removal is higher, in N70 + C condition P is almost completely depleted (∼100%) for both CS1 and CS2 (Table 2). This pattern is consistent with the onset of P limitation for further growth; however, given the potential for P luxury uptake, as well as the possible influence of other limiting factors (e.g., light, inorganic carbon or trace nutrients), the observed P depletion could be interpreted as an indication of P-limited growth, requiring to balance N and P when aiming to maximize growth. Notably, for CS2 in particular, the substantial enhancement of nutrient uptake under carbon-replete conditions is consistent with the five-fold increase in m_bio (Figure 2B), the highest among the strains tested here (Table 2). Indeed, CS2 records the highest accumulation in m_bio, corresponding to 480.8 ± 17.3 mg/L (Table 2).

Two-way ANOVA showed that m_bio was strongly affected by strain, nutrient condition and their interaction (all p < 0.001; Figure 2B). Under N70, G. huxleyi had lower biomass than CS1 and CS2 (G. huxleyi vs. CS1: p = 0.0001, G. huxleyi vs. CS2: p = 0.011), and only strains CS1 and CS2 exhibited a marked increase in biomass under N70C (CS1 and CS2: p < 0.0001; G. huxleyi, p = 0.55). N and P consumption displayed similar patterns (Figure 2A). N70C strongly enhanced N removal in strains CS1 and CS2 (both p < 0.0001) compared to N70 but had little effect on G. huxleyi (p = 0.44), while only CS1 consumed significantly more N than G. huxleyi (p = 0.013) under N70. In contrast, under N70C all three strains differed from one another, with CS2 showing the highest N consumption (all pairwise comparisons p < 0.0001; different letters in Figure 2A). For all strains, P removal was significantly higher under N70C than under N70 (all p < 0.0001). Under N70, P consumption increased from G. huxleyi to CS1, with all pairwise differences being highly significant (all p < 0.0001). Under N70C, G. huxleyi still showed the lowest P removal, whereas strains CS1 and CS2 reached similarly high P consumption (p = 0.99), as reflected by the compact letter display in Figure 2A. P consumption was always different from N70 (p < 0.0001).

The PIC:POC ratio, analyzed on log-transformed data, was also significantly affected by strain, condition and their interaction (all p < 0.05), with generally higher PIC:POC under N70C and strain-specific responses as indicated by the compact letter display in Figure 2C. This points to a particularly strong shift towards calcification in G. huxleyi under the N70C regime.

From the collected data, the amount of CO₂ captured per liter of culture was calculated for the conditions N70 and N70 + C (Table 1). In this initial screening, CO₂ in m_carb was estimated from the inorganic carbon associated with the harvested biomass. Detached coccoliths remaining in the culture medium were not explicitly quantified, and our values should therefore be considered conservative, particularly for species that actively shed coccoliths such as G. huxleyi. In the carbon-replete condition, CO₂ capture is greater in CS2 compared to the others, and approximately one-quarter (∼311 mg/L CO₂) is stored in m_carb. This amount is even higher than that calculated for G. huxleyi (∼225 mg/L CO₂) despite its higher PIC:POC ratio, and thus greater coccolith production (Table 1). Therefore, in our experiments, CS2 combined higher yield in m_bio and stable inorganic carbon storage in m_carb, producing the greatest overall CO₂ capture among the tested strains under N70 + C.

We used daily additions of NaHCO₃ to supply DIC rather than continuous CO₂ bubbling, because coccolithophores are more sensitive to bubbling and bicarbonate addition offers a more stable DIC pool and avoids rapid CO₂ outgassing. At the pH values observed (pH > 8 after daily NaHCO₃ additions), HCO₃ ⁻ is the dominant inorganic carbon species in the medium. Shifts in carbonate chemistry (higher HCO₃ ⁻/CO₂) can change the relative availability of the proximal substrates and thereby differentially constrain photosynthesis and calcification. Experimental work in coccolithophores (Bach et al., 2013; Bach et al., 2015; Brownlee et al., 2015) shows that photosynthesis/growth can be sensitive to CO₂, whereas calcification is closely linked to HCO₃ ⁻ supply, consistent with compartmentation/transport constraints and acclimatory regulation. Therefore, this chemical shift towards HCO₃ ⁻ in our experiments could have favored calcification (PIC formation) over photosynthetic POC production in species that preferentially use HCO₃ ⁻ for calcification while relying on CO₂ for photosynthesis. The discrepancy we observed between strong OD increases and limited POC-based biomass in G. huxleyi suggested interference from detached coccoliths inflating optical readings. We verified this by decalcifying G. huxleyi cultures grown in N70 + C at t7: the measured OD dropped from 1.75 to 0.45 after decalcification (Supplementary Figure S2), confirming that detached coccoliths biased optical-density–based m_bio estimates for G. huxleyi but not (to the same degree) for C. roscoffensis, which has smaller coccoliths and has not the same tendency to shed large numbers of plates.

Taken together, these results emphasize two practical points for coccolithophore cultivation: (1) raising macronutrients alone (N70) is insufficient to substantially increase m_tot without concurrent increases in bioavailable carbon, and (2) while we did not independently manipulate individual inorganic carbon species, the shift in the carbonate chemistry toward HCO₃ ⁻ in N70 + C could have species- and even strain-specific impacts on the balance between photosynthesis (POC) and calcification (PIC), consistently with prior work showing differential sensitivities of photosynthesis and calcification to CO₂ versus HCO₃ ⁻ in G. huxleyi. More broadly, our results motivate targeted control of carbonate chemistry and phosphate availability, together with other growth-relevant constituents (e.g., major ions involved in calcification such as Ca²⁺), when designing cultivation strategies that aim simultaneously to maximize m_bio production and stable inorganic carbon sequestration in m_carb. Finally, and importantly for biotechnological scaling, these experiments illustrate the value of strain selection within a species: CS2 seems to be a promising strain for forthcoming experiments, since it showed higher nutrient conversion efficiency into m_bio compared to G. huxleyi (Figure 2B) and higher stability of the PIC:POC ratio across different conditions (Figure 2C), which are attractive traits for envisioning a future large-scale application. Coupled with literature reports showing promising results for C. carterae in photobioreactors and raceway ponds, our findings corroborate that the genus Chrysotila, and specifically the species C. roscoffensis, can be further studied as a candidate for future large-scale applications where permanent inorganic carbon sequestration is a desired outcome.

In addition to modulating the nutrient supply, light intensity and wavelength are also fundamental for increasing growth efficiency and pigments production in microalgae (Daneshvar et al., 2022 and references therein). Indeed, it has been highlighted that photosynthesis, calcification, as well as growth rate in coccolithophores can be affected by light availability (Zondervan et al., 2002; Ramos et al., 2012; Rokitta and Rost, 2012). To date, among the coccolithophore group, only a few species, such as G. huxleyi, C. carterae and G. oceanica, have been investigated in their response to different light intensities (e.g., Zhang et al., 2023; Zhang et al., 2015; Zondervan, 2007; Zondervan et al., 2002; Heinle, 2014). In coccolithophores, calcification seems to be less light-dependent than photosynthesis (e.g., Balch et al., 1992; Zondervan, 2007 and references therein), with the PIC:POC ratio not significatively or clearly affected by light (Heinle, 2014). The species that shows a higher adaptation to light is G. huxleyi, recording any sign of photoinhibition up to 2,500 μmol m^-2 s^-1 (with an optimum at 900 μmol m^-2 s^-1), whereas C. carterae records its optimum at 500 μmol m^-2 s^-1 (Heinle, 2014). Recently, Zhang et al. (2023) studied the wavelength effects on G. huxleyi, documenting a key role of blue and red lights on cell proliferation and fucoxanthin production. It must be noted that all these studies tested the effects of light through dilute batch experiments, neglecting a biotechnologically oriented approach. In fact, further experiments testing light in high cell density cultures would be useful to better understand coccolithophore response to this crucial parameter.

Besides permanent sequestration, coccolithophores offer diverse potential applications not available with other microalgal groups, especially those without a biosynthesized shell. Unfortunately, to date, many of those uses remain largely theoretical and only partially explored. Indeed, their dual organic–inorganic composition and distinctive morphology make coccolithophores attractive for various technological applications, including biomedicine, construction, nanotechnology, and sustainable materials (Green et al., 2014; Jakob et al., 2018; Moheimani, 2005; Moore et al., 2021; Moreira et al., 2023; Santomauro et al., 2020; Skeffington and Scheffel, 2018; Walsh et al., 2018). Optimizing their productivity is therefore essential to fully exploit their potential, and further effort is needed in this direction for future studies.

For all the three screened strains, nutrient enrichment up to 70 mg/L N and 8 mg/L P (N70 condition) did not lead to an increase in m_tot. Instead, the main differences were observed with carbon addition (N70 + C) compared to the other two conditions, showing a general increase in both OD and DW. Under N70 + C the C. roscoffensis strains—particularly CS2—displayed increased m_bio and overall CO₂ capture potential, whereas G. huxleyi preferentially increased calcification (high PIC:POC) and released abundant coccoliths that biased optical-density measurements. These outcomes indicate that balanced provision of bioavailable carbon together with N and P, as well as careful control of carbonate chemistry and pH, are essential to maximize both POC production and stable inorganic carbon sequestration in scaled coccolithophore cultivation.