Development of an Effective Microalgae Cultivation System Utilizing CO₂ in the Air by Injecting CaCO₃

Abstract

Recognized as the third-generation biomass of the future, microalgae are increasingly viewed as a promising solution for the sustainable production of biofuels, often referred to as “green gold.” Extensive research is being conducted across the upstream, midstream, and downstream sectors to develop fundamental technologies that enable efficient and economical large-scale microalgae cultivation. Recent studies suggest that microalgae-based biofuels have the potential to meet global energy demands. However, challenges such as spatial constraints in site selection and the high cost of transporting CO₂—an essential component for pH regulation and photosynthesis—pose obstacles. Here, this study demonstrates that by supplementing air-only medium with CaCO₃, Chlorella sorokiniana can effectively utilize airborne CO₂ to produce biomass. In laboratory-scale culture conditions supplied only with air, adding 5 mM CaCO₃ (pH 7.8) could maintain the pH stably compared to the untreated conditions (pH 9.5) and improved the biomass concentration and lipid content by 17.68-fold and 9.58-fold, respectively. In bench-scale conditions, cultures supplemented with 5 mM CaCO₃ exhibited a 9-fold increase in the biomass and a 7.15-fold increase in the lipid concentrations compared to those cultivated with air alone. With microalgae emerging as an essential resource for future generations, cultivation technology utilizing CaCO₃ will be a critical technology that enables commercial-scale microalgae cultivation using only air, without artificial CO₂ supply facilities.

1. Introduction

Recent studies indicate that industrialization has led to a roughly 50% increase in atmospheric carbon dioxide levels compared to preindustrial times [1]. According to Dvork et al., this ongoing rise in CO₂ levels is pushing the planet into unprecedented conditions, such as global warming. The effects of global warming include glacial melting, rising sea levels, genetic mutations, and various natural disasters such as typhoons, floods, and droughts, all of which directly impact humanity [2,3]. To mitigate CO₂, which accounts for over 79% of global warming, and to achieve carbon neutrality, more than 195 countries adopted the Paris Climate Agreement. This agreement aims to limit the average global temperature increase to well below 2 °C above preindustrial levels [1,4].

To reach this target, extensive research has been conducted on CO₂ reduction, with many findings being applied to industrial processes. Despite these efforts, global sea levels have continued to rise, and the atmospheric CO₂ concentration, global carbon emissions, and temperatures are still increasing. As a result, ongoing research is focused on developing practical solutions to reduce CO₂ emissions across various sectors, including energy production, industrial processes, construction and architecture, transportation, agriculture, and ocean management. Prominent technologies in this field include carbon capture and storage (CCS), carbon capture and utilization (CCU), and carbon capture, utilization, and storage (CCUS) [5,6].

CCS involves capturing CO₂ emitted from industrial and energy-related activities, transporting it to an underground storage site, such as beneath the ocean, and storing it there. However, this technology carries the risk of the stored carbon being released back into the environment during natural disasters like earthquakes or tsunamis, potentially polluting the local environment [7]. Additionally, storing carbon in the ocean can significantly contribute to ocean acidification, and the limited availability of suitable storage facilities poses another challenge [8,9,10]. An alternative approach, CCU, involves converting CO₂ generated by industry and energy into high-value products. CCU can be divided into chemical processes, which treat CO₂ to transform it into useful chemicals, and biological processes, which use microorganisms or plants to convert CO₂ into valuable biomass. Chemical processes often require complex reactions under high-temperature and high-pressure conditions, and the yield may not justify the associated costs. Conversely, biological processes are more efficient and environmentally friendly, utilizing photosynthetic organisms or electro-synthetic microorganisms to convert CO₂ into valuable substances [11,12,13,14]. Recently, CCUS, which combines the storage aspect of CCS with the utilization aspect of CCU, has gained attention. CCUS technologies, particularly those utilizing microalgae, are increasingly recognized for their sustainability potential. They not only fix carbon but also produce high-value products, making them a promising solution for carbon management [9,15,16].

However, microalgae have a slower growth rate compared to other microorganisms like bacteria and fungi, which limits their potential for large-scale CO₂ reduction. To address this issue, Yu et al. developed a technology that enhances the production of high-value products and biomass from microalgae by incorporating a biomineralization process into microalgal cells. This innovation significantly improves CO₂ removal efficiency by producing additional CaCO₃ through the complete conversion of CO₂, alongside increased biomass production [17]. This approach not only addresses the slow growth rate of microalgae but also yields valuable calcite, a form of CaCO₃ widely used in industries such as cement, plastics, paints, agriculture, and neutralizers [18]. In other studies, to increase the biomass production and carbon fixation capacity of microalgae in response to climate change, the culture conditions were optimized by adjusting the nitrogen and phosphorus concentrations in the cell culture medium and supplying 10–20% CO₂ [19,20].

It is important to note that cultivating microalgae through flue gas injection primarily addresses future emissions, not the CO₂ that has already been released and remains in the atmosphere [21]. The atmosphere already contains a significant amount of CO₂, and while reducing future emissions—such as through alternative energy sources—is crucial, it does little to remove the existing atmospheric CO₂. Despite various strategies devised to lower CO₂ emissions, finding an effective method to remove large quantities of CO₂ from the atmosphere remains a significant challenge. One current approach to atmospheric CO₂ reduction is reforestation, where plants absorb CO₂. However, with ongoing deforestation due to urban development, the capacity of this natural method alone to solve the problem is physically limited. Unlike microalgae, which convert CO₂ into bicarbonate that is quickly absorbed by cells and used for growth, trees utilize CO₂ directly, resulting in a much lower rate of CO₂ reduction [22,23,24,25]. Therefore, developing direct air capture (DAC) technology that can reduce atmospheric CO₂ in conjunction with microalgae cultivation would be a far more effective strategy for mitigating CO₂, the primary driver of global warming [26].

To implement this approach, CaCO₃ produced through biomineralization was injected as a CO₂ replacement source in photosynthetic microalgal cultures. In microalgae, carbonic anhydrase (CA) enzymes are typically activated in low-CO₂ environments, with CAH1 preferentially converting ambient CO₂ to HCO₃⁻ to form the intracellular inorganic carbon (Ci) pool. Since microalgae inhabit aquatic environments where HCO₃⁻ is the dominant form of Ci over CO₂, CA enzymes play a crucial role in this process [27,28,29]. Moreover, in low-CO₂ environments, Ci transport is upregulated by up to 1000-fold through the use of additional adenosine triphosphate (ATP), enabling microalgae to rapidly take up CO₂ or HCO₃⁻ from their surroundings. This process inhibits respiration by preventing CO₂ loss, maintaining the internal pH, and enhancing the photosynthetic efficiency [30,31]. The role of CaCO₃ here is vital, as it allows large amounts of CO₂ to remain available for uptake by the cells for a longer period, a feat that is difficult to achieve using conventional methods.

This study presents an efficient strategy to reduce atmospheric carbon while maintaining biomass and lipid production in microalgal cultures, even when the carbon supply is interrupted. Specifically, we cultured Chlorella sorokiniana, a representative microalgal strain, under ambient CO₂ conditions with the addition of CaCO₃ and compared it to the conventional method of culturing with a 5% CO₂ supply. We measured the biomass concentration, lipid content, and CA activity. Additionally, we monitored the pH and dissolved inorganic carbon (DIC) concentration in the medium to assess the direct impact of these conditions on the culture. Finally, we scaled up the cultures and compared the biomass and lipid production under both conditions. Our results confirmed that this strategy could serve as an innovative DAC technology, potentially applicable to large-scale cultivation for reducing atmospheric CO₂ emissions while producing valuable products.

2. Materials and Methods

2.1. Algal Strains and Culture Conditions

The strain C. sorokiniana UTEX 2714, obtained from the University of Texas Algae Collection in Austin, TX, USA, was used in this study. Tris–acetate–phosphate (TAP-C) medium without acetate was used for cultivation [32]. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 20 mM) was used to prepare a buffer system to maintain an optimal pH between 7 and 7.5. The experiments were conducted indoors at 20–25 °C, and the light intensity was set at 300 µE m⁻² s⁻¹ using LED lights (Lumens, Yongin, Republic of Korea). Cultivation was performed in a 500 mL mass cylinder (height × diameter, 60 cm × 5 cm), and a 2–3 mm hole was drilled in the silicone multipurpose inlet/outlet to install the gas supply and sampling line. Subsequently, two Teflon tubes were cut to the height of the mass cylinder and inserted into the holes. Finally, a stone sparger was connected to the bottom of the Teflon tube to supply 5% CO₂ and ambient air CO₂ (0.04% CO₂) directly to the culture medium. For scale-up, the microalgal cells were centrifuged and inoculated into a 5 L photobioreactor (PBR) made of a glass bottle. CO₂ was supplied at 0.1 vvm (volume of air added to liquid volume per minute) through a gas line connected to a 5 L PBR, and 10 mM potassium hydroxide (KOH) was supplied instead of expensive HEPES to maintain a stable pH, forming a bicarbonate buffer system. Acetazolamide (AZ) (A6011, >99 atom%) powder, purchased from Sigma-Aldrich (St. Louis, MO, USA), was used to inhibit CA enzyme activity in the microalgae cells [33,34].

2.2. Analytical Methods

2.2.1. Analysis of Microalgal Biomass from Mixed Samples

To determine the dry weight of the microalgal biomass, a 10 mL sample was taken from the cultured mass cylinder. The sample containing microalgal cells and CaCO₃ was centrifuged at 3000 rpm for 10 min to obtain a pellet containing both cells and calcite. For the analysis of the microalgal biomass, the cells were tested using a Refrigerated Centrifuge 5430R (Eppendorf, Hamburg, Germany) at the Center for Bio-Medical Engineering Core Facility (Dankook University, Cheonan, Chungnam, Republic of Korea). The pellet was washed 2–3 times with DI water (pH 8.0) and centrifuged at 3000 rpm for 10 min to obtain a pellet containing the biomass and calcite, which was then resuspended in 10 mL of deionized (DI) water (pH 7.5). Subsequently, 5 mL of the sample was used to analyze the dry weight of the cells and calcite. Firstly, 35 mL of DI water (pH 4.5) was added and stirred for approximately 1 min to dissolve CaCO₃, which is soluble under acidic conditions below pH 5.0. The CaCO₃ utilized in the culture was produced through the biomineralization process of Yu et al. [17]. The sample was then centrifuged to obtain CaCO₃ dissolved in the biomass. The remaining 5 mL of the cell sample was used to determine the dry cell weight. To determine the dry cell weight, 5 mL of the remaining cell sample was used. An amount of 5 mL of the sample was loaded onto GF/F glass microfiber filters (Whatman, Cambridge, UK) [35]. Before use, the filters were washed with DI water to remove as much salt as possible and dried overnight in a drying oven at 70 °C. The amount of extracellular calcite was calculated by subtracting the dry weight of the cells from the total dry weights of the cells and calcite. To verify that the sample treated with low-pH DI effectively removed CaCO₃, inductively coupled plasma optical emission spectrometry (ICP-OES; Agilent Technologies, Santa Clara, CA, USA) was used. The results showed that the Ca²⁺ concentration of the sample treated with the low-pH DI was reduced by approximately 99.9% compared to the sample with no pretreatment, and CaCO₃ was still present, proving that the pretreatment method was correct.

2.2.2. Analysis of pH and Dissolved Inorganic Carbon (DIC) Concentration

To measure the pH and dissolved inorganic carbon (DIC) concentration in the culture media, the samples were centrifuged at 3000 rpm for 10 min at 23 °C to separate the supernatant only. The pH of the supernatant was measured using a digital pH meter (Orion Star A211; Thermo Scientific, Waltham, MA, USA). The supernatant was titrated against standardized hydrochloric acid (HCl, 0.03 N), using phenolphthalein and methyl orange as indicators to determine the DIC concentration. An amount of 20 mL of the supernatant was placed in a 50 mL conical tube with a pH meter attached and titrated with HCl using the phenolphthalein (pKa = 8.6) indicator to ensure that the first endpoint appeared colorless. The methyl orange (pKa = 3.8) indicator was used to determine the second endpoint. The first endpoint corresponded to OH⁻ + CO₃²⁻ (total alkalinity) and the second endpoint corresponded to OH⁻ + HCO₃⁻ (total acidity). The DIC concentration was measured by subtracting the volume at the second endpoint from the first endpoint using the formula V₁N₁ = V₂N₂, where V₁ represents the volume of the supernatant, N₁ is the DIC concentration (mg L⁻¹) in the sample, V₂ is the subtracted value, and N₂ is the known concentration of HCl [32].

2.2.3. Analysis of CA Activity

The potentiometric method was used to measure the CA activity of the cells under different conditions, including in the absence of CO₂ [36]. The microalgal species was C. sorokiniana and appropriate cell suspensions (10⁶ cells) were centrifuged for 10 min at 3000 rpm, 4 °C using cells supplied with 5% CO₂, cells supplied with air alone, cells supplied with 5 mM CaCO₃ and air simultaneously, and cells supplemented with AZ to inhibit CA enzyme activity in each condition, and the supernatant was removed. The cells were then washed at least twice with pre-chilled 0.010 M Tris buffer, prepared by mixing 1 mM dithiothreitol (DTT) and 1 mM EDTA at pH 8.3. After confirming that the cells were intact under a microscope, the pellet was resuspended in the same buffer. The enzyme activity was measured as rapidly as possible using a slightly modified potentiometric method [37]. The reaction was continued in a sealed flask maintained at a constant temperature (0 °C) and 1.5 mL of DI water saturated with CO₂ for 30 min at 0 °C was added to 3 mL of buffer with (Ta) or without (Tb) each sample extract. The time required for the CO₂-saturated DI water to lower the pH of both solutions by one unit was measured, and the enzymatic activity was calculated using the following equation:

(Tb Ta − 1) − 1 = UA × 10⁶

(1)

Equation (1) expresses the enzymatic activity as units of activity (UA) per cell (10⁶ cells were employed per measurement of activity, p < 0.05).

2.2.4. Lipid Extraction and Analysis of Total Lipid Content from Microalgal Cells

To measure the total lipid content, lipids were extracted from microalgal cells using the modified Bligh and Dyer method [38,39,40]. Prewashed screw-capped glass vials were prepared, dried in a 60 °C dry oven for 2 h, and weighed using a precision balance (T₁). For lipid extraction, 5 mL of each sample was prewashed with DI water and dried in a screw-cap glass vial. After centrifugation at 3000 rpm for 10 min, 2.6 mL and 1.3 mL of methanol and chloroform were added at a volume ratio of 2:1. The lid of the glass vial was closed, wrapped with parafilm and Teflon tape to prevent evaporation, and heat-extracted in a heat block heated to 50 °C for 1 h. Cell debris was observed as a colorless white pellet at the bottom of a screw-capped glass vial after heating, vortexing, and centrifugation at 3000 rpm for 10 min to separate the supernatant. After transferring the supernatant to a new screw-capped glass vial, 1 mL of DI water and 1.3 mL of chloroform were added, and the solvent was separated. At this time, methanol separated into the water layer, and lipids separated into the chloroform layer. Subsequently, the weight of the pre-dried glass vial was measured and 1 mL of the lower layer (chloroform layer) of the centrifuged sample was transferred to a weighing vial. It was incubated overnight in a 50 °C heat block to remove residual chloroform and the weight of the glass vial (T₂), in which only the lipid remained, was measured. The total lipid content was calculated using Equation (2).

$${\displaystyle \frac{T_2-T_1}{5 \mathrm{mL}}}\times \mathrm{conversion}\mathrm{factor}\left({\displaystyle \frac{1}{2.6}}\right)=\mathrm{Total}\mathrm{Lipid} \left({\displaystyle \frac{\mathrm{g}}{\mathrm{mL}}}\right)$$

(2)

Conversion factor (1/2.6): only 1 mL of the 2.6 mL chloroform layer containing dissolved lipids was taken and weighed.

The total lipid content was expressed as a percentage of the amount of lipid contained in the cell dry weight, measured according to the method described in Section 2.2.1. The total lipid content was calculated using Equation (3).

$${\displaystyle \frac{\mathrm{Total}\mathrm{Lipid}}{\mathrm{Dry}\mathrm{Cell}\mathrm{Weight}}}\times 100=\mathrm{Total}\mathrm{lipid}\mathrm{content}\left(\%\right)$$

(3)

2.2.5. Analysis of ¹³C Isotope Ratios in Culture Media and Cell Biomass

To analyze the dissolved inorganic ¹³C in the culture media, liquid samples were pipetted in duplicate into septum-capped tubes. The tubes were then flushed with 99.995% helium, and phosphoric acid was added to and mixed with the sample medium. The CO₂ gas released from the samples was analyzed using continuous-flow isotope-ratio mass spectrometry (CF-IRMS) [41,42]. CO₂ was resolved on a packed column gas chromatograph, and the resultant chromatographic peak was carried forward into the ion source of a Europa Scientific 20–20 IRMS where it was ionized and accelerated. Gas species with different masses were separated in a magnetic field and simultaneously measured using a Faraday cup collector array to determine the isotopomers of CO₂ at m/z 44, 45, and 46. Analysis of ¹³C in the microalgal biomass was performed in the same way as the culture solution analysis, with the only addition allowing the samples to react with the acid overnight at room temperature after phosphoric acid injection. CO₂ was sampled using a needle as a continuous stream of He flowing in a tube. For reference, ¹³C-labeled CaCO₃ (SKU 492027, >99 atom%, Sigma-Aldrich, St. Louis, MO, USA) was used for isotopic analysis of the cultures and cell biomass.

3. Results and Discussion

3.1. Introduction of CaCO₃ from Biomineralization to Microalgae Cultivation System for Preventing Cell Death from Ambient Air CO₂ Supply

The biological conversion of CO₂ using photosynthetic microorganisms, such as microalgae, is gaining recognition as an economical CO₂ abatement technology that not only reduces CO₂ levels but also enables the production of various value-added materials, including building materials, biofuels, compost, animal feed, biodegradable bioplastics, and, more recently, pharmaceuticals [43,44]. A stable CO₂ supply is essential for this process, as it also plays a significant role in maintaining optimal pH levels in microalgae cultures. However, several challenges exist that confine cultivation facilities to areas near CO₂-emitting sources, such as power plants [45]. The costs associated with transporting CO₂ to a culture facility, whether through pipelines or other means, can be substantial. Transporting CO₂ over long distances increases the risk of leaks or losses, reducing the overall efficiency of microalgal cultivation. Additionally, the installation and maintenance of infrastructure to deliver CO₂ to these facilities can incur further expenses.

As a result, locating microalgae cultivation facilities near CO₂ emission sources is considered a strategy to maximize the economic, operational, and environmental benefits, which are diminished when facilities are situated farther away. However, if a CO₂-free culture is established, the growth rate of photosynthetic microalgae may slow significantly, and maintaining pH regulation becomes a major challenge. Disruptions in the artificial gas supply can destabilize the buffer system regulating the culture’s pH, leading to rapid pH increases and subsequent cell death [46,47]. One proposed solution is to store CO₂ in a tank during normal incubation periods and utilize it when needed, or to inject weak acids or bases to maintain the pH during incubation. However, these methods are time-consuming, expensive, and pose safety concerns due to the constraints of tank storage.

Given these limitations, there is a pressing need to develop new technologies that can replace anthropogenic CO₂ with atmospheric CO₂. Such technologies would offer significant environmental and industrial benefits by harnessing the CO₂ already present in the atmosphere. In previous research, Yu et al. introduced a technology that utilizes urea and KH₂PO₄ to minimize biomass and lipid productivity losses during overhaul periods in CO₂-fed culture facilities. However, these substances must be continuously injected during CO₂ supply interruptions, and the primary focus is on minimizing productivity losses rather than actively removing CO₂ [32].

In this context, we present a strategy for effectively operating a culture system by utilizing airborne CO₂ without the need for artificial flue gas injection. In a previous study, Hong et al. performed biomineralization to increase the biomass productivity of Neochloris oleoabundans and C. sorokiniana [48]. In our study, CaCO₃, which can be obtained at no cost through biomineralization, was injected into the C. sorokiniana culture medium, and ambient air CO₂ (AC) was supplied. The results demonstrated that this approach successfully maximized the biomass and lipid content while maintaining stable pH levels (Figure 1). CaCO₃ played a key role by effectively adsorbing CO₂ onto its crystal surface, prolonging the residence time of CO₂ in the culture medium, and allowing cells to utilize the CO₂ or, in an aquatic environment, convert it to bicarbonate for growth and lipid accumulation.

3.2. Effect of Cost-Effective Materials CaCO₃ on Microalgal Biomass Production

3.2.1. Effect of CaCO₃ Concentration on Biomass Production

CaCO₃ is known for its relatively low solubility in culture media. However, it dissociates into Ca²⁺ and CO₃²⁻ ions, and its solubility can slightly increase in the presence of CO₂, producing Ca²⁺ and HCO₃⁻. This characteristic allows CaCO₃ to act as a marginal carbon source for microalgal cells when the artificial CO₂ supply is interrupted. To investigate whether C. sorokiniana cells could rely solely on CaCO₃ for growth, we analyzed the biomass production as a function of the concentration of injected CaCO₃ under AC supply conditions. C. sorokiniana cells were cultured with CaCO₃ concentrations ranging from 0 to 10 mM, added in 1 mM increments. The CaCO₃ used in this study was produced through a biomineralization process. The results showed that as the concentration of CaCO₃ increased, the biomass concentration also increased, reaching a peak at 5 mM CaCO₃. Each injected CaCO₃ concentration was treated by quantifying the corresponding amount based on the total volume of the culture solution. Beyond this concentration, no significant changes in the biomass were observed, and there was even a reduction in the biomass at 7 mM. Specifically, cultures injected with 5 mM CaCO₃ demonstrated a substantial increase in biomass production, yielding 2.563 g L⁻¹, which was 17.68 times higher than the biomass produced in cultures with ambient CO₂ alone, without the addition of CaCO₃ (0.145 g L⁻¹) (Figure 2a). These findings suggest that under autotrophic conditions with AC, C. sorokiniana cells achieved optimal growth when supplemented with 5 mM CaCO₃.

3.2.2. Effect of Carbonic Anhydrase Enzyme on Biomass Production and Resulting pH Changes

Mishra et al. [30] demonstrated that in low-ambient CO₂ conditions, photosynthetic bacteria and marine algae optimize photosynthesis by enhancing CO₂ availability through a CCM. This mechanism involves increased activity of the CA enzyme, which facilitates the uptake of more ambient CO₂ and reduces competitive inhibition by oxygen, resulting in a higher CO₂ concentration at the Rubisco site [30,49,50,51]. This process ensures efficient carbon fixation and photosynthesis even under low-CO₂ conditions. To investigate this, we compared the biomass productivity of C. sorokiniana grown under 5% CO₂, 5 mM CaCO₃, and AC with the addition of AZ to inhibit the CA enzyme [52]. After 7 days of autotrophic induction, Chlorella cells cultured with 5 mM CaCO₃ and AC (2.563 g L⁻¹) showed a significant difference in biomass concentration of 2.261 g L⁻¹ compared to cells cultured under the same conditions with the addition of AZ alone (0.302 g L⁻¹). Conversely, under the 5% CO₂ conditions, there was minimal difference in the biomass concentration with or without AZ, as CO₂ is delivered to Rubisco via diffusion. This indicates the role of CA enzymes in low-CO₂ conditions, but also highlights that under air conditions alone, cells eventually die despite the carbon capture mechanism (CCM) being active. Typically, when using a bicarbonate buffer system, the pH is regulated through the interconversion of bicarbonate (an acid) and carbonate (a base). Adding additional DIC to the buffer increases its capacity. However, when the CO₂ supply is interrupted, the bicarbonate buffer system cannot replenish DIC, leading to an imbalance and eventual buffer system failure. As shown in Figure 2b, under conditions with air alone, the buffer system failed, resulting in cell death as the pH exceeded 9 after only 2 days, peaking at 9.5 on day 4. In contrast, adding 5 mM CaCO₃ to the air conditions maintained a stable pH up to 7.8, consistent with Hong et al.’s findings that N. oleoabundans and C. sorokiniana can sustain biomass production up to pH 8 without significant loss [46]. The pH increased to 9.42 when AZ was added to conditions with 5 mM CaCO₃ and air, suggesting that CA enzymes are crucial for effectively utilizing CO₂ from the air. The addition of CaCO₃ likely extends the residence time of CO₂ in the culture, enhancing the effectiveness of the CCM even with the low-CO₂ concentration of 0.04% in the air.

3.3. Analysis of the Cause of Biomass Increase When 5 mM CaCO₃ Was Injected with AC

To determine the contribution of dissolved CaCO₃ to the final biomass concentration of C. sorokiniana under ambient CO₂ (AC) conditions, CaCO₃ was weighed before and after cultivation (

Table 1. Biomass and lipid production and productivity of C. sorokiniana cultured for 12 days under different growth conditions, as well as CaCO₃ consumption, CO₂ removal rate, and conversion rates from CaCO₃ and CO₂ to reach the respective biomass concentrations. Data shown represent mean values obtained from three independent experiments. Abbreviation: AZ, acetazolamide; AC, ambient air CO₂ (0.04%).

Parameters (during Autotrophic Induction)	Chlorella sorokiniana (UTEX 2714)
	5% CO2	AZ with 5% CO2	5 mM CaCO3 with AC	5 mM CaCO3 and AZ with AC	AC
CaCO3 consumption (g)	-	-	0.272	0.056	-
Biomass conversion rate (g) from dissolved calcite	-	-	0.066	0.014	-
Biomass conversion rate (g) from dissolved CO2	2.829	2.814	2.497	0.288	0.145
Biomass concentration (g L–1)	2.829	2.814	2.563	0.302	0.145
Biomass productivity (g L–1 day–1)	0.404	0.402	0.366	0.043	0.021
Lipid content (g g–1)	0.274	0.269	0.249	0.028	0.026
Lipid productivity (g L–1 day–1)	0.111	0.108	0.091	0.001	0.001
CO2 removal rate (g L–1 day–1) *	1.029	1.024	0.932	0.11	0.053

* CO₂ removal rates of biomass and calcite were calculated as follows: CO₂ removal rate (biomass) (g L^–1 day^–1) = [biomass concentration (g L^–1) × carbon content in biomass (49.6%, C. sorokiniana) × 44/12 (conversion factor: CO₂/C)]/culture time (day).

). The initial amount of CaCO₃ used was 0.272 g L⁻¹, which corresponds to a potential yield of 0.120 g L⁻¹ of CO₂, calculated using the molecular weight ratio of CO₂ to CaCO₃ (0.272 × CO₂ molecular weight/CaCO₃ molecular weight). Using a conversion factor of 1.83 (as outlined in the CO₂ removal rate formula in Table 1), the amount of biomass that could theoretically be produced from this CO₂ is 0.066 g L⁻¹. This calculation reveals that the dissolved CaCO₃ contributed only 2.58% to the final biomass, with the majority (97.42%) of the biomass being derived from CO₂ in the air. This confirms that CO₂ from the atmosphere was the primary carbon source for the cells, even under conditions where CaCO₃ was supplemented. To further analyze the biomass accumulation, we measured the total inorganic carbon throughout the cultivation period. The total inorganic carbon increased slightly from 629.3 mg L⁻¹ to 633.04 mg L⁻¹ between days 2 and 8, before starting to decrease. Given that the biomass yield for a carbon source typically ranges from 50% to 60%, we assumed a 55% conversion of carbon to biomass on each cultivation day. Interestingly, from days 4 to 8, the actual biomass increase was up to twice the theoretically possible biomass accumulation (Figure 3a). This discrepancy led us to investigate the changes in carbonic anhydrase (CA) activity over the cultivation period. We found that the CA activity was significantly higher when 5 mM CaCO₃ and AC were supplied, compared to conditions with either 5% CO₂ or AC alone. Specifically, on day 6, the CA activity was 82.6% higher than under the 5% CO₂ conditions and 29.64% higher than under the AC conditions alone. This sharp increase in the CA activity between days 4 and 8 indicates that the CCM was much more active during this period, allowing C. sorokiniana cells to utilize more CO₂ from the air and maximize their internal Ci pools (Figure 3b). This enhanced CA activity and CCM likely contributed to the higher-than-expected biomass accumulation observed during the latter part of the cultivation period, emphasizing the crucial role of CA enzymes in optimizing carbon utilization under low-CO₂ conditions.

3.4. Comparison of Microalgal Lipid Content and Productivity According to Different Culture Systems

Lipids were extracted on day 7 of the induction phase, when all nitrogen sources in the culture had been depleted and the intracellular lipids had reached their maximum accumulation. As shown in Figure 4, the lipid content in C. sorokiniana peaked when supplied with 5% CO₂ and was comparable to that observed with AZ injection under these conditions, consistent with the biomass results. In contrast, the lipid content drastically decreased by a factor of 10.54 in the culture with AC alone compared to the 5% CO₂ condition. However, the lipid content increased to 24.9% when 5 mM CaCO₃ was added to the AC condition, which was 9.58 times higher than the lipid content with AC alone, and only 2.5% (0.025 g g⁻¹) less than the content observed under the 5% CO₂ condition. To assess whether the increase in the lipid content with 5 mM CaCO₃ was due to the CCM facilitated by the CA enzyme activity, the culture was further treated with AZ. The lipid content under these conditions was 8.89 times lower than without AZ and nearly identical to that of the AC-only condition. This confirms that the CA enzyme activity effectively utilized the 0.04% CO₂ present in the air. Zhao et al. have noted that Chlorella can effectively accumulate lipids when the pH is below 7.8–8.0, with significant decreases in lipid accumulation above this pH range [53]. Thus, the addition of 5 mM CaCO₃ is crucial for efficient CO₂ utilization from the air, maintaining the pH below 8 and delivering CO₂ effectively to the intracellular Rubisco. The lipid productivity followed a similar trend to the lipid content. Under the AC conditions, the lipid productivity with 5 mM CaCO₃ was 0.091 g L⁻¹ day⁻¹, which was 128 times higher than the productivity without CaCO₃ (0.001 g L⁻¹ day⁻¹). This difference was only 1.21 times greater than that observed with 5% CO₂. Therefore, it can be concluded that the addition of 5 mM CaCO₃ under AC conditions is an effective technique for enhancing the biomass productivity and lipid content (Table 1, Figure 4).

3.5. Isotopic Analysis to Determine Whether the Carbon Source Utilized by C. sorokiniana Cells for Biomass Accumulation Was from Air or CaCO₃

Isotope analyses showed that C. sorokiniana effectively used atmospheric CO₂ rather than CaCO₃ as a carbon source for biomass accumulation. Previously, calcium carbonate with ¹²C, which is biomineralized through Ca²⁺ and CO₂, was used. However, in this experiment, CaCO₃ with ¹³C was purchased and used in the culture. Since airborne CO₂ has ¹²C (δ¹³C_PDB = −20.2‰) and purchased CaCO₃ has ¹³C (δ¹³C_PDB = 4.58‰), determining which isotopic ratio is higher in the biomass can reveal whether the carbon source is from air or CaCO₃. The carbon content within the biomass of C. sorokiniana cultured in 5% CO₂ was 47.33% with a δ¹³C_PDB value of −49.53‰, indicating that the ¹²C present in the CO₂ was utilized. If the CaCO₃ was dissolved and used, the δ¹³C_PDB value inside the C. sorokiniana biomass under the condition that AC was supplied with CaCO₃ should be positive, indicating the presence of ¹³C. However, the internal carbon content was 48.332%, and the δ¹³C_PDB value was −48.27‰, which was not significantly different from the culture conditions supplied with 5% CO₂ (Figure 5). Thus, we demonstrated that in an AC environment without an artificial CO₂ supply, the cells did not grow on the carbon source CaCO₃ but instead utilized airborne CO₂. This also indicates that the supplied CaCO₃ prolongs the time that airborne CO₂ remains in the culture, allowing it to be used efficiently in CCMs.

3.6. Analyzing the Effect of CaCO₃ on Microalgal Cultures and DIC Concentration Changes and the Role of CA Enzymes

To determine whether CaCO₃ effectively delivered HCO₃⁻ into the cells by extending the residence time of airborne CO₂ in the culture, we conducted experiments using N₂ gas instead of air. Under the N₂ conditions, the biomass concentration with 5 mM CaCO₃ was significantly reduced to 0.20 g L⁻¹ (Figure 6a). The addition of AZ under the N₂ conditions resulted in a greater loss of biomass compared to AC alone, indicating that the contribution of CaCO₃ to cell growth was minimal. This suggests that AZ plays a crucial role in cell growth by trapping CO₂ from the air. Additionally, we examined the change in the DIC concentration in the TAP-C medium used for the culture. Initially, the DIC concentration was 100 mg L⁻¹. Under the AC conditions, when only 5 mM CaCO₃ was added, the DIC concentration increased to 200 mg L⁻¹. With the addition of C. sorokiniana, the DIC concentration improved by 97.5%, reaching 395 mg L⁻¹. This increase was attributed to the activation of the CA enzyme in C. sorokiniana, which rapidly converts CO₂ to HCO₃⁻, thereby enhancing the Ci pool. Furthermore, the maximum DIC concentration with 5 mM CaCO₃ and C. sorokiniana was 3.87 times and 3.59 times higher compared to the conditions without cells and those cultured under AC conditions, respectively (Figure 6b). These findings suggest that cultivating with 5 mM CaCO₃ under AC conditions effectively utilizes CO₂ from the air as a carbon source for cell cultures. This approach could offer significant industrial benefits by addressing challenges related to CO₂ emission and establishing large-scale microalgae cultivation facilities.

3.7. Analyzing the Effect of CaCO₃ on Biomass Production and Resulting pH Changes in Microalgal Cells with AC under Bench-Scale Culture

To assess the impact of the CaCO₃ addition on the biomass concentration and lipid content in bench-scale cultures, C. sorokiniana was cultured in a 5 L PBR. The initial inoculum concentration of C. sorokiniana was consistent across all conditions, set at 0.05 g L⁻¹ based on the biomass concentration. Under the 5% CO₂ conditions, CO₂-enriched air was directly supplied to the PBRs. In contrast, under the AC conditions, air was introduced into the PBRs using an air pump throughout the 20-day cultivation period. The culture conditions were altered from an HEPES buffer system to a bicarbonate buffer system using only KOH.

As shown in Figure 7a, the bench-scale cultures mirrored the laboratory-scale results, with the highest biomass concentration achieved under the 5% CO₂ conditions. Under the AC conditions, biomass growth occurred until day 4 of incubation but then plateaued. Specifically, the biomass concentration under 5% CO₂ (2.49 g L⁻¹) was 9.94 times higher than that under air alone (0.25 g L⁻¹). However, in the cultures supplemented with 5 mM CaCO₃ under the AC conditions, the biomass increased steadily throughout the cultivation period, peaking at 2.25 g L⁻¹ on day 20—approximately 9 times higher than the biomass under the AC conditions alone.

The lipid content was also significantly higher under the AC conditions with 5 mM CaCO₃, measuring 0.286 g g⁻¹, which was 7.15 times greater than that of the cells cultured under the AC conditions alone (0.04 g g⁻¹). The pH measurements confirmed that under the 5% CO₂ conditions and with 5 mM CaCO₃ and AC, the final pH remained stable at 7.3 and 7.74, respectively. In contrast, the pH under AC alone was 9.89, indicating a collapse of the buffer system that inhibited cell growth (Figure 7b). This study demonstrates that high biomass and lipid production can be achieved without CO₂ supply, even in bench-scale cultures. These findings suggest the potential for establishing a cost-effective biofuel production process that could significantly reduce the carbon footprint.

4. Conclusions

Biological conversion using microalgae has emerged as a promising method due to its dual benefits of CO₂ reduction and the production of valuable substances. However, current research has primarily targeted reducing future CO₂ emissions rather than addressing the CO₂ already accumulated in the atmosphere. Additionally, transporting CO₂, essential for maintaining the pH and supplying carbon sources in cultivation, is expensive, which restricts microalgae cultivation facilities to locations near CO₂ emission sources. To address these challenges, we developed an innovative culture system that significantly enhances biomass and lipid productivity while maintaining pH stability, without relying on an artificial CO₂ supply. This technology utilizes air and 5 mM CaCO₃ to increase the CA enzyme activity in C. sorokiniana, rapidly converting CO₂ in the air to bicarbonate, allowing cells to utilize it to the maximum. The culture system utilizing CaCO₃ provides a practical and industrially applicable method for producing useful materials while removing CO₂ in the air without constraints on site selection for constructing culture facilities. This advancement is significant as it provides an effective solution for utilizing atmospheric CO₂ and can potentially overcome the limitations of traditional CO₂ transport and supply methods.