Biological Carbon Capture Using Spinach Carbonic Anhydrase Immobilized on Magnetite Nanoparticles

Abstract

Bio-based carbon capture, utilization, and storage (CCUS) presents a promising alternative to conventional CCU methods, primarily due to its inherent potential for valorization. In the present study, carbonic anhydrase extracted from spinach leaves (Spinacia oleracea) was immobilized onto citric acid-functionalized magnetite nanoparticles (Fe₃O₄@CA NPs). This bio-nano hybrid functions as an efficient catalyst for enhancing CO₂ solubility by accelerating its conversion to bicarbonate (HCO₃ ⁻), thereby overcoming the low aqueous solubility of gaseous CO₂, a known limiting factor in photosynthetic autotrophs. Growth experiments using Escherichia coli cultures supplemented with these NPs demonstrated a ~62% increase in biomass production compared to the control group when the culture was sparged with atmospheric air, demonstrating that carbonic anhydrase-immobilized NPs effectively facilitated the uptake of atmospheric CO₂ and redirected it into cellular biomass. Considering that 1 g E. coli dry cell weight can capture ~86 mg CO₂, this approach can be used for carbon capture and production of fermentation-derived value-added products. Moreover, such systems hold significant potential for applications in algal biofuel production and the cultivation of slow-growing organisms, such as cyanobacteria, where efficient carbon assimilation is crucial for their growth.

Keywords

Carbon capture, utilization, and storage - Carbonic anhydrase - Magnetite nanoparticles - Enzyme immobilization

Significance

The present study explores the feasibility of linking CO₂ capture to microbial biomass production using carbonic anhydrase as a biocatalyst. Carbonic anhydrase increases the aqueous availability of CO₂ for organisms to utilize. This sustainable and economically viable system can be scaled up to meet the needs of large-scale CO₂ capture. In the current context, significant research is focused on employing stable and reusable forms of carbonic anhydrase for carbon capture and its conversion into biomass and other value-added products. This study explores such an approach, aiming to provide experimental validation for its potential.

Introduction

Climate change, driven by anthropogenic activities, such as burning of fossil fuels for electricity and transportation, large-scale industrial manufacturing, deforestation for agriculture and urban expansion, and improper waste-management practices like open dumping and methane-emitting landfills, continues to intensify, necessitating urgent and coordinated efforts from both government bodies and scientific institutions. A major contributor to this crisis is the rise in greenhouse gas (GHG) emissions, with global GHG levels reaching 53.0 gigatonnes CO₂-equivalent in 2023.[1]

Although CO₂ plays an important role in photosynthesis as well as the carbon cycle, excessive accumulation of carbon dioxide in the atmosphere can promote the greenhouse effect, which ultimately leads to global warming. It has been observed that there has been a significant rise in the atmospheric carbon dioxide levels in a span of 60 years, from 320 ppm to approximately 420 ppm,[1] with excessive combustion of fossil fuels being the primary contributor to the increase in CO₂ levels in the atmosphere. On a global scale, the world’s largest GHG emitters are China, the United States, India, the EU, Russia, and Brazil. These countries collectively contribute to more than half of the GHG emissions (62.7%), and their fossil fuel consumption is estimated to be 64.2% when compared globally.[2] The emission of greenhouse gases into the atmosphere can result in elevated global temperature, melting of polar ice caps, a rise in sea level, disruption of food webs, and an imbalance in the biogeochemical cycles.[3]

To combat this threat, many technology-driven innovations have been introduced that can help reduce CO₂ levels. In order to reduce our reliance on fossil fuels, sustainable alternatives such as wind turbines, hydroelectric power, photovoltaic cells, and carbon capture technologies are employed.[4] Carbon capture, utilization, and storage (CCUS) has been identified as one of the key components in combating climate change. The implementation of CCUS aligns with the United Nations’ Sustainable Development Goals (SDGs). Reducing global emissions is essential to limit global warming to 1.5 °C, which was the goal of the Paris Agreement. Carbon capture technologies thus have an important role in reducing global emissions.[5]

Industrial sites and power plants represent point sources of CO₂ emissions. CCUS technologies can be employed at such sites. However, this may require major modifications to be made to existing infrastructure, making it costly and time-consuming. Hence, carbon capture technologies have evolved around the postcombustion stage. This reduces operational costs by implementing the capture systems as a retrofit to the existing infrastructure.[5] Postcombustion capture technologies can be broadly categorized into physical, chemical, and biological methods. Among these, a commercialized technology is the absorption method, which can be either physical, chemical, or a combination of both, using mixed solvents. Currently, the most mature technology available is the chemical scrubbing method. It involves CO₂ capture using an amine-based solvent (alkanolamine solvents such as monoethanolamine), desorption, dehydration, and compression of the CO₂ for storage and transport. However, a limitation of these methods is the additional cost and energy required for the CO₂ to be released from its captured form, its storage, and transportation. This can be mitigated by integrating the capture step with the utilization step. Thus, the captured CO₂ can be directed toward the synthesis of valuable products, eliminating the previously mentioned energy-intensive steps.[6] [7]

One approach for CCUS is the biological fixation of CO₂, which leverages the carbon-fixing ability of various organisms to utilize the captured CO₂. Microorganisms like algae, cyanobacteria, fungi and yeast, as well as certain fast-growing and resource-efficient plants, can convert atmospheric CO₂ into biomass through photosynthetic and nonphotosynthetic pathways.[8] [9]

Flue gas from boilers and burners, which are point sources of CO₂ emissions, typically has a CO₂ concentration of 3–15%. Thus, CO₂ in the gas phase has to be captured and transferred to an aqueous phase so that the carbon-fixing organism can effectively utilize it. Instead of the conventional amine-based scrubbing, using enzymes could be considered as a sustainable alternative with less environmental impact. Carbonic anhydrase is one such metalloenzyme (with Zn²⁺ as the metal ion cofactor) that catalyzes the reversible hydration of CO₂. The uncatalyzed dissolution of CO₂ (equilibrating with bicarbonate) in an aqueous medium is very slow. Carbonic anhydrase accelerates its hydration and makes it available in its dissolved form (bicarbonate) for the cells to take up. It has a high turnover number, making it a potential candidate for improving CO₂ absorption kinetics in the capture process. The stability of the enzyme and its reusability are major hurdles in implementing carbonic anhydrase-based carbon capture and biomass production systems. Several approaches have been explored to produce robust carbonic anhydrase-based CCU systems, including protein engineering to improve the enzyme’s natural stability, immobilization onto support systems, and obtaining resistant forms of the enzyme from extremophiles.[10] [11] [12] [13]

Algae and photoautotrophic cyanobacteria represent crucial biological models for CO₂ sequestration and utilization.[14] [15] [16] [17] In the present study, we have used the model prokaryote Escherichia coli to study the potential of immobilized enzyme systems for carbon capture and integrating them with biomass production. E. coli requires bicarbonate as a metabolic substrate during normal aerobic growth, which is sourced from the hydration of CO₂. It is essential for the biosynthesis of fatty acids and small molecules like arginine, purines, and pyrimidines. While the supply of CO₂ is adequately fulfilled endogenously under most conditions, the demand for bicarbonate increases significantly when cells are grown in a minimal medium. Hence, bicarbonate supply to the organism under such conditions would impact its growth and biomass production.[18]

In the present study, biomass production by E. coli was used as a metric to assess the hypothesized carbon capture potential of carbonic anhydrase-immobilized nanoparticles (NPs). Our findings highlight the potential of enzyme-immobilized systems for carbon capture and sequestration into biomass.

Results and Discussion

Multiple strategies have been reported to improve the range of working conditions and recovery of carbonic anhydrase. Immobilization can enable quick and easy recovery of the enzyme. Covalent bonding of the enzyme to the surface of a carrier reduces its structural flexibility and increases its stability. It also reduces the diffusional resistance to CO₂ at the gas–liquid interface.[10] The selection of a suitable carrier with functional groups that support covalent binding of the enzyme without losing its activity is essential. Other important parameters include ease of separation of the immobilized enzyme, optimum surface area of the carrier to allow for maximum enzyme loading, and compatibility and uniform dispersibility of the immobilized enzyme.

Carbonic anhydrases of different microbial origins have been covalently immobilized on various support materials such as NPs (such as silanized iron oxide NPs and aminated paramagnetic Fe₃O₄ NPs), metal-organic frameworks, and microcrystalline cellulose beads, leading to significant enzyme recovery and reusability. Carbonic anhydrase has also been stabilized via the formation of cross-linked enzyme aggregates (CLEAs).[19] For instance, magnetic CLEAs using magnetic NPs cross linked to bovine carbonic anhydrase have been employed as catalysts for carbon capture in slurry absorbers.[20] A recent study by Liu et al. has reported an increase in thermal stability and catalytic efficiency of carbonic anhydrase upon immobilization onto ZnO NPs while maintaining 85.36% of initial activity after 5 recycling cycles.[21]

Vinoba et al. developed gold NPs immobilized with human carbonic anhydrase and assembled onto a silica-based mesoporous material functionalized with either amine or thiol. This system was demonstrated to carry out carbon capture and sequestration into CaCO_3. [22] Synthetic mimics of carbonic anhydrase have also been produced that rapidly hydrate CO₂, like its natural counterpart. Fan et al. developed zinc-coordinated hydrogel polymer NPs with superior catalytic properties to those of the natural enzyme.[23]

In the present study, we used carbonic anhydrase extracted and purified from spinach leaves (Spinacia oleracea),[24] an underutilized biological resource for CCUS studies. We synthesized surface-functionalized magnetite (Fe₃O₄) NPs as carriers for the enzyme. The magnetic property of the carrier can enable magnetic recovery of the enzyme from its products, thereby simplifying the downstream processing activity. A colloidal dispersion of nano-scale particles allows for a high surface area that can be utilized for efficient enzyme loading. However, the large surface-to-volume ratio of the NPs leads to higher surface energy of the particles and the tendency to agglomerate to reduce the total surface energy. Hence, functionalization of NPs and selection of the appropriate solvent system are crucial.

Water-stable Fe₃O₄ NPs were synthesized using the conventional coprecipitation approach. The synthesized NPs were coated using citric acid (CA). The coordination of carboxylate functionalities of CA on the surface of the particles serves the purpose of forming a stable suspension. It also aids in forming covalent linkages of the enzyme with the carrier.[16] The synthesized CA-functionalized magnetite NPs, Fe₃O₄@CA, were characterized by several techniques to verify their properties. The crystalline structure of the synthesized NPs was characterized by X-ray diffraction crystallography (XRD). Well-defined peaks were obtained in the XRD pattern of the Fe₃O₄ NPs, Fe₃O₄@CA NPs, and enzyme-immobilized Fe₃O₄@CA NPs, indicating high crystallinity ([Fig. 1A]). The peaks obtained at the 2𝛳 values of 30.12°, 35.50°, 43.01°, 53.32°, 57.36°, and 62.64° correspond to lattice planes (220), (311), (400), (422), (511), and (440) of magnetite, respectively. Consistent with the reported XRD characterization of magnetite, it was determined that the synthesized Fe₃O₄ NPs have a cubic spinel structure.[25] It has also been reported that CA functionalization of the NPs does not result in phase change in the XRD spectra of uncoated NPs of Fe₃O₄.[25]

Fig. 1 Characterization of synthesized NPs: XRD pattern (A) and FTIR spectra (B) of Fe₃O₄ NPs, Fe₃O₄@CA NPs and enzyme-immobilized Fe₃O₄@CA NPs.

The FTIR spectra of the synthesized Fe₃O₄ NPs, Fe₃O₄@CA NPs, and enzyme-immobilized Fe₃O₄@CA NPs ([Fig. 1B]) determined the presence of CA on the surface of Fe₃O₄, confirming successful CA functionalization, with reference to the reported FTIR spectrum.[25] The peak obtained at 1618 cm⁻¹ represents the C=O stretching from the COOH group of the adsorbed carboxylate ions of CA. Another peak observed at the range of 548 cm⁻¹ could be designated to the Fe–O bond stretching.[25] Particle size analysis by dynamic light scattering (DLS) was carried out for the Fe₃O₄ NPs, Fe₃O₄@CA NPs, and enzyme-immobilized Fe₃O₄@CA NPs, and their corresponding values are presented in [Table 1]. The zeta potential values aid in understanding the dispersion stability of Fe₃O₄@CA NPs. The negative charge of the zeta potential may be due to electrostatic stabilization owing to the CA functionalization of the NPs. An increase in the zeta potential of the Fe₃O₄@CA NPs as compared to Fe₃O₄ NPs can be attributed to the adsorption of citrate ions on the surface of the NPs, leading to a negative surface charge that prevents agglomeration and improves stability of the suspension.

Table 1 Particle size analysis of Fe₃O₄ NPs, Fe₃O₄@CA NPs, and enzyme-immobilized Fe₃O₄@CA NPs.

Parameters	Fe3O4 NPs	Fe3O4@CA NPs	Enzyme-immobilized Fe3O4@CA NPs
Effective diameter	653.53 nm	225.65 nm	590.73 nm
Zeta potential	−23.60 mV	−39.93 mV	−39.22 mV
Polydispersity index	0.83	0.34	0.27

The Wilbur-Anderson (WA) assay was performed to determine the activity of carbonic anhydrase in spinach extract.[26] Based on the assay procedure described in the Experimental section, the enzyme activity was found to be 40 WA units/mL in the spinach extract, corresponding to a yield of 0.8 WA units per gram of spinach leaves. Successful immobilization of the enzyme onto the Fe₃O₄@CA NPs was also confirmed using this assay. Postimmobilization, the enzyme activity was determined to be 190 WA units/g of the NPs ([Fig. 2]).

Fig. 2 Carbonic anhydrase activity determined using the Wilbur–Anderson method. Time taken by (A) spinach extract and (B) 75 mg/mL enzyme-immobilized Fe₃O₄@CA NPs suspension to drop the pH of the assay mixture from 8.3 to 6.3.

The carbon-capture efficiency of carbonic anhydrase immobilized onto the Fe₃O₄@CA NPs was assessed by monitoring the growth of E. coli in the presence of these NPs. Growth analysis was performed for two sets of cultures, viz., control (medium containing NPs without carbonic anhydrase) and test (medium with carbonic anhydrase-immobilized NPs), under similar conditions of temperature, aeration, and growth media, as described in the Experimental section. Significantly higher cell density was observed in the test as compared to the control (P < 0.05) ([Fig. 3A]). The enhanced growth may be due to increased availability of carbon in the form of soluble bicarbonate, which can be utilized by the cells.

Fig. 3 E. coli cell growth in the presence of (A) fresh and (B) recycled Fe₃O₄@CA NPs (control) and carbonic anhydrase immobilized-Fe₃O₄@CA NPs (test). Mean ± SD values are plotted.

E. coli cultivated in the presence of carbonic anhydrase exhibited a 62.5% increase in cell biomass as compared to cells cultivated in the presence of the NPs without the enzyme (0.52 and 0.32 average OD₆₀₀, respectively). A similar effect on growth (P < 0.01) was observed when the NPs were recovered and reused ([Fig. 3B]). Carbonic anhydrase facilitates the conversion of atmospheric CO₂ into soluble bicarbonate ions, enhancing bicarbonate availability. Bicarbonate is utilized as a substrate for reactions catalyzed by the enzymes carbamoyl phosphate synthetase, phosphoenolpyruvate carboxylase, 5-aminoimidazole ribotide carboxylase, and biotin carboxylase.[18] Thus, the increase in cell biomass may be attributed to increased bicarbonate availability in the medium.

In E. coli, carbon contributes 47% of the total biomass.[27] Considering that 1–5% of this is contributed by dissolved inorganic carbon,[28] 1 g of E. coli dry cell weight (DCW) can capture ~86 mg CO₂. Thus, 23 kg E. coli DCW would be required to capture 1 tonne CO₂, which may be achieved by operating ~500 high cell density 1 kL bioreactors (assuming that 100 OD corresponds to 23 g/L DCW of E. coli).[29]

Experimental Section

Materials

For the synthesis of magnetite NPs (Fe₃O₄ NPs), FeCl₃·6H₂O, FeCl₂·4H₂O, and NaOH were used (SRL Pvt Ltd., India). Carboxyl functionalization/coating of the surface of the NPs was carried out using CA (SD Fine Chemicals, India). Fresh spinach leaves were obtained from the local vegetable market. Dialyzing membrane-150 (12–14 kDa cut-off), Tris base, and ammonium sulfate (Himedia, India) were used for carbonic anhydrase enzyme extraction from spinach leaves.

Synthesis and Characterization of Carboxyl-functionalized Fe₃O₄@CA NPs

Synthesis of carboxyl-functionalized Fe₃O₄@CA NPs was carried out using the coprecipitation method.[25] The iron salts FeCl₃·6H₂O and FeCl₂·4H₂O were dissolved in deionized water in a 2:1 molar ratio in a 500 mL round-bottom flask in an inert atmosphere (achieved by purging the flask with N₂ gas). An aqueous solution of 0.1 M NaOH was added dropwise until a pH of 10 was obtained. The formation of a blackish-brown precipitate indicated the formation of a suspension of magnetite (Fe₃O₄) NPs that settled with time. The following reaction represents the stoichiometry of magnetite synthesis:

$${2\mathrm{Fe}}^{3+}+{\mathrm{Fe}}^{2+}+{8\mathrm{OH}}^{-}⟶{\mathrm{Fe}}_3{\mathrm{O}}_4\left(\mathrm{s}\right)+4{\mathrm{H}}_2\mathrm{O}$$

The magnetite NPs were synthesized with a yield of 80.57%.[30]

A solution of 1 M CA was added to the suspension until a pH of 3 was achieved. The mixture was heated to 65 °C, followed by stirring for 10 min, allowing the CA to coat the NPs and introduce carboxyl groups on their surface. A stable suspension of CA-coated NPs (Fe₃O₄@CA) was obtained. The Fe₃O₄@CA NPs were magnetically separated, followed by multiple washes with deionized water to remove any unreacted materials. The synthesized Fe₃O₄@CA NPs were dried in a Petri dish, and the resulting powder was used for FTIR and XRD analyses. The dried powder was finely ground using a mortar and pestle, suspended in distilled water (1% w/v), and sonicated for 15 min. This suspension was used for particle size analysis and zeta potential measurements.

The crystalline structure of the synthesized NPs was determined from the XRD pattern (λ = 1.5406 Å) obtained using a Rikagu Smartlab X-ray diffractometer. The scan was performed from a 2θ value of 10°–80° at a rate of 10° per minute. Surface functionalization of the NPs by CA was determined using Fourier transform infrared spectroscopy (Perkin Elmer Spectrum 2) in ATR mode from 4000 cm⁻¹ to 400 cm⁻¹. DLS analysis was performed using Malvern Zetasizer (Nanoseries: Nano-ZS) to determine the particle size distribution of the synthesized NPs.

Extraction of Carbonic Anhydrase Enzyme from Spinach Leaves and Immobilization on Fe₃O₄@CA NPs

Spinach leaves were de-stemmed, cleaned with deionized water, and dried at room temperature to remove excess water. A modified procedure of Pocker and Ng[24] was followed for enzyme extraction. The cleaned leaves were homogenized with 20 mM Tris–HCl buffer (pH 8), adding 1.5 mL of buffer for each gram of leaves used. The homogenized mixture was filtered and centrifuged for 30 min at 4000 rpm. The supernatant was subjected to ammonium sulfate precipitation. Ammonium sulfate was added slowly until a concentration of 30% w/v was achieved; the resulting solution was incubated for 1 h at 4 °C with constant stirring. A crude enzyme extract was obtained after centrifuging the suspension for 30 min at 4000 rpm, and the pellet was discarded. To this supernatant, more ammonium sulfate was added until a final concentration of 55% w/v was achieved with constant stirring for 1 h. Precipitate was recovered after centrifugation. The pellet was dissolved in 5 mL of 20 mM Tris–HCl buffer (pH 8) and dialyzed overnight against the same buffer using a dialyzing membrane to remove excess salts. After dialysis, the solution was incubated with the Fe₃O₄@CA NP suspension at 4 °C for 5 h while being agitated. The enzyme-immobilized NPs were washed with Tris–HCl buffer (pH 8) to remove any unbound enzyme. The entire procedure was carried out at 4 °C. Using the assay procedure as described in the following subsection, the enzyme activity in the extract obtained from 250 g of spinach leaves was quantified to be 200 WA units.

Enzyme Activity Assay

Carbonic anhydrase enzyme activity was assessed using the electrometric method developed by Wilbur and Anderson.[26] The assay was performed with the dialyzed enzyme extract and with the NPs, postimmobilization. The entire procedure was carried out under chilled conditions (0 °C).

The test solution was prepared by adding 6 mL of 20 mM Tris–HCl buffer (pH 8.3) to 100 μL of the enzyme solution. To this, 4 mL of ice-cold CO₂-saturated water was added, and the drop in pH from 8.3 to 6.3 (2 units) was monitored. In the control solution, chilled deionized water was added instead of the enzyme solution. To determine the activity of the immobilized enzyme, a similar test solution was prepared, with the enzyme solution being replaced by a suspension containing 75 mg of the enzyme immobilized NPs.

The time taken (in seconds) for the pH drop of 2 units in the control and test solutions was compared, and enzyme activity was determined in both cases. The calculations depicted in equation ([2]) were used to determine the enzyme activity:

$$\text{Units of enzyme}=\frac{2\times \left(T_{\mathrm{o}}-T\right)}{T\times \left(\text{Volume of enzyme solution added}\right)}$$

where, T = Time taken for pH drop (by 2 units) in the test solution (s). T _o = Time taken for pH drop (by 2 units) in the control solution (s).

A pH drop of a 20 mM Tris–HCl buffer from 8.3 to 6.3 per minute at 0 °C is defined as one Wilbur–Anderson unit.

Bacterial Growth

E. coli DH5α was grown in minimal medium of the following composition: (NH₄)₂SO₄ 8.75 g/L; KH₂PO₄ 13 g/L; MgSO₄·7H₂O 4.5 g/L; CaCl₂·2H₂O 0.5 g/L; CO(NH₂)₂ 5 g/L; yeast extract 2.5 g/L; and glycerol 1% (w/v). Cultivation was carried out in triplicate in 250 mL Erlenmeyer flasks containing 100 mL minimal medium. The cultures were incubated at 37 °C in a rotatory incubator (180 RPM). Cultures were continuously aerated with sterile air (filtered using a 0.2 μm filter), ensuring a constant supply of CO₂.

75 mg NPs and 75 mg enzyme immobilized NPs were placed in dialysis bags (1.5 cm × 1.5 cm). To evaluate the effect of carbonic anhydrase on E. coli biomass production, cell growth was monitored in the presence of either NPs (control) or enzyme-immobilized NPs (test) enclosed in dialysis bags to prevent their bio-fouling. Growth was monitored by optical density measurement at 600 nm (OD₆₀₀) up to 22 h. The enclosed NPs were recovered, washed with sterile water, and reused to determine their recyclability. Student’s unpaired t-test was used to determine statistical significance.

Conclusions

This study demonstrates the potential of carbonic anhydrase immobilized on CA-functionalized magnetite NPs as an effective biocatalytic system for enhancing biological carbon capture. Additionally, the magnetic properties of the NPs may facilitate easy separation and reuse while minimizing enzyme loss. The low-cost synthesis of the NPs and the use of less toxic chemicals make this system both scalable and biocompatible.

The system can be improved by sourcing carbonic anhydrase from a more reliable and scalable source, such as recombinant expression in a heterologous host. This would ensure a cost-effective source of the enzyme and eliminate the time and energy-intensive enzyme extraction step.

Contributorsʼ Statement

H.R.A.: Data curation, Formal analysis, Investigation, Writing – original draft. A.C.: Data curation, Formal analysis, Investigation, Writing – original draft. D.Z.: Formal analysis, Supervision, Writing – review & editing. D.S.: Investigation. E.G.: Investigation. N.J.: Resources, Supervision, Writing – review & editing. S.R.: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Received: 10 October 2025

Accepted after revision: 26 January 2026

Accepted Manuscript online:
26 January 2026

Article published online:
11 February 2026