Influence of enzyme concentration on bio-sequestration of CO₂ in carbonate form using bacterial carbonic anhydrase

Highlights

• Influence of enzyme concentration on CaCO₃ precipitation by bacterial CA.

• CA concentrations of 0.2–2.0 U/mL were beneficial to CaCO₃ precipitation.

• Overhigh CA concentration was not conducive to CaCO₃ precipitation.

• CA concentration influenced the polymorph and morphology of CaCO₃ crystals.

• Electrostatic adsorption of Ca²⁺ on CA enzyme protein promoted CaCO₃ precipitation.

Abstract

One of the most promising biological sequestration technologies of CO₂ is the enzyme catalyzed CO₂ sequestration into stable and environmentally friendly mineral carbonates. The present manuscript focused on the biocatalytic precipitation of CaCO₃ by extracellular carbonic anhydrase (CA) which was extracted and purified from the culture of Bacillus cereus. The kinetics of CaCO₃ precipitation catalyzed by the bacterial CA in the presence of different enzyme concentrations was investigated through the gaseous diffusion system. The polymorph and morphology of CaCO₃ crystals obtained in the precipitation process were also analyzed using XRD, FTIR and FESEM. The results showed that the change in the amount of deposited Ca²⁺ during the process of CaCO₃ precipitation catalyzed by bacterial CA of different concentrations was well fitted with the exponential model. The enzyme concentrations of 0.2–2.0 U/mL were beneficial to CaCO₃ precipitation, however, overhigh enzyme concentration (8.0 U/mL) was not conducive to CaCO₃ precipitation. The integrated results of XRD, FTIR and FESEM analysis showed that there were significant differences in the morphologies of CaCO₃ crystals among different enzyme concentrations. Vaterite was present at the lower concentration of CA, and the higher concentration of CA favored the formation of calcite. Therefore, different polymorphs and morphologies of CaCO₃ crystals can be produced in the presence of different concentrations of CA. Furthermore, the role of bacterial CA in CaCO₃ precipitation was related to the electrostatic adsorption of Ca²⁺ on CA enzyme protein and the preferential adsorption of CA enzyme protein on the crystal faces, in addition to the enzymatic catalysis.

Introduction

The concentration of atmospheric carbon dioxide (CO₂) has been obviously increased by anthropogenic activities since the rapid industrialization. CO₂ is well known to be a major contributor to the warming of the earth among all the greenhouse gases [1], therefore, the capture and sequestration of atmospheric CO₂ is paid more and more attention. There are various approaches to CO₂ sequestration, such as use of chemical/physical solvents, adsorption onto solids, membranes, cryogenic/condensation systems, geological sequestration, and deep ocean sequestration [1], [2]. The utilization cost for the previously mentioned approaches has proven to be highly expensive [2]. It is regarded that bio-sequestration of CO₂ in the form of a stable carbonate solid holds much promise, because it is a viable and environmentally benign technology and it offers obvious appeal for long-term storage of CO₂ [1], [3], [4].

Carbonate minerals such as calcite, aragonite, dolomite and dolomitic limestone, constitute the earth’s largest carbon reservoir, which is estimated to account for 99.55% of the total global carbon [5]. The geological record demonstrates the feasibility of a large amount of CO₂ stored in carbonate form. However, the precipitation process of carbonate minerals is very slow in nature. The reaction ${\text{CO}}_2+{\text{H}}_2\text{O}\to {\text{H}}^{+}+{\text{HCO}}_3^{-}$ is considered to be a rate-limiting step for the precipitation of carbonate minerals [6]. In order to make the above reaction faster under a conducive environment, an enzyme known as carbonic anhydrase (CA, EC4.2.1.1) is employed [7], [8]. CA is a metalloenzyme containing zinc metal ion in its active site, widespread in animals, plants and prokaryotes, and can dramatically catalyze the reversible hydration of CO₂ into ${\text{HCO}}_3^{-}$ [9], [10], with typical catalytic rates as high as k_cat = 10⁶ s⁻¹, or a million times a second [1], thus CA would promote the precipitation of carbonate minerals. This enzyme has been studied as a catalyst in both free and immobilized forms [7], [11], [12].

At present, quite a few studies have shown the effect of bovine CA on the sequestration of CO₂ in carbonate form. Favre et al. [7] investigated the biocatalytic capture of CO₂ by bovine CA to form solid CaCO₃ in basic conditions, and found that the initial precipitation rate of solid CaCO₃ increased significantly by the addition of the enzyme. Vinoba et al. [11] reported the sequestration of CO₂ and conversion to CaCO₃ using bovine CA immobilized on mesoporous silica. However, the effect of microbial CA on the sequestration of CO₂ in carbonate form has not been elucidated in depth. Since microorganisms are ubiquitous in the natural environment, elucidating how the sequestration of CO₂ in carbonate form mediated by microbial CA and its influencing factors may have far-reaching implications. It may lead to a better understanding of microbial carbonate precipitation, and help establish a promising approach for biomimetic sequestration of CO₂ using microbial CA.

In previous studies we found that CA from bacteria could significantly promote carbonate precipitation [13], [14]. Based on the previous studies, we further investigated the influences of initial pH [15], initial Ca²⁺ concentration [16] and enzyme concentration on the precipitation and crystal morphology of CaCO₃ induced by bacterial CA. This manuscript focused on the kinetics of CaCO₃ precipitation catalyzed by bacterial CA at different enzyme concentrations through the gaseous diffusion method. The change in CaCO₃ polymorph and crystal morphology was also observed during the precipitation process.