The Effects of Amino Acids on the Polymorphs and Magnesium Content of Calcium–Magnesium Carbonate Minerals

Abstract

Calcium–magnesium (Ca–Mg) carbonates are among the most widely distributed carbonates in the Earth’s surface environment, and their formation mechanisms are of great significance for revealing geological environmental changes and carbon sequestration processes. In this study, the gas diffusion method was employed with L-glutamic acid, L-glycine, and L-lysine as nucleation templates for carbonate minerals to systematically investigate their regulatory effects on the mineralization of Ca–Mg carbonates. The results demonstrated that L-glycine, with the shortest length, was more conducive to forming aragonite, whereas acidic L-glutamic acid, which contains more carboxyl groups, was more beneficial for the structural stability of aragonite. The morphology of the Ca-Mg carbonate minerals became more diverse and promoted the formation of spherical and massive mineral aggregates under the action of amino acids. Moreover, the amino acids significantly increased the MgCO₃ content in Mg calcite (L-glutamic acid: 10.86% > L-glycine: 7.91% > L-lysine: 6.63%). The acidic L-glutamic acid likely promotes the dehydration and incorporation of Mg²⁺ into the Mg calcite lattice through the preferential adsorption of Mg²⁺ via its side-chain carboxyl groups. This study shows how amino acid functional groups influence Ca–Mg carbonate mineralization and provides insights into biogenic Mg-rich mineral origins and advanced mineral material synthesis.

1. Introduction

Ca–Mg carbonate minerals are widely distributed in surface environments on Earth, including marine and lake sediments, soil, rocks, and living organisms [1,2]. They are among the most abundant mineral types in the surface environment and are widely involved in key geological processes, such as the early diagenesis of marine, lake, and river sediments, the regulation of the chemical evolution of the water environment, and the formation of travertine deposits in caves [3]. As the largest carbon reservoir on the Earth’s surface, carbonate rocks containing Ca–Mg carbonate minerals play a crucial role in the global carbon cycle and the evolution of life by regulating the migration and transformation of carbon, in the lithosphere, hydrosphere, and atmosphere [4]. Furthermore, Ca–Mg carbonate minerals and rocks, as key industrial raw materials, have significant application value in fields such as building materials, chemical manufacturing, and environmental remediation [5]. Therefore, they have become the focus of global attention in recent decades. Analyzing the sedimentary mechanisms of Ca–Mg carbonate minerals is scientifically important for revealing geological environmental changes and carbon sequestration processes and synthesizing biomimetic mineral materials.

The Ca–Mg carbonate minerals include vaterite, aragonite, calcite, calcium carbonate monohydrate, dolomite, huntite, and magnesite. Among these, calcite and dolomite are the most common minerals. Calcite is an essential component in many organisms and plays an important role in the geological environment. Many marine organisms (e.g., sea snakes, oysters, foraminifera, and shellfish) use calcite to form exoskeletons or shells [6]. Dolomite is primarily found in ancient rock strata (such as Precambrian and Paleozoic strata). However, it is almost impossible to synthesize dolomite in modern marine environments or precipitate dolomite abiotically under normal temperature and pressure conditions in laboratories. Its scarcity in the modern ocean (the “dolomite problem”) has long remained unresolved [7]. Researchers suggest that Mg-rich carbonate minerals, particularly high-Mg calcite and huntite, may be precursor phases of dolomite [8]. Exploring the influence mechanism of the polymorphs and MgCO₃ content of Ca–Mg carbonate minerals will provide ideas for magnesium incorporation in Mg-rich carbonates, which is beneficial for solving the classic “dolomite problem.” Furthermore, the unique polymorphs and Mg content of these minerals contribute to their excellent mechanical properties, demonstrating their great potential for material applications. For instance, Mg calcite is frequently used in construction due to its impressive compressive strength and stability [9]. Mg calcite can effectively enhance the load-bearing capacity and stability of soil, thereby preventing problems such as foundation settlement and landslides. Additionally, Mg calcite is an effective adsorbent in water treatment processes, where it helps remove pollutants and improve water quality [10]. Therefore, research on the influence mechanism of polymorphs and Mg content of Ca–Mg carbonate minerals will also provide a scientific theoretical basis for the synthesis of excellent mineral materials.

An increasing number of studies have demonstrated that microorganisms play a significant role in the formation of Ca–Mg carbonate minerals [11,12]. Organic molecules secreted by microorganisms, such as polysaccharides, amino acids, and lipids, play important roles in the formation of Ca–Mg carbonate minerals [13]. Research has revealed that polymorphs of Ca–Mg carbonate minerals formed with organic molecules secreted by microorganisms as nucleation templates are related to the properties of the organic substances, including heterogeneity, electrical charge, and molecular structure [14]. Moreover, the secretion of extracellular organic molecules by microorganisms can modulate the physicochemical properties of the surrounding microenvironment, including ion concentration, pH, and saturation, which creates favorable conditions for the deposition of Ca–Mg carbonate minerals [12]. Furthermore, these organic molecular interactions directly affect the evolution of mineral types and morphologies [15]. However, the mechanism by which organic molecules affect the polymorphs and Mg content of Ca–Mg carbonate minerals is unclear.

Numerous studies have demonstrated that sugars, alcohols, and amino acids containing functional groups, such as -COOH, -OH, and -NH₃, presumably provide nucleation sites for the crystallization of Ca–Mg carbonate minerals [16,17,18]. Moreover, these soluble biological macromolecules can form diverse regulatory templates by changing the type of residue, connection positions, or rotation in their secondary structure. Therefore, in this study, acidic amino acids (L-glutamic acid), neutral amino acids (L-glycine), and basic amino acids (L-lysine) were used as regulatory templates for the mineralization of Ca–Mg carbonates to explore their effects on the polymorphs and Mg content of Ca–Mg carbonate minerals.

2. Materials and Methods

2.1. Selection and Properties of Amino Acids

In this study, L-glycine, L-lysine, and L-glutamic acid (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China; AR, 99%) were selected as representative amino acids of neutral, basic, and acidic character, respectively, to perform biomimetic mineralization experiments on Ca–Mg carbonates. The differences in the specific properties of the three amino acids are compared in

Table 1. Physicochemical properties of the selected amino acids.

	L-Glycine	L-Lysine	L-Glutamic Acid
Isoelectric Point (pl)	5.97	9.74	3.22
Charge state (Physiological pH)	Neutral	Positively charged (basic)	Negatively charged (acidic)
Side Chain Structure	-H	-(CH2)4NH2	-(CH2)2COOH

.

2.2. Biomimetic Mineralization Experiments of Ca–Mg Carbonates

A gas diffusion method for ammonium carbonate decomposition was employed to accomplish biomimetic mineralization [19]. The general setup of the reaction chamber is illustrated in Figure 1. Crystallization reactions occur in a closed container; therefore, a 10 L desiccator was used in this study. Ammonia and CO₂ diffuse from the lower section into the upper space, where a reaction occurs. In this study, Petri dishes containing 25 mL experimental solutions were placed in the upper space and sealed with a 0.22 μm cellulose acetate filter. The 25 mL experimental solutions contained 0.01 M CaCl₂, 0.01 M MgCl₂, and 1.5 mg solid amino acid powder, and their initial pH was adjusted to 5.5. Two beakers containing 10 g of ammonium carbonate and 100 mL of concentrated sulfuric acid (to absorb NH₃) were placed at the bottom of the desiccator. Subsequently, aseptic static cultures were established, and the Petri dishes were observed at specific intervals. Finally, the reaction solution and sediment in the Petri dishes were transferred to a sterilized centrifuge tube, centrifuged (5000 rpm, 10 min), rinsed with deionized water, air-dried, and subjected to mineralogical and morphological analyses. All experiments were repeated twice.

2.3. Observation and Analysis

The pH of the solutions was measured using a pHS-3C pH meter (precision: 0.01). The mineralogical compositions of the precipitates were determined by X-ray diffraction (XRD; Rigaku D/MAX-B, Tokyo, Japan). The samples were prepared by dropping the dried precipitate onto glass slides. The measurement parameters were as follows: Cu Kα radiation, 35 kV tube voltage, 20 mA current, scanning rate of 2°/min, step size of 0.02°, and scanning range of 20–60° (2θ). The FT-IR (Nicolet 380, Thermo Fisher Scientific Inc., Waltham, MA, USA) experiment was conducted by using the potassium bromide method in a scanning range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹. The morphologies of the mineral products were observed using a field-emission scanning electron microscope (FESEM; Zeiss, Oberkochen, Germany) equipped with an energy-dispersive X-ray spectrometer (EDS). Sample preparation involved washing the precipitates three times with distilled water, soaking them in absolute ethanol, and depositing them onto SEM stubs. After air-drying, the samples were sputter-coated with ~8 nm gold film and imaged at 20 kV and 60 μA. The precipitated minerals were ground in an agate mortar and sieved to obtain uniform grains, and their thermal decomposition characteristics were analyzed using a thermal analyzer (TGA/DSC1/1600LF, METTLER TOLEDO Co., Zurich, Switzerland) from 20 to 800 °C at a constant heating rate (20 °C min⁻¹) with pure nitrogen as a protective gas to prevent oxidation.

When the precipitated minerals contained calcite and aragonite, the proportions of aragonite and calcite were calculated using the following formulas [20]:

X_A = 3.157 I_A(221)/(I_C(104) + 3.157 I_A(221))

X_C = 1 − X_A

where X_A and X_C represent the proportions of aragonite and calcite minerals (%), respectively. I_{A (221)} and I_C(104) are the diffraction intensities of the (221) and (104) crystal planes of aragonite and calcite, respectively.

The Mg content of calcite is often expressed as a molar fraction of MgCO₃. When the molar fraction of MgCO₃ in the calcite was less than 4 mol%, it was considered to be Mg-containing calcite or low-Mg calcite; when the MgCO₃ content in the calcite was between 4 and 35 mol%, it was high-Mg calcite; and when the MgCO₃ content in the calcite was between 36 and 52 mol%, it was Ca–dolomite or dolomite. The molar fraction of MgCO₃ in the calcite was calculated using the empirical formula proposed by Lumsden [21]:

MgCO₃ (mol%) = −333.33d + 1011.99

where d denotes the distance (nm) of the crystal face (104 nm) of the Mg calcite family.

3. Results

3.1. XRD and FT-IR Analyses of Ca-Mg Carbonate

The XRD pattern (Figure 2) illustrates that the Ca–Mg carbonate minerals formed throughout the mineralization experiment period (from 24 to 120 h) were Mg calcite (d₁₀₄ ≈ 3.00, d_11-3 ≈ 2.28, d_11-6 ≈ 1.85) and aragonite (d₁₁₁ ≈ 3.39, d₀₁₂ ≈ 2.70, d₂₂₁ ≈ 1.97) regardless of which amino acid was used. However, the Ca–Mg carbonate minerals formed throughout the mineralization experiments without amino acids were single calcite, and no aragonite was precipitated. Calculating the proportion of aragonite in the deposited Ca–Mg carbonate minerals revealed that under the action of L-glycine, the proportions of aragonite in the deposited Ca–Mg carbonate minerals at 24, 48, 72, and 120 h were 54.13, 66.11, 59.61, and 83.51%, respectively. Under the action of L-glutamic acid, the proportions of aragonite in the sedimentary Ca–Mg carbonate minerals at 24, 48, 72, and 120 h were 56.35, 51.00, 65.83, and 60.53%, respectively. Under the action of L-lysine, the proportions of aragonite in the sedimentary Ca-Mg carbonate minerals at 24, 48, 72, and 120 h were 75.80, 72.21, 53.53, and 43.10%, respectively. Characteristic peaks of the minerals were obtained from the FTIR spectra (Figure 3). It can be observed from Figure 3 that the peaks at 712, 1403, and 2532 cm⁻¹ are the characteristic peaks of calcite, and the peaks at 699, 853, and 1082 cm⁻¹ prove the existence of aragonite, which is consistent with the results of the XRD analyses.

3.2. Morphological Changes of Ca-Mg Carbonate

In the biomimetic mineralization experiment of Ca–Mg carbonates involving different amino acids, the morphologies of the Ca–Mg carbonate minerals formed at 120 h were observed using FE-SEM. The particle size of Ca–Mg carbonate minerals formed under the action of amino acids was between 10 and 100 μm, presenting the morphology of prismatic, spherical, and irregular mineral aggregates (Figure 4). The particle size of the formed Ca–Mg carbonate minerals in the control experiment without amino acids ranged between 1 and 10 μm and presented the morphology of relatively typical rhombohedral mineral aggregates.

3.3. Magnesium Content in Calcite

Figure 5 illustrates the calculation of the Mg content (molar fraction of MgCO₃) in calcite induced by L-glycine in sequence, which was 10.17% (24 h), 8.71% (48 h), 8.02% (72 h), and 7.91% (120 h). The Mg content of the calcite formed under the action of L-glutamic acid gradually increased from 7.29% at 24 h to 10.86% at 120 h. The Mg content of the calcite formed in the presence of L-lysine decreased from 11.59% at 24 h to 6.53% at 120 h. However, in the control experiment without amino acids, the Mg content of the calcite was maintained at approximately 4%.

3.4. Thermal Decomposition Characteristics of the Different Precipitates

A thermogravimetric differential scanning calorimetry (TG-DSC) analysis of the precipitated minerals was performed to verify the influence of amino acids on the structural stability of aragonite. As illustrated in Figure 6, the decomposition temperature in the first stage was 28–458 °C, and the mass loss rate was approximately 4%, indicating that approximately 4% of amino acid organic molecules and a small amount of water molecules were adsorbed or incorporated into the mineral. The weight-loss temperature during the second stage of thermal decomposition was 341–800 °C. At this stage, aragonite is transformed into calcite, and the minerals gradually decompose to release CO₂. The mass loss rate was approximately 35%, which is lower than the theoretical value of 44% (in the system of pure calcium carbonate). Obviously, the precipitated aragonite and Mg calcite were more stable in the amino acid systems, and some minerals required higher temperatures (>800 °C) for transformation and decomposition. Thus, the TG-DSC curves of Ca–Mg carbonate minerals induced by different amino acids still showed a downward trend at 800 °C and too small mass loss owing to the incomplete decomposition of minerals below 800 °C. Furthermore, the initial temperatures of thermal decomposition for minerals induced by L-lysine, L-glycine, and L-glutamic acid were 341, 406, and 458 °C, respectively. The mineral phase stability of aragonite induced by glutamic acid was better than that of L-glycine and L-lysine, which is consistent with the variation law of the proportion of aragonite in the mineral phase composition throughout the previous mineralization cycle (Figure 2).

4. Discussion

4.1. Control of Reaction Liquid and Mineral Precipitation

In the biomimetic mineralization experiment of Ca–Mg carbonates, the continuous dissolution of (NH₄)₂CO₃ released large amounts of CO₂ and NH₃ (Reaction (1)), and the pH increased (Figure S1) owing to the dissolution of NH₃ in the reaction liquid and the release of OH- (Reaction (2)). Meanwhile, the continuous dissolution of CO₂ in the alkaline solution increased the CO₃^2- concentration in the solution (Reaction (3)). Ultimately, mineral precipitation occurred quickly when supersaturation was attained because the concentrations of cations and anions in the reaction solution increased rapidly (Reaction (4)).

(NH₄)₂CO₃ → 2NH₃ + CO₂ + H₂O

(1)

NH₃ + H₂O → NH₄⁺ + OH^-

(2)

CO₂ + H₂O → CO₃^2- + 2H⁺

(3)

xCa²⁺ + (1 − x) Mg²⁺ + CO₃^2- → Ca_xMg_1−xCO₃ (0 ≤ x ≤ 1)

(4)

Compared to the control experiment, the Ca–Mg carbonate minerals mainly underwent heterogeneous nucleation in the biomimetic mineralization system involving amino acid molecules [22]. Heterogeneous nucleation enables crystals to form preferentially on the surfaces of amino acid molecules by reducing the nucleation energy barrier rather than spontaneous nucleation in solution (homogeneous nucleation) [23]. Amino acids carry different charges in aqueous solutions, where the pH is not equal to their isoelectric point (pl), thereby affecting the nucleation of Ca–Mg carbonate minerals [24]. When the pH of the aqueous solution is greater than the pl value of the amino acids, the amino acids (L-glycine and L-glutamic acid) release protons and acquire negative charges. The negatively charged amino acids attract Ca²⁺ and Mg²⁺ ions, increasing the local ion concentration and leading to local supersaturation, thereby promoting the nucleation of Ca–Mg carbonate minerals. When the pH of the aqueous solution was lower than the pH value of the amino acid, the amino acid (L-lysine) was protonated and acquired a positive charge. Similarly, positively charged amino acids attract CO₃²⁻ ions, causing the solution to reach a local supersaturated state, thereby promoting the nucleation of Ca–Mg carbonate minerals [25].

4.2. The Effect of Amino Acids on Polymorphs of Ca–Mg Carbonates

In addition to calcite, metastable aragonite was formed under the action of amino acid molecules, compared with the control experiment. This result implies that amino acid molecules are conducive to aragonite formation. According to the proportion of aragonite in the precipitated minerals under the action of different amino acid molecules, the proportion of L-glycine aragonite increased steadily, reaching a maximum of 83.51% with the extension of mineralization time. The effect of L-glutamic acid on the proportion of sedimentary minerals was relatively stable, with approximately 58.43% aragonite in the precipitates. Under the influence of L-lysine, the proportion of aragonite in the sedimentary minerals gradually decreased to 43.10%. Compared with L-lysine and L-glutamic acid, L-glycine is more conducive to the formation of aragonite. Glycine is the simplest amino acid and has a single hydrogen atom (–H) as its side chain group; therefore, the length of the side chain can be regarded as approximately 0. The ionic radius of Ca²⁺ is approximately 0.174 nm [26]. According to the template theory analysis (

Table 2. Relationship between the radius ratio of positive/negative ions and the coordination number of crystals.

R+/R−	0.155–0.225	0.225–0.414	0.414–0.732	0.732–1	≥1
Coordination number	3	4	6	8	12

Note: R⁺ represents positive ions, and R⁻ represents negative ions.

), glycine is more likely to form ligand templates with coordination numbers of 8 or 12 with Ca²⁺ [27]. Therefore, the amount of aragonite with a coordination number of 9 in the mineralization products induced by glycine was significantly higher than that induced by glutamic acid and lysine.

The results of TG-DSC analysis of the precipitated minerals further confirmed the influence of amino acids on the structural stability of aragonite (Figure 6). Amino acid molecules are conducive to the formation and stability of aragonite and inhibit the phase transition process of its transformation into calcite. The orthogonal lattice parameters of aragonite match relatively well with the molecular arrangement of amino acids, thereby preferentially inducing aragonite nucleation rather than the thermodynamically more stable trigonal calcite [28]. However, the adsorption of amino acid molecules on the surface of aragonite can reduce its surface energy and delay its phase transition kinetics [29]. Amino acids combine with Ca²⁺ and Mg²⁺ through their carboxyl functional groups to form a protective layer on the surface of aragonite, preventing its structural reorganization into calcite.

Compared to the control experiment, the morphology of the Ca–Mg carbonate minerals formed under the action of amino acid molecules was more diverse. It has been established that many fibrous and needle-like mineral aggregates formed with amino acids are likely to be relatively stable aragonite growing along the C-axis direction, as supported by the XRD (Figure 2) and EDS (Figure 7) analysis results. This finding is consistent with previous studies, which demonstrated that spherical and needle-like aragonite forms with the participation of amino acids [30]. Amino acid molecules also had a significant influence on calcite morphology. Amino acids can guide the orderly arrangement of mineral ions through specific functional groups or structures, match the lattice parameters of calcite minerals, and promote the preferred growth of specific crystal planes [31]. Second, the geometric arrangement of the amino acid side chains may match the lattice spacing of calcite minerals (for example, the carboxylic acid group spacing is similar to the Ca²⁺ spacing in calcite), inducing epitaxial growth and thereby causing the morphological evolution of calcite [32]. Furthermore, the Mg contents of the calcite mineral phases formed under the action of different amino acids varied significantly. Mg²⁺ replaced Ca²⁺ in the calcite mineral lattice, resulting in the accumulation of lattice strain and inducing changes in particle size and morphology (Figure 7).

4.3. The Effect of Amino Acids on Mg Content in Calcite

As illustrated in Figure 5, the Mg content in the calcite mineral phase induced by amino acids was significantly higher than that in the control experiment (without amino acid participation). Furthermore, the Mg content of the calcite formed by the action of different amino acid molecules varied significantly. As illustrated in Figure 5, the Mg content in the calcite (120 h) induced by the acidic amino acid (L-glutamic acid) was higher than that in the calcite formed under the action of neutral L-glycine and basic L-lysine. Characteristic peaks of the minerals and organic functional groups were obtained from the FTIR spectra (Figure 3). In addition to the characteristic peaks of Ca-Mg carbonate minerals induced by amino acids, certain peaks representing other organic functional groups present in/on the minerals also exist, such as O–H and N–H stretching (2925 cm⁻¹) and C=O stretching (1787 cm⁻¹; 1636 cm⁻¹) illustrated in Figure 3, which indicates that these organic functional groups of amino acids have a close relationship with the nucleation and growth of the minerals, especially the influence of the carboxyl group on Mg content in calcite.

The carboxyl group of L-glutamic acid carries a negative charge in an alkaline solution and preferentially adsorbs Mg²⁺, which has a higher charge density (compared to Ca²⁺, Mg²⁺ has a smaller ionic radius and a higher charge density). This selective adsorption enables more Mg²⁺ to be enriched in the local microenvironment of the mineral, thereby increasing the possibility of Mg²⁺ entering the calcite lattice [33]. Indeed, Mg²⁺ has a stronger hydration ability than Ca²⁺ and is prone to form hydrated Mg²⁺ in a water environment, making it difficult for Mg²⁺ to enter the calcite lattice [34]. It may only partially dehydrate when Mg²⁺ is involved in the growth of calcite, and the residual hydration spheres doped with Mg²⁺ inhibit the further incorporation of surface Mg²⁺ into the calcite lattice [35]. However, the numerous carboxyl groups in acidic amino acids (L-glutamic acid) tend to form complexes with Mg²⁺, thereby reducing the stability of hydrated Mg²⁺ and promoting dehydration. Therefore, L-glutamic acid causes more Mg²⁺ to enter the calcite lattice and replace Ca²⁺, which accelerates the incorporation of Mg²⁺ at the growth interface of the calcite crystals.

As the ionic radius of Mg²⁺ is significantly smaller than that of Ca²⁺, the gradual substitution of Mg²⁺ for Ca²⁺ in the calcite lattice results in lattice distortion [36]. Acidic amino acids can stabilize the distorted calcite lattice by forming complexes with Mg and adhering to the crystal surface, thereby reducing the calcite surface energy. Therefore, more Mg²⁺ in the solution could replace Ca²⁺, forming relatively thermodynamically stable calcite. Furthermore, it has been reported that biomolecules rich in carboxyl groups in biological minerals (for example, L-glutamic acid) can temporarily stabilize the amorphous precursor phase (such as amorphous calcium carbonate), delaying its transformation to calcite. Therefore, more Mg²⁺ was integrated into the final calcite during the reorganization process from amorphous calcium carbonate to calcite [37]. In conclusion, amino acid molecules, especially acidic amino acids, can induce the formation and stability of Mg-rich calcite minerals through multiple mechanisms, such as ion selectivity, coordination stability, and thermodynamic regulation. This process has important applications in geology, environmental science, and biomimetic material synthesis.

5. Conclusions

In this study, acidic (L-glutamic acid), neutral (L-glycine), and basic (L-lysine) amino acids were selected to systematically explore their effects on the mineral composition, morphology, and Mg²⁺ incorporation efficiency of Ca–Mg carbonates using the gas diffusion method. The results demonstrated that L-glycine, with the shortest side chain, was more conducive to forming aragonite, whereas acidic L-glutamic acid, containing more carboxyl groups, was more beneficial to the structural stability of aragonite. The morphology of the Ca-Mg carbonate minerals became more diverse and promoted the formation of spherical and massive mineral aggregates under the action of amino acids. Acidic amino acids (L-glutamic acid) preferentially adsorb Mg²⁺ through the carboxyl group and promote its dehydration, which increases the MgCO₃ content in calcite to 10.86%. Research has revealed the mechanism by which biological macromolecules directly regulate mineral formation through ion-selective adsorption or incorporation induced by functional groups, offering new insights into the biogenesis of Ca–Mg carbonate minerals and the design of biomimetic mineralized materials.