# The Monomolecular-Layer Selective Adsorption of Amino Acids on Microbial Proto-Dolomite Induced by the Cyanobacterium Leptolyngbya Boryana

Yanyang Zhao, Yishui Han, Xiao Gao, Xiangyu Wei, Chao Han, Zhaopeng Wang, Karim Benzerara, Maurice Tucker, Zuozhen Han

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## To cite this version:

Yanyang Zhao, Yishui Han, Xiao Gao, Xiangyu Wei, Chao Han, et al. The Monomolecular-Layer Selective Adsorption of Amino Acids on Microbial Proto-Dolomite Induced by the Cyanobacterium Leptolyngbya Boryana. ACS Earth and Space Chemistry, 2025, 9 (7), pp.1946-1957. 10.1021/acsearthspacechem.5c00142. http://dx.doi.org/10.1021/acsearthspacechem.5c00142

HAL Id: hal-05395312

https://hal.science/hal-05395312v1

Submitted on 4 Dec 2025

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# The Monomolecular-Layer Selective Adsorption of Amino Acids on Microbial Proto-Dolomite Induced by the Cyanobacterium *Leptolyngbya boryana*

Yanyang Zhao, Yishui Han, Xiao Gao, Xiangyu Wei, Chao Han, Zhaopeng Wang, Karim Benzerara, Maurice E. Tucker, and Zuozhen Han

**ABSTRACT:** Microbially mediated mineralization is widely recognized as a key pathway for proto-dolomite formation, with extracellular polymeric substances (EPS), especially amino acids, considered important mediators. Yet, the surface interactions between amino acids and proto-dolomite remain insufficiently characterized. In this study, we examined the adsorption behavior of four representative amino acids as partic acid (Asp), arginine (Arg), glycine (Gly), and proline (Pro) on organic-free proto-dolomite (Cpdol) synthesized by the cyanobacterium *Leptolyngbya boryana*. Experiments were conducted across a range of pH and salinity conditions, and amino acid concentrations were measured using high-performance liquid chromatography. The adsorption data were well described by Langmuir isotherms, indicating a monomolecular-layer selective adsorption model. Adsorption capacity varied among amino acids (Asp > Gly > Pro > Arg) and showed a positive correlation with pH and a negative

correlation with salinity. Based on density functional theory (DFT) calculations, the adsorption energies of the four amino acids were evaluated on seven crystal surfaces of proto-dolomite. The results indicate that surface complexation between carboxyl groups and surface metal ions is the primary mechanism driving adsorption. Under higher pH conditions, the deprotonated amino group can further enhance adsorption by lowering the adsorption energy. These findings suggest that, beyond acidic amino acids, surface complexation of various environmentally responsive amino acids may play an important role in regulating the early-stage formation of proto-dolomite in microbial environments.

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**KEYWORDS:** microbial proto-dolomite, amino acids, selective adsorption, monomolecular-layer adsorption, surface complexation

## 1. INTRODUCTION

Dolomite ($CaMg[CO_3]_2$) is a critical mineral, extensively found in carbonate reservoirs and playing a key role in global carbon cycling and hydrocarbon storage.¹,² Its prevalence in ancient sedimentary formations has fostered intensive investigations to understand its formation processes, offering valuable insights into both past environmental conditions and contemporary biogeochemical cycles.³,⁴ In particular, the low-temperature formation of dolomite remains

one of the most intriguing questions in geochemistry.⁵,⁶ While dolomite can be found in abundance in older geological records, the precise mechanisms that drive its precipitation, especially in modern low-temperature environments, are still not fully understood.⁷,⁸

The microbial dolomite model has emerged as one of the most promising explanations for dolomite precipitation in natural environments.⁹,¹⁰ This model
suggests that microbial activity, particularly sulfate-
reducing bacteria, cyanobacteria, and halophilic
bacteria, alters water chemistry through metabolic
processes, promoting precipitation of proto-dolomite,
the precursor of dolomite.¹¹,¹² Additionally, microbial
extracellular polymeric substances (EPS) are
believed to facilitate the crystallization of proto-
dolomite by providing nucleation sites and altering
local chemical conditions, sometimes through
nonclassical crystallization pathways.¹³⁻¹⁶ Amino
acids, the fundamental building blocks of proteins
and key constituents of EPS, exhibit structurally
diverse functional groups and can be categorized as
acidic (e.g., aspartic acid, glutamic acid), basic (e.g.,
arginine, lysine), or neutral (e.g., glycine, proline).¹³
Acidic amino acids carry additional carboxyl groups
that readily dissociate in alkaline conditions, whereas
basic amino acids possess functional groups such as
guanidinium or primary amines that tend to retain
protons under near-neutral pH, resulting in variable
protonation states.¹³ Neutral amino acids, often in
zwitterionic form at circumneutral pH, display less
pronounced net charges. These differences in
molecular structure and protonation behavior
significantly influence their adsorption
characteristics on mineral surfaces.¹³,¹⁷⁻²⁰

Recent studies have highlighted that amino acids can
influence carbonate minerals like calcite and
dolomite, modulating their dissolution, growth rates,
and crystallization behaviors.²¹,²² Experimental
evidence suggests that amino acids form coordination
complexes with cations (e.g., Ca²⁺, Mg²⁺), alter
surface free energy, and selectively interact with
specific crystal faces, thereby influencing crystal
habit and potentially promoting the formation of
high-Mg carbonate phases.²²,²³ These processes are
especially relevant in microbial environments where
selective adsorption may steer the nucleation of
dolomite-like precursors.¹⁷ Functional groups such as
carboxyl and hydroxyl groups show strong affinity
toward carbonate surfaces, and their interactions can
be energetically favorable,²²,²⁴ contributing to
adsorption strength and site-specific binding.

## 2. MATERIALS AND METHODS

**2.1. Initial Mineral Material.** Microbially precipitated proto-dolomite was selected as the initial mineral material for this study.

Our preliminary studies identified both the
cyanobacterium *Leptolyngbya boryana*¹⁷ and the
halophilic bacterium *Vibrio harveyi* QPL2¹⁸ as
effective inducers of proto-dolomite precipitation.
*Leptolyngbya boryana* is a photosynthetic autotroph,

Moreover, organic molecules can modulate surface
charge and zeta potential of carbonate minerals,
further shaping amino acid-mineral interfacial
dynamics.²⁵ These processes were also confirmed by
recent Density Functional Theory (DFT)
calculations, including amino acids and gypsum,²⁶
Ca-montmorillonite,²⁷ kaolinite,²⁸ and
hydroxyapatite.²⁹

However, the precise molecular process involved and
the nature of the interactions between amino acids
and proto-dolomite remain unclear: (1) on the one
hand, in previous microbial proto-dolomite
precipitation experiments exogenous proteins or
amino acid components (such as beef extract and
peptone) were artificially added,¹⁸,³⁰ posing
challenges in deciphering the interactions between
microbes-derived amino acids and microbial proto-
dolomite crystals; (2) on the other hand, while
environmental factors such as pH and salinity play
crucial roles in influencing mineral nucleation and
crystal growth in carbonate systems, they have not
been taken into account carefully. Overall,
understanding how these variables affect amino acid
adsorption onto proto-dolomite surfaces is essential
for replicating natural conditions that promote proto-
dolomite formation. To address this gap, we
conducted a controlled adsorption experiment using
organic-free, cyanobacterially induced proto-
dolomite (Cpdol) as a mineral substrate. Four amino
acids-aspartic acid (Asp), arginine (Arg), glycine
(Gly), and proline (Pro)-were chosen based on their
distinct isoelectric points and chemical properties,
and were added separately to the proto-dolomite
surface under various pH and salinity conditions. By
isolating these variables, we aim to elucidate the
differential adsorption behaviors of these amino acids
and their potential influence on proto-dolomite
crystallization. Specifically, this study addresses two
key questions: (1) do different amino acids exhibit
distinct adsorption patterns on proto-dolomite
surfaces? and (2) how pH and salinity modulate the
strength of these interactions?

cultured in a medium containing only inorganic salts,
allowing to specifically assess the influence of
microbially derived organic compounds on mineral
formation and morphological changes. Importantly, the
composition of the mineral phases induced by
*Leptolyngbya boryana* is highly homogeneous, with no
mineral impurity detected, even upon examination via
scanning electron microscopy (SEM). In contrast,
proto-dolomite precipitated by *Vibrio harveyi* QPL2
contains mixed phases, including huntite (CaMg₃[CO₃]₄). Considering these facts, the present study utilized protodolomite induced by *Leptolyngbya boryana* as the initial mineral material. The protodolomite was synthesized under controlled conditions, with a salinity of 1.5% and a Mg/Ca ratio of 5, as described in literature.¹⁷

## 2.2. Preprocessing of Initial Mineral Material.

Prior studies have indicated that cyanobacterially induced protodolomite (Cpdol) contains significant amounts of organic matter,¹⁷ which could interfere with adsorption simulation experiments. To mitigate this, we employed a four-step preprocessing procedure aimed at removing as much organic material as possible from the Cpdol. The first step involved physical agitation. The Cpdol samples were immersed in deionized water in plastic centrifuge tubes and subjected to 15 min of ultrasonic shaking. This washing procedure was repeated three times, effectively removing larger organic particles adsorbed onto the crystal surfaces. In the second step, the physically treated Cpdol was soaked in a 15% acetone solution for 2 h, followed by three rounds of washing. This step successfully dissolved and removed smaller organic microparticles from the crystal surfaces. In a third step, the acetone-treated Cpdol was soaked in a 5% hydrogen peroxide (H₂O₂) solution for 1 h. This process of oxidative degradation, repeated three times, led to the decomposition of residual organic matter. Finally, the Cpdol was heated at low power in a microwave for 1 h, facilitating the breakdown of any remaining organic material trapped within the crystal structure.

## 2.3. Adsorption Experiments and Analytical Methods for Adsorption Data.

### 2.3.1. Adsorption Experiments.
To investigate the adsorption behavior of amino acids onto the surface of Cpdol, a series of controlled experiments were conducted. Amino acid solutions with the desired concentrations were prepared by dissolving amino acids in distilled water. Since each amino acid solution initially had a distinct pH, the pH of all solutions was uniformly adjusted to 7 using HCl or NaOH. Subsequently, a measured amount of Cpdol crystals was added to the amino acid solutions to ensure adequate contact between the minerals and amino acids. The pH of the resulting mixtures was then fine-tuned as per experimental requirements.

The adsorption reactions were carried out under oscillation on a shaker to promote homogenization of the suspensions. After the specified adsorption time, the Cpdol crystals were separated via centrifugation

and subjected to further analysis. To determine the post-adsorption concentration of residual amino acids, the supernatant was filtered through a 0.45 µm membrane to remove the Cpdol in solution, and the amino acid content was quantified using high-performance liquid chromatography (HPLC; Agilent 1200, USA) coupled with a mass spectrometer (MS; Agilent 6460A, USA).

Four distinct amino acids were selected for the experiments, representing different chemical properties: aspartic acid, an acidic amino acid (HOOC-CH(NH₂)-CH₂-COOH, isoelectric point = 2.85); arginine, a basic amino acid (NH₂-C(NH)-NH-(CH₂)₃-COOH, isoelectric point = 11.15); glycine, a hydrophilic amino acid (NH₂-CH₂-COOH, isoelectric point = 5.97); and proline, a hydrophobic branched-chain amino acid (NH-CH₂-CH₂-COOH, isoelectric point = 6.3). Considering the pivotal role of pH and salinity in Cpdol precipitation,¹³ a range of pH values (8, 9, 10, and 11) and salinities (NaCl, wt %) were also applied in the experiments to evaluate the potential impact of these conditions on amino acid adsorption (Supplementary Table 1).

### 2.3.2. Adsorption Kinetics Study of Amino Acids on Cpdol.
A 150 mg/L amino acid solution was prepared, and 100 mL of this solution was transferred into a series of conical flasks. Adsorption experiments were initiated by adding 0.15 g of Cpdol to each flask, and the reaction time was recorded immediately. Oscillation was conducted at 26 °C with a shaking frequency of 120 rpm for varying durations of 10, 20, 30, 50, 70, 90, 110, 150, 200, 250, and 500 min (Supplementary Table 1). Following each designated time interval, the amino acid concentrations in the solution were determined by separating the Cpdol through centrifugation and dilution. The amount of amino acid adsorbed on Cpdol at time t (Qt) was calculated using eq 1:

$$Q_t = \frac{(C_0 - C_t) \times V}{m} \quad (1)$$

where: Qt is the adsorption capacity at time t (mg/g); C₀ and Ct are the concentrations of the amino acid solution (mg/L) at time 0 and time t; V is the volume of the solution (L), and m is the mass of Cpdol (g). When the amount of adsorbed amino acid no longer increases with time, the system is considered to have reached equilibrium, and the adsorption capacity at this point is denoted as Qe.
The pseudo-second-order kinetic model, a mathematical framework commonly used to describe adsorption processes by analyzing the interaction between adsorbates and adsorbent surfaces in solution,³¹ was applied to analyze the adsorption process. The equation for this model is shown in eq 2:

$$ \frac{t}{Q_t} = \frac{1}{kQ_e^2} + \frac{1}{Q_e} \quad (2) $$

where $Q_e$ is the equilibrium adsorption capacity (mg/g), and k is the pseudo-second-order rate constant (g/mg/min).

### 2.3.3. Adsorption Isotherm Study of Amino Acids on Cpdol.

For the adsorption isotherm study, amino acid solutions with initial concentrations of 15, 100, 150, 200, 300, 400, 500, and 800 mg/L were prepared (Supplementary Table 1). A 100 mL aliquot of each solution was placed in a series of conical flasks. Upon the addition of 0.15 g of Cpdol, the solutions were shaken at 26 °C with a frequency of 120 rpm for 500 min to achieve the equilibrium state. The equilibrium adsorption capacity ($Q_e$) was calculated as described in eq 1.

Adsorption isotherms offer key insights into the interaction mechanisms between the adsorbent and adsorbate, showing how active molecules adhere to the adsorbent surface.³² In this study, two classical adsorption isotherm models were tested: (1) the Langmuir model, referred to as the monomolecular layer adsorption model, which characterizes adsorption occurring on a homogeneous surface where each adsorption site holds one molecule,³³ and (2) the Freundlich model, which accounts for adsorption on heterogeneous surfaces with non-uniform energy distributions, allowing for multilayer adsorption.³² The Langmuir model is described by the following eq 3:

$$ \frac{c_e}{Q_e} = \frac{c_e}{b} + \frac{1}{k_L b} \quad (3) $$

where $C_e$ is the equilibrium concentration of the amino acid in solution (mg/L). $Q_e$ is the equilibrium adsorption capacity (mg/g). b is the saturation adsorption capacity (mg/g). $k_L$ is the Langmuir constant (L/mg).

The Freundlich model is described by the following eq 4:

$$ \ln Q_e = \ln k_F + n \ln C_e \quad (4) $$

where $C_e$ is the equilibrium concentration of the amino acid in solution (mg/L). $Q_e$ is the equilibrium adsorption capacity (mg/g). $k_F$ and n are Freundlich constants.

## 2.4. Characterization of Cpdol.

To characterize the Cpdol samples before and after amino acid adsorption, several analytical techniques were employed. X-ray diffraction (XRD) was conducted using a Scintag X1 powder diffractometer (Bruker D8 Advance, Germany) using Cu Kα radiation as an X-ray source (40 kV, 35 mA), and scanning across a 2θ range between 5° and 65° at 2°/min. The XRD data were refined using the Rietveld method and processed with the MDI JADE 6.0 software.

The XPS C 1s spectra were collected using a Thermo ESCALAB 250XI system, which was equipped with an aluminum (Al) target to identify the organic signal in Cpsol. Surface morphology and microstructure were further examined using scanning electron microscopy (SEM; SU8010, Hitachi, Japan) at an accelerating voltage of 5–15 kV. Additionally, elemental analyses were carried out using energy-dispersive spectrometry (EDS) coupled with the SEM (EX-350, Horiba, Japan). The thermal characteristics of proto-dolomite were examined using a thermogravimetric analyzer (TG-DSC, STA449F5, Germany). The sample was subjected to heating from 25 °C to 1,000 °C at a rate of 15 °C·min⁻¹ in a nitrogen atmosphere. Amino acid signals in proto-dolomites after adsorption were analyzed through Raman spectroscopy was performed using a 20 mW, 532 nm Ar⁺ laser on a DXR2 laser Raman spectrometer (Thermo Fisher, USA). Spectral calibration was achieved using the 520.5 cm⁻¹ line of a silicon wafer.

## 2.5. DFT Calculations.

Density functional theory (DFT) calculations with periodic boundary conditions were conducted to model the adsorption behavior of amino acids on proto-dolomite. A disordered dolomite crystal structure (CaMg[CO₃]₂) (a = b = 4.805 Å, c = 16.022 Å; α = β = 90°, γ = 120°) with stoichiometry same that of protodolomite,³⁴ was used as a representative model in the simulations.

In previous studies on carbonate-organic matter interactions, the (104) surface has often been selected due to its natural cleavage in bulk carbonate minerals.²⁵,³⁵⁻³⁷ However, the proto-dolomite examined in this study is a microbially induced carbonate that forms via spherulitic growth from nanoscale crystal nuclei, lacking well-developed crystal faces. This suggests that a variety of crystallographic orientations could participate in growth and surface interactions. To
reflect this, we selected seven representative crystallographic planes with distinct orientations(001), (100), (010), (110), (101), (011), and (111)to systematically investigate amino acid adsorption behavior across different growth directions (Figure S1). Each of these planes was cleaved from a proto-dolomite unit cell and expanded into 2 × 2 supercells to ensure adequate surface area for adsorption studies. A vacuum layer of 20 Å was added perpendicular to the surface to prevent interlayer interactions.

Under the experimental conditions, the carboxyl groups of all amino acids are deprotonated, while the protonation state of the amino groups varies with pH. Therefore, computational simulations must account not only for the charge state of the amino groups, but also for the diverse initial spatial configurations of amino acids to determine the lowest adsorption energy. A uniform neutralizing background charge was employed to counterbalance the net charge of the system. The electronic exchange-correlation functional was described using the generalized gradient approximation (GGA) with the Perdew-Burke-Ernzerhof (PBE) formulation. For the basis set, an all-electron double numerical augmented with polarization (DNP) was applied, with all electrons considered in the calculations. Convergence criteria for the total energy, maximum force, and maximum displacement were set at 10⁻⁵ Hartree, 0.001 hartree/Å, and 0.001 Å, respectively. The Brillouin zone was sampled using a Γ-centered k-point grid of 3 × 3 × 1. During the relaxation process, DIIS and thermal smearing methods were employed to accelerate the selfconsistent field (SCF) convergence. All calculations were performed using the *dmol3* package. The adsorption energy (*E*<sub>ads</sub>) of the amino acids on the proto-dolomite surface was calculated using the following equation:

$$E_{ads} = E_{system} - E_{proto-dolomite} - E_{amino acid}$$

Where *E*<sub>system</sub>, *E*<sub>proto-dolomite</sub> and *E*<sub>amino acid</sub> are the total energy of the optimized system with adsorbed amino acid, the isolated dolomite, and amino acid in its isolated state, respectively.

# 3. RESULTS

## 3.1. State of Cpdol Before and After Adsorption Experiments.

The proto-dolomite precipitate mediated by *Leptolyngbya boryana* exhibited a distinct double-

spheroid morphology, with cell filaments clearly visible between the mineral spherulites (Figure 1A,B). Following the 4-step preprocessing, cell remnants were completely removed from the interstitial spaces (Figure 1C), revealing the development of numerous nanopores on the surface of the mineral particles (Figure 1D, red arrows).

<figure><img src="image_4.png" /><figcaption>Figure 1. Morphology of cyanobacterially induced proto-dolomite (Cpdol) before (A,B) and after preprocess (C,D).</figcaption></figure>

Thermogravimetric analysis (TGA) of the Cpdol before and after preprocessing revealed four distinct stages of weight loss (Figure S2A). The first stage (~30–205 °C) corresponds to the evaporation of adsorbed water, while the second stage (~205–431 °C) reflects the loss of crystalline water and the combustion of organic matter.¹⁹ The third stage (~431–617 °C) represents the decomposition of MgCO₃, and the fourth stage (~617–794 °C) corresponds to the decomposition of CaCO₃. Notably, in the unpreprocessed Cpdol, weight loss in the first two stages was significantly higher compared to the preprocessed mineral, indicating partial loss of crystalline water due to temperature exposure and effective removal of organic matter during preprocessing.

These findings were further corroborated by X-ray photoelectron spectroscopy (XPS) data (Figure S2B). Both processed and unprocessed samples exhibit a high binding energy CO₃ peak at 289.6 ± 0.2 eV.¹⁸,³⁸ Additionally, a C–C peak is observed at 285.1 ± 0.2 eV, which is commonly associated with samples prepared and stored in solution.³⁸ Significant changes were also observed between the two: in the unprocessed Cpdol, distinct organic carbon peaks were detected, specifically at ~286.4 eV (C–O–C) and ~287.9 eV (C–OH)¹⁸ (Figure S2B, shaded areas), attributed to proteins, amino acids, and polysaccharides from microbial metabolites and cell debris. After
preprocessing, these organic carbon peaks disappeared, confirming the effective removal of organic matter. This result demonstrates that the processed Cpdol is well-suited for subsequent amino acid adsorption experiments.

**3.2. Adsorption of Amino Acids on Cpdol.**

The kinetics of amino acid adsorption on Cpdol was investigated at a temperature of 26 °C, with an amino acid concentration of 150 mg/L, a pH of 10, and a NaCl salinity of 5‰ (Supplementary Table 1). The dynamic adsorption curves for the four amino acids on Cpdol in aqueous solution are presented in Figure 2A. During the initial stages, the adsorption capacity of Cpdol for amino acids increased rapidly with time, indicating a fast adsorption rate. After approximately 200 min, adsorption reached equilibrium, and the rate gradually decreased. Subsequently, the adsorption capacity remained relatively stable over time. This suggests that the adsorption equilibrium for amino acids on proto-dolomite in aqueous solution is achieved in approximately 200 min under the present experimental conditions. Under these conditions, the R² values for fitting the data using the pseudo-second-order kinetic model ranged from 0.9853 to 0.9931, indicating very good fits of the models to the experimental data (Figure 2A). The calculated adsorption capacities and corresponding rate constants for aspartic acid, arginine, glycine, and proline on Cpdol are presented in Supplementary Table 2. Overall, under the current experimental conditions, the adsorption of all amino acids follows the pseudo-second-order kinetic model, with the acidic amino acid Asp exhibiting the highest adsorption capacity on proto-dolomite.

To investigate the impact of different concentrations of amino acids on the adsorption behavior of Cpdol, adsorption isotherm studies were conducted at a temperature of 26 °C, an adsorption time of 500 min, a salinity of 5‰, and amino acid initial concentrations ranging from 15 to 800 mg/L (Supplementary Table 1 and Figure 2B). Overall, at lower amino acid concentrations, the adsorption capacity of Cpdol for amino acids increased rapidly with the rise of equilibrium concentration. As the equilibrium concentration of amino acids increased, the incremental increase in adsorption capacity gradually diminished. At a certain equilibrium concentration, the adsorption capacity of Cpdol for amino acids stabilized (Figure 2B). For aspartic acid, glycine, proline, and arginine, these equilibrium concentrations ($C_e$) were determined to be 169 mg/L, 177.9 mg/L, 178.2 mg/L, and 192.77 mg/L, respectively.

The experimental data were fitted using both the Langmuir and Freundlich isotherm equations, with the results presented in Figure 3. Upon comparison, the fitting accuracy of the Langmuir isotherm exceeded 0.98, dramatically outperforming the Freundlich isotherm (Supplementary Table 2), suggesting a monomolecular-layer adsorption mechanism for amino acids on proto-dolomite surfaces. This indicates that amino acid adsorption by Cpdol in aqueous solution follows a monomolecular-layer adsorption process, with varying strengths for different amino acids.

<figure><img src="image_5.png" /><figcaption>Figure 2. Adsorption kinetics curves of cyanobacterially induced proto-dolomite (Cpdol) for different amino acids. (A) Variation in amino acid adsorption capacity of Cpdol over time at an initial concentration of 150 mg/L, with a pH value of 10 and a salinity of 5‰. (B) The relationship between equilibrium amino acid concentration ($C_e$) and equilibrium adsorption capacity ($Q_e$) at varying initial amino acid concentrations, with the solution pH set at 10 and a salinity of 5‰.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

Figure 3. Linear fitting results of the Langmuir isotherm equation (A) and Freundlich isotherm equation (B) for the adsorption of amino acids on cyanobacterial proto-dolomite. In the experiments depicted in the figure, the concentrations of various types of amino acids were set from 15 mg/L to 800 mg/L, with a fixed pH value of 10 and a salinity of 5‰. In this figure, C<sub>e</sub> is the concentration of amino acid in the solution at equilibrium. Q<sub>e</sub> is the adsorption quantity of amino acids by Cpdol at equilibrium.

<figure><img src="image_7.png" /></figure>

Figure 4. Cross-plot of amino acid adsorption quantity on cyanobacterially induced proto-dolomite (Cpdol) vs solution pH (A) and salinity (B).

**3.3. Impact of pH and Salinity on Amino Acid Adsorption.**

The adsorption behavior of the four amino-acids: aspartic acid (Asp), glycine (Gly), proline (Pro), and arginine (Arg) on the Cpdol surface was systematically evaluated under varying pH and salinity conditions (Figure 4). The pH range tested was 8–11, and salinity ranged from 5‰ to 200‰ (Supplementary Table 1). The adsorption capacities of the four amino acids on proto-dolomite increased with rising pH in the range of 8–11. Specifically, the adsorption of Asp increased from 10.26 mg/g to 31.25 mg/g, Gly from 7.32 mg/g to 23.25 mg/g, Pro from 6.68 mg/g to 22.49 mg/g, and Arg from 5.98 mg/g to 10.59 mg/g. Among the tested amino acids, Asp consistently exhibited the highest adsorption capacity, while Arg showed the lowest.

Figure 4B illustrates the relationship between salinity and amino acid adsorption. As the salinity of the solution increased from 5‰ to 200‰, the adsorption capacity of all four amino acids decreased significantly. At the lowest salinity level (5‰), all four amino acids-Asp, Gly, Pro, and Arg-reached their maximum adsorption capacities, with Asp showing the highest value (Q<sub>e</sub> ~ 28 mg/g) and Arg the lowest (Q<sub>e</sub> ~ 7 mg/g).

**3.4. Characterization of Cpdol After Adsorption.**

Figure 5 presents the XRD and Raman analyses of Cpdol following the adsorption experiments at an amino acid concentration of 150 mg/L, pH 11, and a salinity of 5‰.

<figure><img src="image_8.png" /></figure>

Figure 5. X-ray diffraction (XRD) patterns (A) and Raman spectra (B) of cyanobacterially induced proto-dolomite (Cpdol) after adsorption with four amino acids. The minerals were collected from experiments with 150 mg/L amino acid concentrations, pH 11, and 5‰ salinity. Gray curves in (B) represent the reference Raman spectra of the four amino acids.

The XRD results (Figure 5A) indicate that the characteristic diffraction peaks, particularly the (104) peak at approximately 30.7° 2θ, remain unchanged after the adsorption of various amino acids. This absence of new diffraction peaks suggests that Cpdol retained its structural integrity throughout the adsorption process, without undergoing significant alterations in its composition or lattice structure.
In the Raman spectra (Figure 5B), in addition to the peaks associated with the inorganic components of proto-dolomite such as the external modes at 306 cm⁻¹, resulting from interactions between cations and CO₃²⁻ ions, and the higher frequency modes, v₄ at 733 cm⁻¹ and v₁ at 1102 cm⁻¹, representing internal modes of the CO₃²⁻ group³⁹new absorption bands attributable to organic matter were observed, confirming the adsorption of amino acids on the proto-dolomite surface. Notably, these peaks are most prominent after the adsorption of aspartic acid, which aligns with the experimental data showing a higher adsorption capacity for this amino acid. For example, the band at 939 cm⁻¹ is attributed to the γ(OH) vibration, which is notably strong in aspartic acid's Raman spectrum.⁴⁰ Additionally, the bands at 1084 cm⁻¹ and 1125 cm⁻¹ correspond to -NH₂ wagging vibrations, while the bands at 877 cm⁻¹ and 779 cm⁻¹ are assigned to C–C stretching and out-of-plane OCO⁻ vibrations, respectively.⁴⁰ Meanwhile, the functional groups of amino acid molecules adsorbed onto the proto-dolomite surface-particularly those of Asp, which exhibits a higher adsorption capacity-tend to shift toward higher binding energies (Figure S3). These

results provide direct evidence of amino acid adsorption and the presence of organic functional groups on Cpdol after interaction with amino acids.

SEM analysis reveals notable surface morphological changes in proto-dolomite (Cpdol) following amino acid adsorption. Overall, the Cpdol particles retain their characteristic dumbbell-shaped morphology; however, their surfaces become significantly rougher compared to those prior to amino acid treatment. Specifically, among the four amino acids, aspartic acid (Figure 6A,B) induces the most pronounced surface roughening, characterized by densely distributed, well-defined nanopores approximately 100 nm in diameter. In contrast, glycine and proline (Figure 6C–F) produce slightly larger but less densely packed surface pores ranging from 100 nm to 1 µm. Arginine-treated surfaces (Figure 6G,H) exhibit minimal porosity, with fewer and less distinct surface features. These variations in surface texture likely reflect different extents of mineral-organic interactions, with the bidentate coordination capability of aspartic acid contributing to more substantial surface modification.

<figure><img src="image_9.png" /><figcaption>Figure 6. SEM images of cyanobacterially induced proto-dolomite (Cpdol) after adsorption of different amino acids and corresponding local enlargements (white boxes). (A, B) Aspartic acid; (C, D) Glycine; (E, F) Proline; (G, H) Arginine. The minerals were collected from experiments conducted with amino acid concentrations of 150 mg/L, pH 11, and a salinity of 5‰.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 7. DFT simulations of the interaction between an Asp molecule and the proto-dolomite (001) surface. (A) Charge distribution of Asp with deprotonated carboxyl groups and a protonated amino group. (B) Optimized adsorption configuration of this Asp form on the proto-dolomite (001) surface. (C) Electron density map of the adsorbed system in this configuration. (D) Charge distribution of Asp with both carboxyl and amino groups deprotonated. (E) Optimized adsorption configuration of this fully deprotonated Asp on the proto-dolomite (001) surface. (F) Electron density map of the adsorbed system in this configuration. Green, yellow, gray, red, white, and blue spheres represent calcium, magnesium, carbon, oxygen, hydrogen, and nitrogen atoms, respectively.</figcaption></figure>

**3.5. DFT Calculations of Amino Acid Adsorption on Cpdol Crystal Faces.**

Under the experimental conditions of pH > 8, the four selected amino acids exhibit different dissociation states due to their distinct $pK_a$ values. Aspartic acid (Asp) has a $pK_{a1}$ (α-carboxyl group) of approximately 2.1, $pK_{a2}$ (α-amino group) of ~9.8, and $pK_{a3}$ (side-chain carboxyl group) of ~3.7. Glycine (Gly) exhibits $pK_{a1}$ and $pK_{a2}$ values of ~2.3 and ~9.6, respectively; proline (Pro) has $pK_{a1}$ ~ 2.0 and $pK_{a2}$ ~ 10.6. Arginine (Arg) presents $pK_{a1}$ ~ 2.2, $pK_{a2}$ ~ 9.0, and $pK_{a3}$ (guanidinium group) ~12.5.⁴¹ Within the pH range of the current experiments, the carboxyl groups of all amino acids are almost fully deprotonated to form –COO⁻, while the amino groups gradually deprotonate to –NH₂ as the pH increases, especially above pH ~ 10.

To elucidate the adsorption mechanisms of amino acids on proto-dolomite (Cpdol) surfaces, we performed density functional theory (DFT) calculations to determine the adsorption energies of four representative amino acids proline (Pro), glycine

(Gly), aspartic acid (Asp), and arginine (Arg) on seven low-index crystallographic surfaces: (001), (010), (011), (100), (101), (110), and (111). For each amino acid, two protonation states were considered, corresponding to different pH conditions. The adsorption energies are summarized in Figure S4. Among the four amino acids, Asp consistently exhibited the most negative adsorption energies across all surfaces, reaching values as low as –6.6 eV and –5.5 eV on the (011) and (101) surfaces, respectively. These results indicate a particularly strong interaction between Asp and the proto-dolomite surfaces, which is consistent with its experimentally observed highest adsorption capacity. Comparative analysis of the two protonation states revealed that all amino acids generally exhibit more negative adsorption energies at high pH. For example, the adsorption energy of Gly on the (001) surface changes from approximately –1.9 eV (with the –NH₃⁺ group) to –2.5 eV (with the deprotonated –NH₂ group), and similar trends are
observed for the other amino acids across different surfaces.

To further clarify the roles of deprotonated carboxyl and amino functional groups during adsorption, we carried out charge density difference calculations using Asp, the most strongly adsorbing amino acid, as a representative case. The two carboxyl groups of Asp are deprotonated, enabling chelation with surface metal ions of proto-dolomite. As shown in Figure 7, the deprotonated carboxyl groups act as the primary binding moieties, forming strong coordination bonds with surface cations, as evidenced by significant electron redistribution at the carboxyl-metal interfaces.

In addition to the dominant role of carboxyl groups, the protonation state of the amino group also influences adsorption strength. At relatively lower pH (Figure 7A–C), the amino group remains protonated as $-NH_3^+$ and shows limited overlap with surface metal ions. In contrast, under higher pH conditions (Figure 7D–F), the amino group is deprotonated to $-NH_2$, allowing for extra coordination with surface sites. This is further supported by the more pronounced electron density redistribution near the nitrogen atom (Figure 7F), indicating that $-NH_2$ can act as an auxiliary binding site. These results demonstrate that although the carboxylate groups are the primary contributors to adsorption, the $-NH_2$ group provides additional stabilization at high pH, enhancing the overall affinity of amino acids for the proto-dolomite surface.

# 4. DISCUSSION

## 4.1. Mechanism of Amino Acid Adsorption on Proto-Dolomite.

The findings from the current adsorption study are generally consistent with prior research on the interaction between amino acids and carbonates. Various researchers investigating the interaction of carbonate with amino acids have reported a preference for the adsorption of aspartic acid (Asp) onto carbonate surfaces.42–44 In an experiment conducted on calcite surfaces,25 the adsorption ratios of Asp/Gly were reported as 0.96/0.97. In contrast, the present study yielded data indicating Asp/Gly ratios of approximately 28.86/20.2 (Figure 2), suggesting potential variations attributable to the specific crystal face (104) employed in their study. Under the current experimental conditions, all types of amino acids (acidic, basic, and neutral) exhibit adsorption capabilities on the surface of Cpdol (Figures 2–4), resembling previous findings with calcite (a structural analogue to Cpdol).25 Furthermore, we demonstrate that the adsorption quantities on Cpdol significantly

vary between different types of amino acids for a given salinity and pH (Figure 4).

The surface complexation between amino acids and proto-dolomite is identified as the principal mechanism driving adsorption. This interaction is substantiated by the Raman spectral shifts of functional groups toward higher wavenumbers upon adsorption, indicating stronger bonding environments. SEM observations further support this mechanism, revealing surface roughening and pore development consistent with enhanced mineral-organic interactions. These experimental findings are further corroborated by density functional theory (DFT) calculations, which demonstrate strong surface complexation driven by both carboxylate and amine functional groups.22,45 Overall, the carboxylate group ($-COO^-$) serves as the key coordinating functional moiety, forming strong surface complexes with exposed surface cations.46,47 Under higher pH conditions, the deprotonated amino group ($-NH_2$) can provide additional stabilization through secondary coordination, thereby enhancing the overall adsorption affinity. This mechanistic framework explains the experimentally observed increase in amino acid adsorption capacity with rising pH across all amino acid types.

The observed differences in amino acid adsorption under identical conditions are primarily attributable to variations in their molecular structures and functional groups. Aspartic acid (Asp), which contains an additional carboxyl group as its side chain (R group), is the only amino acid in this study capable of forming bidentate chelation complexes with the surface. This dual coordination ability likely contributes to the enhanced adsorption of Asp, a behavior also reported for Asp on other substrates such as rutile and magnesium-based alloys.48,49 In the case of arginine (Arg), both the carboxylate and deprotonated amine groups can participate in surface complexation. However, its relatively large molecular size may introduce steric hindrance, limiting effective adsorption.49 Moreover, the guanidinium group of Arg, which remains protonated under the experimental conditions, may exert a repulsive electrostatic effect or interfere sterically with optimal coordination, thus reducing its adsorption efficiency.50 Glycine (Gly) and proline (Pro) exhibit comparable adsorption capacities, with Gly slightly higher. This similarity is attributed to their shared functional groups — one carboxyl and one amino group — and closely aligned dissociation constants ($pK_a$). The slightly stronger adsorption of Gly may result from its small, linear molecular structure, which minimizes steric hindrance and allows closer
and more effective coordination of its functional
groups with surface cations.⁵¹ In contrast, Pro contains
a cyclic pyrrolidine ring, which restricts the spatial
flexibility of the amino group and may hinder the
formation of optimal surface complexes.

Changes in salinity also impact the adsorption of
amino acids. The observed decrease in amino acid
adsorption with increasing salinity is likely driven by
the salting-out effect,⁵²,⁵³ where elevated ionic strength
reduces complexation interactions between the amino
acid molecules and the Cpdol surface, leading to
diminished adsorption efficiency. The increase in salt
concentration exerts a certain inhibitory effect on the
adsorption of various amino acids, which may be
related to changes in the solubility of proto-dolomite
and amino acids. Aspartic acid has been considered a
key catalytic substance for proto-dolomite
precipitation, and its production in bacterial systems
increases with an increase in salinity,¹⁸ suggesting the
existence of a possible salinity threshold favorable for
proto-dolomite formation. This has been confirmed by
experimental observations: microbially induced proto-
dolomite precipitation can be induced at a salinity of
200‰, whereas no proto-dolomite is produced when
salinity increases to 360‰.¹³

## 4.2. The Possible Role of Non-acidic Amino Acids in Proto-Dolomite Precipitation.

Acidic amino acids (aspartic acid and glutamic acid) are pivotal components within microbial extracellular polymeric substances (EPS), repeatedly demonstrated to play a central role in the microbial-induced formation of proto-dolomite.13,15,17,30 The increase in the salinity of the culture medium significantly enhances the expression of acidic amino acids in the EPS of halophilic bacteria,¹³ and a similar phenomenon occurs with the elevation of the Mg/Ca ratio in the solution.¹⁸ These amino acids, featuring two carboxyl groups, are deemed effective in promoting the dehydration of magnesium ions and the precipitation of proto-dolomite. The core mechanism by which acidic amino acids catalyze proto-dolomite precipitation lies in their ability to remove water molecules surrounding magnesium ions through carboxyl groups,¹³,⁵⁴,⁵⁵ In the proposed water solution, both Ca²⁺ and Mg²⁺ ions form hydrous complexes with H₂O, and subsequently undergo a dehydration process prior to entering the anhydrous mineral structure. The dehydration of Mg²⁺ is comparatively more challenging than that of Ca²⁺, attributed to at least two factors. First, the enthalpy of Mg²⁺ dehydration (ΔH = 351.8 kcal/mol for Mg[H₂O]₆²⁺ at 298 K, 1 atm) is significantly higher than that of Ca²⁺ dehydration (ΔH = 264.3 kcal/mol for

Ca[H₂O]₂⁺ at 298 K, 1 atm).⁵⁶ Second, the half-life of Mg²⁺H₂O (600 ps) is considerably longer than that of Ca²⁺H₂O (19 ps).⁵⁷ The promotion of proto-dolomite precipitation by acidic amino acids occurs through a mechanism wherein their abundant carboxyl groups replace water molecules in Mg²⁺H₂O complexes.⁵⁸ This substitution process demands lower energy for carbonation and the subsequent growth of ions,¹³,¹⁹,⁵⁸ Neutral amino acids and some basic amino acids can exhibit similar effects under specific pH conditions. Hence, the role of other types of amino acids (basic and neutral amino acids), which predominate in quantity within EPS,¹³,¹⁸ may have been overlooked in the microbial precipitation of proto-dolomite.

The adsorption experiments in this study confirm the amino acid monomolecular-layer adsorption on proto-dolomite. Results from adsorption experiments and simulation calculations suggest that even on crystal faces predominantly exhibiting a positive charge, such as (001), amino acids carrying positive charges (arginine) can undergo a certain degree of adsorption. In summary, alkaline amino acids with carboxyl groups and neutral amino acids can also facilitate the dehydration of magnesium ions at the crystalline interface, thereby promoting the further growth of proto-dolomite. Thus, beyond acidic amino acids, certain neutral and basic amino acids may also play a non-negligible role in modulating mineral growth through interfacial interactions.

## 5. IMPLICATIONS FOR PROTO-DOLOMITE GROWTH

Abundant experimental studies have indicated that proto-dolomites induced by different types of microorganisms exhibit various morphologies, including single-spheroid, dumbbell-shaped, double-spheroid, and other distinct shapes.¹²,¹⁷,⁵⁹,⁶⁰ These morphologies significantly differ from the typical rhombohedral dolomite crystals formed through high temperature chemical routines.⁵,⁶¹ Researchers have considered these varied morphologies as a biological signature, in some cases successfully identifying them in ancient carbonate rocks dating back millions to even billions of years.³,⁴,⁶² However, the mechanisms underlying the formation of spheroidal morphologies are currently not well understood. Proto-dolomite synthesized under high-temperature conditions also displays a spherulitic morphology akin to that of microbially induced proto-dolomite formed at ambient temperatures. This similarity implies the existence of specific components in microbial systems that potentially mimic the crystallization-promoting effects
of elevated temperatures on dolomite formation.⁶³,⁶⁴
Some organic molecules are known to alter the habit of
crystals,⁴⁴,⁶⁵,⁶⁶ Considering the crucial role of amino
acids in the stoichiometry of proto-dolomite,¹³,¹⁸ we
propose that they also play a role in the formation of
spheroidal crystal morphology. In typical precipitation
environments, crystals are often surrounded by smooth
crystal faces, resulting in euhedral crystals.⁶⁷ This is
primarily due to differences in the growth rates of
different crystal faces: those faces with faster growth
rates gradually disappear during crystallization, and
the faces with relatively slower growth rates eventually
dominate the crystal surface.⁶⁷,⁶⁸ The adsorption of
amino acids on the surface of proto-dolomite, in
addition to being influenced by their own charge, is
also related to the surface properties of the crystal faces
themselves.²⁵ Different crystal faces possess distinct
adsorption sites, leading to variations in the types and
quantities of adsorbed amino acids. It is assumed that
amino acids may be adsorbed at specific crystal planes
or active sites at the steps, hindering growth there.
Consequently, even high-energy crystal facets, which
would not naturally occur, can be grown. Therefore,
the differences observed in morphologies may be
correlated with the extent of interactions at the
interface.⁶⁹

Amino acids display property-dependent adsorption
on proto-dolomite, with simulations revealing marked
differences in their interactions even on identical
crystal surfaces. When amino acid molecules adsorb
onto crystal faces through a monomolecular-layer
model, variations in adsorption energy lead to
structural differences, significantly altering crystal
morphology and inhibiting the formation of flat crystal
faces. A similar phenomenon is observed in solutions
with varying types and concentrations of amino acids.
Previous studies have shown this effect in calcite,
where the addition of charged electrolytes containing
benzene rings (e.g., polystyrenesulfonate) resulted in
the formation of unstable, high-energy (001) faces
instead of more stable (104) faces.⁷⁰⁻⁷²

Moreover, amino acid adsorption can also affect the
crystallization direction through intercalation. For
example, chiral acidic amino acids have been shown to
induce chiral hierarchical structures in calcium
carbonate crystals.⁷³ Thus, the demonstrated
monomolecular-layer selective adsorption of amino
acids may be related to the formation of the spheroidal
morphology observed in microbial proto-dolomite.
Future studies may explore how tailored combinations
of amino acids in microbial EPS can be leveraged to
regulate proto-dolomite crystal morphology and

nucleation pathways under different geochemical
conditions.

6. CONCLUSIONS

In this study, we investigated the adsorption behavior
of four amino acids with different isoelectric points on
cyanobacterial proto-dolomite (Cpdol) surfaces under
varying initial conditions, including amino acid
concentration, pH, and salinity. The experimental
results demonstrate that under these conditions, acidic,
basic, and neutral amino acids can all adsorb onto the
surface of proto-dolomite. This adsorption follows a
monomolecular-layer selective adsorption mechanism,
and the adsorption capacity is strongly influenced by
both pH and salinity. Specifically, the adsorption
efficiency of amino acids increases as the pH and
decreases with rising salinity. Additionally, density
functional theory (DFT) calculations showed that
amino acids were adsorbed mainly through surface
complexation of carboxyl groups with dolomite, and
deprotonated amino groups could provide additional
adsorption energy under high pH conditions. Our
findings provide new insights into the role of amino
acids in microbial mineralization processes and
suggest that environmental factors, such as pH and
salinity, play a crucial role in shaping proto-dolomite
formation.

Author Contributions

Y.Z.: Conceptualization, Methodology, Investigation, Writing/Original Draft. Y.H.: Data Curation. X.G.: Formal Analysis. X.W.: Formal Analysis. C.H.: Formal Analysis. Z.W.: Formal Analysis. K.B.: Writing/review and editing. M.E.T.: Writing/review and editing. Z.H.: Conceptualization, Supervision, Funding Acquisition. Each author has contributed significantly to the research presented in this paper. All authors have read and agreed to the published version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by grants from the Open
Research Fund of State Key Laboratory of Deep Oil
and Gas (SKLDOG2024-KFZD-01), National Natural
Science Foundation of China (42472163, 42402130
and 42272122), China Postdoctoral Science
Foundation (2024M751858), Qingdao Natural Science
Foundation (24-4-4-zrjj-52-jch), and Shandong
Provincial Natural Science Foundation
(ZR2022MD053). Yanyang Zhao acknowledges
financial support from the China Scholarship Council
(No. 202308370205).

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Supporting Information

**The monomolecular-layer selective adsorption of amino acids on microbial proto-dolomite induced by the cyanobacterium *Leptolyngbya boryana***

Yanyang Zhao ¹,²,³, Yishui Han ¹, Xiao Gao ¹, Xiangyu Wei ¹, Chao Han ¹, Zhaopeng Wang ¹, Karim Benzerara ⁴, Maurice E. Tucker ⁵, Zuozhen Han ¹,²*

1 Shandong Provincial Key Laboratory of Depositional Mineralization and Sedimentary Minerals, College of Earth Science and Engineering, Shandong University of Science and Technology, Qingdao 266590, China

2 Laboratory for Marine Mineral Resources, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China

3 Key Laboratory of Deep Oil and Gas, China University of Petroleum, Qingdao 266580, China

4 Sorbonne Université, Muséum National d'Histoire Naturelle, CNRS, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), 4 Place Jussieu, 75005 Paris, France

5 School of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK

*corresponding author: hanzuozhen@sdust.edu.cn

The following are the supplementary materials for the manuscript, including: the specific proto-dolomite crystal faces used in the DFT calculations; adsorption energy data of amino acids with different dissociation states on the crystal surfaces; TG and XPS data of pretreated proto-dolomite; and a magnified view of the Raman spectra showing the adsorption of Asp on the proto-dolomite surface.
<table><caption>Table S1 Initial conditions of the amino acid-Cpdol adsorption experiments.</caption><thead><tr><th>Group</th><th>Additive</th><th>R side chain group</th><th>Class</th><th>Solubility in water (g/100 mL H<sub>2</sub>O 25°C) (Xing et al. 2005)</th><th>Batc h number</th><th>Amino acid concentration (mg/L)</th><th>pH value</th><th>Salinity (NaCl, wt‰)</th></tr></thead><tbody><tr><td rowspan="12">Run 1</td><td rowspan="12">Asp</td><td rowspan="12">HOOCC H<sub>2</sub>-</td><td rowspan="12">Acidic</td><td rowspan="12">0.54</td><td>1</td><td>15</td><td>10</td><td>5</td></tr><tr><td>2</td><td>100</td><td>10</td><td>5</td></tr><tr><td>3*</td><td>150</td><td>8</td><td>5</td></tr><tr><td>4*</td><td>150</td><td>9</td><td>5</td></tr><tr><td>5**</td><td>150</td><td>10</td><td>5</td></tr><tr><td>6*</td><td>150</td><td>11</td><td>5</td></tr><tr><td>7*</td><td>150</td><td>10</td><td>10</td></tr><tr><td>8*</td><td>150</td><td>10</td><td>100</td></tr><tr><td>9*</td><td>150</td><td>10</td><td>200</td></tr><tr><td>10</td><td>200</td><td>10</td><td>5</td></tr><tr><td>11</td><td>300</td><td>10</td><td>5</td></tr><tr><td>12</td><td>500</td><td>10</td><td>5</td></tr><tr><td>13</td><td>800</td><td>10</td><td>5</td></tr><tr><td rowspan="12">Run 2</td><td rowspan="12">Gly</td><td rowspan="12">H-</td><td rowspan="12">Hydrocarbon</td><td rowspan="12">25</td><td>14</td><td>15</td><td>10</td><td>5</td></tr><tr><td>15</td><td>100</td><td>10</td><td>5</td></tr><tr><td>16*</td><td>150</td><td>8</td><td>5</td></tr><tr><td>17*</td><td>150</td><td>9</td><td>5</td></tr><tr><td>18**</td><td>150</td><td>10</td><td>5</td></tr><tr><td>19*</td><td>150</td><td>11</td><td>5</td></tr><tr><td>20*</td><td>150</td><td>10</td><td>10</td></tr><tr><td>21*</td><td>150</td><td>10</td><td>100</td></tr><tr><td>22*</td><td>150</td><td>10</td><td>200</td></tr><tr><td>23</td><td>200</td><td>10</td><td>5</td></tr></tbody></table>
<table><tbody><tr><td></td><td></td><td></td><td></td><td></td><td>24</td><td>300</td><td>10</td><td>5</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>25</td><td>500</td><td>10</td><td>5</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>26</td><td>800</td><td>10</td><td>5</td></tr><tr><td rowspan="10">Run 3</td><td rowspan="10">Pro</td><td rowspan="10">-CH<sub>2</sub>CH<sub>2</sub>CH<sub>2</sub>-</td><td rowspan="10">Hydrocarbon</td><td rowspan="10">162</td><td>27</td><td>15</td><td>10</td><td>5</td></tr><tr><td>28</td><td>100</td><td>10</td><td>5</td></tr><tr><td>29*</td><td>150</td><td>8</td><td>5</td></tr><tr><td>30*</td><td>150</td><td>9</td><td>5</td></tr><tr><td>31**</td><td>150</td><td>10</td><td>5</td></tr><tr><td>32*</td><td>150</td><td>11</td><td>5</td></tr><tr><td>33*</td><td>150</td><td>10</td><td>10</td></tr><tr><td>34*</td><td>150</td><td>10</td><td>100</td></tr><tr><td>35*</td><td>150</td><td>10</td><td>200</td></tr><tr><td>36</td><td>200</td><td>10</td><td>5</td></tr><tr><td>37</td><td>300</td><td>10</td><td>5</td></tr><tr><td>38</td><td>500</td><td>10</td><td>5</td></tr><tr><td>39</td><td>800</td><td>10</td><td>5</td></tr><tr><td rowspan="15">Run 4</td><td rowspan="15">Arg</td><td rowspan="15">HN=C(NH<sub>2</sub>)-NH(CH<sub>2</sub>)<sub>3</sub>-</td><td rowspan="15">Basic</td><td rowspan="15">15</td><td>40</td><td>15</td><td>10</td><td>5</td></tr><tr><td>41</td><td>100</td><td>10</td><td>5</td></tr><tr><td>42*</td><td>150</td><td>8</td><td>5</td></tr><tr><td>43*</td><td>150</td><td>9</td><td>5</td></tr><tr><td>44**</td><td>150</td><td>10</td><td>5</td></tr><tr><td>45*</td><td>150</td><td>11</td><td>5</td></tr><tr><td>46*</td><td>150</td><td>10</td><td>10</td></tr><tr><td>47*</td><td>150</td><td>10</td><td>100</td></tr><tr><td>48*</td><td>150</td><td>10</td><td>200</td></tr><tr><td>49</td><td>200</td><td>10</td><td>5</td></tr><tr><td>50</td><td>300</td><td>10</td><td>5</td></tr><tr><td>51</td><td>500</td><td>10</td><td>5</td></tr><tr><td>52</td><td>800</td><td>10</td><td>5</td></tr></tbody></table>

* Represents the batches reflecting the relationship between adsorption and pH values.

* Represents the batches reflecting the relationship between adsorption and salinities.
<caption>Table S2</caption>

Fitting parameters of the adsorption isotherm of amino acids on proto-dolomite.

<table><thead><tr><th rowspan="3">Amino acids</th><th colspan="3">Calculated pseudo-second-order kinetic parameters</th><th colspan="6">Adsorption isotherm models</th></tr><tr><th rowspan="2">Qe (mg/g)</th><th rowspan="2">k (g/mg/mi n)</th><th rowspan="2">R<sup>2</sup></th><th colspan="3">Langmuir model</th><th colspan="3">Freundlich model</th></tr><tr><th>b (mg/g)</th><th>K<sub>L</sub> (L/m g)</th><th>R<sup>2</sup></th><th>n</th><th>K<sub>F</sub></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Arg</td><td>21.44</td><td>0.0025</td><td>0.9853</td><td>7.29</td><td>0.19</td><td>0.9953</td><td>0.123</td><td>15.48</td><td>0.966</td></tr><tr><td>Gly</td><td>20.25</td><td>0.0026</td><td>0.9876</td><td>21.83</td><td>0.11</td><td>0.9901</td><td>0.092</td><td>13.01</td><td>0.928</td></tr><tr><td>Pro</td><td>7.19</td><td>0.00354</td><td>0.9905</td><td>22.32</td><td>0.23</td><td>0.9975</td><td>0.166</td><td>8.16</td><td>0.887</td></tr><tr><td>Asp</td><td>28.86</td><td>0.00139</td><td>0.9931</td><td>32.36</td><td>0.11</td><td>0.9838</td><td>0.191</td><td>2.41</td><td>0.733</td></tr></tbody></table>
<figure><img src="image_11.png" /><figcaption>Figure S1 The proto-dolomite (disorder dolomite) crystal faces employed for DFT calculations. (A) Seven representative crystal faces with different spatial positions for simulation computing. (B) Atomic arrangement diagrams on seven crystallographic planes ((101), (110), (111), (001), (010), (011), (100)). Green spheres represent calcium atoms; yellow spheres represent magnesium atoms; gray spheres represent carbon atoms; red spheres represent oxygen atoms. For each crystal plane, the x-axis is perpendicular to the page, the z-axis is vertically upward, and the y-axis is horizontally to the right.</figcaption></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

Figure S2 Thermogravimetric analysis (TGA) (A) and X-ray photoelectron spectroscopy (XPS) C 1s spectra (B) of cyanobacterially-induced proto-dolomite (Cpdol) before and after preprocess.
<figure><img src="image_14.png" /></figure>

Figure S3 Comparison of Asp functional groups adsorbed on the proto-dolomite surface and free Asp functional groups at wavenumbers 850-980 cm⁻¹. The minerals were collected from experiments with 150 mg/L amino acid concentrations, pH 11, and 5‰ salinity.
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

Figure S4 The minimum adsorption energies of four amino acids with protonated and deprotonated amino groups on seven selected crystal planes under current experimental conditions. The white bars are the calculation results for the protonated amino group state, and the gray bars are the calculation results for the deprotonated amino group. (A) Pro, (B) Gly, (C) Asp, (D) Arg.