# Effect of MICP treatment on the mechanical properties of clay soils

Olivier Maston, Tariq Ouahbi, Said Taibi, Ahmad El Hajjar, Leslie Sapin,

Annette Esnault-Filet, Benoit Duchemin, Jean-Marie Fleureau

► To cite this version:

Olivier Maston, Tariq Ouahbi, Said Taibi, Ahmad El Hajjar, Leslie Sapin, et al. Effect of MICP treatment on the mechanical properties of clay soils. Transportation Geotechnics, 2025, 50, pp.101483. 10.1016/j.trgeo.2025.101483. hal-04864533

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Original Article

# Effect of MICP treatment on the mechanical properties of clay soils

Olivier Maston<sup>a</sup>, Tariq Ouahbi<sup>a,* ID</sup>, Said Taibi<sup>a ID</sup>, Ahmad El Hajjar<sup>a ID</sup>, Leslie Sapin<sup>b</sup>, Annette Esnault-Filet<sup>b ID</sup>, Benoit Duchemin<sup>a ID</sup>, Jean-Marie Fleureau<sup>c ID</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

MICP

Clay

Mechanical properties

Soil improvement

FTIR / XRD

ABSTRACT

The presence of drying cracks can significantly affect soil hydromechanical behavior, which has effects on soil performance in civil engineering. One innovative approach that has received much interest in the last decade is using microbially induced calcium carbonate precipitation (MICP) for soil reinforcement and stabilization. A series of clay specimens with varying moisture levels and concentrations of cementation solution were carefully prepared. All these samples were subjected to a series of mechanical tests to assess the improvement in the clay's mechanical characteristics. These tests covered various conditions, ranging from unsaturated to saturated states of the clays. The results showed that the strength of clays was significantly improved, and the most significant increase in mechanical strength was observed with the 1.4 M MICP solution. The precipitation of CaCO₃ was quantified using a calcimeter. In addition, composition analysis by X-ray diffraction and attenuated total reflectance infrared spectroscopy confirmed the presence of calcium carbonate crystals and indicated residual urea and calcium acetate.

Introduction

Climate change is a major environmental challenge of the 21st cen-
tury, with greenhouse gases, especially CO2, contributing significantly
to global warming. The construction sector plays a key role in this issue,
as its materials consume energy, emit pollutants, and deplete non-
renewable resources. Using bio-based materials with low environ-
mental impact is essential for the ecological transition. Raw earth ma-
terials are a promising option for future construction, since a third of the
world's population already lives in earthen buildings. However, to be
widely adopted, these materials need to meet mechanical, hygrothermal
and durability standards. The mechanical strength of raw earth depends
on the construction technique used [1], and can be improved through
hypercompaction [2], the use of biopolymers [3,4] or vegetable fibres
[5,6]. Latex or acrylic emulsions surface treatments of raw earth walls,
enhance durability by reducing water infiltration, but may negatively
affect the material's hygrothermal properties.

Some studies focus on compromise solutions. For example, Keboa
and kagi [7] demonstrated that adding a silicone water-repellent can
reduce water absorption in cement-stabilized rammed earth without
compromising vapor permeability.

Raw earth materials contain a complex clay fraction. Clay behavior must be considered in all related engineering fields. In fact, clays exhibit significant chemical sensitivity due to the complexity of their mineral structures and surface properties. The crystalline structure of clay mineral sheets consists of a stacking of one octahedral layer and one or two tetrahedral layers. The apical oxygens of the tetrahedra, which replace hydroxyls at the vertices of the octahedra, ensure the bond between these two types of layers [8]. Clays behavior is largely influenced by their chemical composition. Several factors affect the chemical sensitivity of clays, such as cation exchange capacity (CEC), specific surface area, pH, and oxidation state [9]. Additionally, clays are sensitive to specific elements such as calcium, which often alters their properties by increasing their strength and reducing their dispersion [10]. Thus, the chemical sensitivity of clays arises from the complex interactions between their mineral composition, surface properties, and environmental conditions. It is essential to consider these sensitivities to understand the behavior of clays in various applications.

Furthermore, the osmotic effect influences the behavior of clays. Sathyapriya et al. [11] demonstrated that the difference in salt concentration between the free pore fluid and the fluid adjacent to clay particles leads to osmotic pressure in clay soils. Where, Jones [12]

<footnote><sup>a</sup> Université Le Havre Normandie, Normandie Université, LOMC, UMR 6294 CNRS, 53 rue de Prony, 76058 Le Havre Cedex, France</footnote>

<footnote><sup>b</sup> Soletanche Bachy, 280 Av. Napoléon Bonaparte, 92500 Rueil-Malmaison, France</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, CentraleSupélec, Laboratoire de Mécanique des Sols, Structures et Matériaux, UMR 8579 CNRS, Gif-sur-Yvette, France</footnote>

<footnote>* Corresponding author at: Université Le Havre Normandie, Normandie Université, LOMC, UMR 6294 CNRS, 53 rue de Prony, 76058 Le Havre Cedex, France.
E-mail address: tariq.ouahbi@univ-lehavre.fr (T. Ouahbi).</footnote>
<table><caption>**Table 1**

Characteristics of kaolin P300.</caption><thead><tr><th>Characteristics</th><th>Designation</th><th>Unit</th><th>Kaolin P300</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>Granular Form</td><td>—</td><td>Rigid hexagonal plates</td><td>[73]</td></tr><tr><td></td><td>Clay family</td><td>—</td><td>Phyllosilicates 1/1 (TO)</td><td>[74]</td></tr><tr><td></td><td>Specific Area</td><td>Sp (m²/g)</td><td>32</td><td>[75]</td></tr><tr><td></td><td>Cation Exchange Capacity</td><td>CEC (meq/hg)</td><td>15</td><td>[75]</td></tr><tr><td></td><td>Granulometry</td><td>(%) &lt; 2 µm</td><td>60</td><td>[76]</td></tr><tr><td></td><td>Grain density</td><td>Gs (g/cm³)</td><td>2.65</td><td>[77]</td></tr><tr><td></td><td>Liquid Limit LL</td><td>(%)</td><td>40</td><td>This study</td></tr><tr><td></td><td>Plastic Limit PL</td><td>(%)</td><td>20</td><td></td></tr><tr><td></td><td>Plasticity Index PI</td><td>(%)</td><td>20</td><td></td></tr><tr><td></td><td>USCS classification</td><td>—</td><td>CL</td><td></td></tr></tbody></table>

highlighted the behavior of clays in response to osmotic effects, which depend on their cation exchange properties. These properties favor the adsorption of calcium and magnesium over sodium. As a result, clays containing sufficient adsorbed calcium and magnesium are more resistant to dispersion by water due to the aggregation of their particles. Aware of these facts, Jamsawang et al. [13] have considered this behavior to stabilize natural swelling soils, by valorizing bottom ash. This waste acts as an alternative stabilizer, reducing the sodium content of swelling clays and increasing their calcium content. These authors established a correlation to predict free swell potential using the stabilized thickness ratio at field scale. Various techniques have been developed to stabilize swelling soils, such as the addition of cement [14], or lime [15]. Recent advances have led to the development of simple constitutive models aimed at predicting the behavior of clay soils, particularly their one-dimensional deformation, based on factors such as hydric variations over time and under stress [16], loading conditions and saturation degree [17], or as a function of cracking rate, water content, dry density and loading [18].

Several authors studied soil enhancement solutions to allow to use it in engineering. Biocalcification by microbially induced calcium carbonate precipitation (MICP) has emerged as a potential and ecological solution for soil enhancement over the last two decades. It is inspired by bacterial activity in natural environments, harnessing the ability to catalyze the precipitation of calcite (CaCO₃), [19]. Researchers have expressed significant interest in using MICP to enhance the mechanical properties of soils through the formation of calcite bridges between soil particles while controlling alterations to the medium's permeability [20]. In the first large-scale trials, MICP was observed to improve the

stability of 1000 m³ of gravelly soil prior to drilling [21], and to reinforce 100 m³ of sandy soil [22]. Nonetheless, the complexity of biogeochemical reactions has resulted in a wide dispersion of results, prompting investigations into treatment protocols and the impact of the MICP process on soil microstructure. [23] studies established links between improvements in sand mechanical strength, modifications in permeability, and microstructural changes observed in 3D.

The field of use of MICP processing is increasingly expanding. Several research studies focus on it, particularly for the treatment against internal erosion of dikes and embankments. [24] studied the bio-cementation in marine conditions, which prevents soil liquefaction [25]. It is used to improve the durability of roads [26] and, more broadly, the durability of cohesive soils under dry-wet and freeze-thaw cycles [27]. Some companies use the process for the treatment against internal erosion of dikes and embankments, soil liquefaction, deepening of quay walls, soil reinforcement under vibration-sensitive structures and stone regeneration in historic monuments.

Research on the use of MICP in environmental fields are therefore expanding, as evidenced by the growing number of publications and recent literature reviews. These include studies on the impact of hydrodynamics, as detailed by Omoregie et al. [28] which explores global research trends; the immobilization of heavy metals [29,30]; self-repair of concrete and carbon sequestration [31–33]; soil erosion mitigation [34]; nanoparticle-enhanced biocementation [35]; using frass for MICP in soil biocementation [36]; and the broader optimization of CaCO₃ precipitation processes [37]. These highlights future research directions and the extensive work required, particularly regarding the impact of environmental factors on MICP, which complicates scaling from laboratory experiments to field applications. Given the wide range of potential environmental applications, it is evident that the effects of MICP on clays is a key factor in many engineering challenges that require in-depth study.

Punnoi et al. [38] conducted a comparison between vegetative cells and bacterial spores. Their findings demonstrated that both vegetative cells and bacterial spores can enhance the strength and modulus of clays by inducing MICP. Vegetative cells are more effective at enhancing clay strength in the early stages, whereas bacterial spores provide more significant improvements in long-term clay strength. Furthermore, [39] have demonstrated that bacterial spores repair shrinkage cracks in cementations through the formation of calcite crystals on the crack surface. Similarly, Nuaklong et al. [33] have optimized cement healing by coupling a minimal amount of bacterial spores with a superabsorbent polymer.

In addition, Xiao et al. [40] used MICP to strengthen clay samples by mixing the soil with the bacterium and cementation solution. This study

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** CaCO₃ precipitation process in clay particles.</figcaption></figure>
<table><caption>**Table 2**
Summary of parameters used in shear and UCS tests.</caption><thead><tr><th>Type of test</th><th>K100 sample class</th><th>Bacterial presence</th><th>Cementation solution concentration (M)</th><th>Calcium source</th><th>w (%)</th><th>γ<sub>d</sub> (kN/m<sup>3</sup>)</th></tr></thead><tbody><tr><td rowspan="12">Shear test<br>(Laboratory vane test)<br>D = 50 mm<br>H = 70 mm<br>T = 20 ± 0.2 °C</td><td rowspan="12">Treated</td><td rowspan="12">None</td><td rowspan="12">1.4</td><td>None</td><td>28</td><td>15.1</td></tr><tr><td rowspan="3">Calcium Nitrate</td><td>32</td><td>14.5</td></tr><tr><td>36</td><td>13.6</td></tr><tr><td>40</td><td>12.9</td></tr><tr><td rowspan="3">Calcium Chloride</td><td>60</td><td>10.2</td></tr><tr><td>Calcium Acetate</td><td>32</td><td>14.9</td></tr><tr><td>None</td><td>32</td><td>15.0</td></tr><tr><td rowspan="3">Calcium Nitrate</td><td>32</td><td>14.7</td></tr><tr><td>32</td><td>15.3</td></tr><tr><td>Calcium Chloride</td><td>32</td><td>15.4</td></tr><tr><td rowspan="3">Calcium Acetate</td><td>28</td><td>15.2</td></tr><tr><td>32</td><td>13.2</td></tr><tr><td>40</td><td>10.8</td></tr><tr><td rowspan="12">UCS test<br>D = 50 mm<br>H = 100 mm<br>T = 20 ± 0.2 °C</td><td rowspan="12">Untreated</td><td rowspan="12">None</td><td rowspan="12">None</td><td rowspan="12">None</td><td>32</td><td>15.4</td></tr><tr><td>40</td><td>13.4</td></tr><tr><td>60</td><td>10.4</td></tr><tr><td>32</td><td>14.6</td></tr><tr><td>40</td><td>12.8</td></tr><tr><td>60</td><td>10.8</td></tr><tr><td>32</td><td>14.9</td></tr><tr><td>40</td><td>12.9</td></tr><tr><td>60</td><td>10.6</td></tr><tr><td rowspan="3">Calcium Nitrate</td><td>7.5</td><td>15.2</td></tr><tr><td>12</td><td>15.4</td></tr><tr><td>18</td><td>15.2</td></tr><tr><td rowspan="3">Calcium Chloride</td><td>7.5</td><td>15.2</td></tr><tr><td>12</td><td>15.0</td></tr><tr><td>18</td><td>15.1</td></tr></tbody></table>

observed the mechanical effectiveness of it on clays by conducting UCS tests. Also, Liu et al. [41] used surface spray MICP to strengthen clay samples against desiccation cracking. Here, the study showed the effectiveness of a spray technique in mitigating this phenomenon by decreasing the number, thickness, and length of cracks. [42] used MICP at different bacterial and nutrient solution dosages to mitigate cracking during bentonite desiccation. They observed the deformations at the surface of their samples using a camera, and were able to highlight the effectiveness of the treatment in delaying crack initiation and mitigating them. [43] also demonstrated an increase in bacteria growth and urease activity when clay minerals were added to aqueous solutions. MICP also provided better calcite precipitation and UCS resistance for sand samples containing a clay fraction compared to sand samples without a clay fraction. However, MICP treatment by injection is less effective in low-porosity media such as clays [44]. Geometrical compatibility between the size of the bacteria and the pore size of a clayey soil is essential. Clay soil compaction density must be controlled to ensure pore sizes are larger than bacterial cells 1.5 µm diameter [45,46] demonstrated that MICP enhanced the mechanical properties of expansive clay soil, indicating that the bacterial size was appropriate for clay pore size. The comparative study by Zhang et al. [47] highlighted an improvement in erosion resistance due to the effect of MICP, more significant on sandy than clayey slopes. However, MICP still reduced erosion on clay slopes. Despite variations in techniques for implementing MICP treatments, the process can be further optimized by combining it with other methods. In this respect, Wang et al. [48] demonstrated the promising results achieved by combining biochar and the MICP-BIN technique for reinforcing soft ground. Vail et al. [49] also showed the benefits of using bottom ash and MICP in reinforcing clayey soils for cyclic wetting-drying conditions.

The common thread running through all this research on MICP treatment in clays is using CaCl₂ as a calcium source. [50] showed that calcium acetate is more suitable for MICP treatment of reinforced concrete because it does not degrade steel, unlike calcium chloride. In addition, it provides better mechanical strength and better pore size distribution.

The calcium source used for MICP can affect mechanical strength [51], precipitated crystalline form [52], hydromechanical properties [53], and pollutant emissions. Calcium acetate could reduce NH₃ production in cement by half compared to calcium chloride and calcium nitrate [54]. Specifically, a part of CH₃COO⁻ reacts with NH₃ to generate CH₃COONH₄, reducing NH₃ emissions.

The main objective of using MICP in clay is to limit cracking due to desiccation. Before conducting further studies on this phenomenon, the aim was to confirm the precipitation of CaCO₃ crystals in clay, which is a non-inert and complex environment, and to examine the mechanical improvement it provides. Although similar studies have already been conducted in clay environments, an innovative approach was taken by utilizing calcium acetate as the calcium source, which emits less ammonia during MICP and seems more suitable than calcium chloride for the P300 clay material. In this work, specimens were prepared experimentally using the soil mixing procedure, which involves mixing a clay powder with a bacterial and cementation solution containing an equimolar calcium acetate and urea solution. Samples are mechanically tested 3 days after preparation in order to evaluate the increase in strength of a clay in the saturated and unsaturated domains. This also allowed to identify the optimal concentration of cementation solution to maximize the strength gain of the material. Chemical composition analysis by ATR-FTIR demonstrated that it is simple and effective to detect the presence of CaCO₃ and other chemical bonds without altering the material through high-temperature drying. Furthermore, X-ray diffraction analysis confirmed that, although present in small quantities, the precipitation of CaCO₃ occurs in the form of calcite.

## Materials and methods

### Clayey soils

One clay was used in this study. An industrial kaolin called “P300” containing 96 % pure kaolinite with low content of quartz and illite [55]. The choice of P300 refers to the one used by El Hajjar et al. [56], who studied its cracking behavior. This study aims to confirm the
<figure><img src="image_4.png" /><figcaption>Fig. 2. Preliminary results obtained from the laboratory vane shear test (a) Representative undrained cohesion-time curve of K100 treated by MICP (b) Effect of MICP calcium sources on undrained cohesion of K100 specimens at 32% of water content.</figcaption></figure>

successful implementation of MICP in this clay to subsequently assess its impact on desiccation cracking.

For mixtures, pure kaolin P300 will be referred to as K100.

According to ASTM D4318-17e1, Table 1 summarizes some characteristics of this clay.

## Bacteria

This study uses a urease-producing bacterium called *Sporosarcina pasteurii*, supplied in powder form. This recognized and ubiquitous bacteria is often used for MICP treatment due to its high urease activity and negative surface charge, which favors calcium ion binding [57].

To cultivate these bacterial colonies, the typical process involves inoculating a strain of *S. pasteurii* into a nutrient-rich medium, which is then sterilized using an autoclave [58]. Subsequently, the bacterial cells were incubated in this medium at an optimal temperature, typically between 30 and 37 °C. To assess bacterial growth, the optical density (OD) measured using a UV spectrophotometer at a wavelength of 600 nm (OD600). The obtained bacteria were then used in the MICP process.

In this study, the used OD falls within the range of 0.8 to 1.2. It's important to note that the enzymatic activity of the bacteria has been verified to adjust the bacterial mass concentration. This activity considers the explanations several authors like Stocks-fischer et al. [57]; Okwadha et Li [59] and Al salloum et al. [60]) to allow a theoretical total precipitation of CaCO₃ within a defined time.

Calcification reaction can be described through two equations (Eq. (1) and Eq. (2), which correspond to two distinct steps: (Eq. (1) urea is hydrolyzed by microbial urease, resulting in the formation of ammonium and carbonate ions; (Eq. (2) free calcium ions react with the carbonate ions produced to generate calcite precipitation (Fig. 1).

$$CH_4N_2O + 2H_2O \rightarrow 2NH_4^+ + CO_3^{2-} \quad (1)$$

$$Ca^{2+} + CO_3^{2-} \rightarrow CaCO_3 \downarrow \quad (2)$$

## Sample preparation

Samples were prepared using a “Soil-mixing” technique, which
<figure><img src="image_5.png" /><figcaption>Fig. 3. Representative undrained cohesion – water content curve of treated MICP K100 specimens with various MICP concentrations obtained from the laboratory vane shear test.</figcaption></figure>

involves the creation of two solutions for the biocementation process. The first solution consists of bacteria from S. pasteurii diluted in water while the second is an equimolar cementation solution containing urea and calcium source diluted in water. Both solutions were mixed to form a MICP solution composed of 75 % cementation solution and 25 % bacterial solution. The soil mixing technique consists of two steps. Firstly, the K100 clay powder was dried in an oven at 105 °C for 48 h to remove the pore water and reach a zero-water content [61]. Then, the solution (MICP, cementation or bacterial) is mixed with dried K100 clay powder, until reaching a uniform clay slurry with a homogeneous water content. In this case, as the interstitial fluid is composed solely of this solution, there is no chemical concentration gradient within the material, and therefore no osmotic effect.

The concentration of the MICP solution refers to the concentration of the urea and calcium source in solution. The water content of the samples preparation only considers the liquids from the MICP solution.

**For the shear test using laboratory vane test:** The clay slurry was poured in layers into hermetic cylindrical jars of D = 50 mm diameter and H = 70 mm height. For each sample, the water content and the MICP solution concentration were monitored. Samples were fully saturated, with water contents ranging from 28 % to 60 %. The MICP solution concentration vary from 0.4 M to 1.4 M. Between 28 % and 32 % of water content, the dry unit weight (γd) was set to 15 kN/m³. This parameter decreases as the water content of the saturated material increases, between 32 % and 60 %.

For each given water content, a reference sample without MICP treatment was prepared. For each parameters condition, three similar samples were prepared.

It's important to note that to select the most suitable calcium source for MICP treatment on clay, eight distinct K100 samples were prepared at a water content of 0.8 LL. Three specimens were treated with a 1.4 M MICP solution using Ca(NO₃)₂, CaCl₂, and Ca(CH₃COO)₂ as calcium sources, denoted MICP-Nitrate, MICP-Chloride, and MICP-Acetate respectively. Three other specimens used only a cementation solution with the same sequence of calcium sources and were called CS-Nitrate, CS-Chloride and CS-Acetate respectively. One specimen was based solely on a bacterial solution, labeled as BS, while a reference sample containing only water was designated as untreated.

**For the Unconfined Compressive Strength (UCS) test:** soil samples were prepared in an unsaturated state, with water content ranging from 7.5 % to 18 %. The MICP solution concentration was set at 1.4 M.

The soil mixture was compacted in a double-piston cylindrical mold with a diameter of D = 50 mm and a height of H = 100 mm to reach a γd of 15 kN/m³. The compacted samples were then sealed to prevent drying for 72 h, to ensure calcium carbonate precipitation and water content homogeneity.

For the shear and UCS tests, a reference untreated K100 sample was prepared for each target water content. For each condition, three similar samples were prepared. All samples were prepared and tested in a thermostatic room maintaining the temperature at T = 20 °C with a variation of ±0.2 °C.

For both tests, water contents were defined based on the plastic and liquid limits of the clay material, then added points based on sample and/or test feasibility. Samples with 12 % and 32 % water content were prepared to refine the curve illustrating the evolution of mechanical strength.

The summary of parameters used in shear and UCS tests is presented in Table 2.

## Testing methods

The test procedure involved mechanically testing samples three days after their preparation to evaluate the strength increase of clay in both saturated and unsaturated conditions. Samples were taken from the top, middle, and bottom sections of each specimen, and their composition was analyzed using FTIR. These sample sections were then dried in an oven at 105 °C and subsequently ground into powder form for further compositional analysis via XRD and FTIR. Finally, the dried powders were tested using a Dietrich-Fruhling calcimeter to determine the calcium carbonate content in each sample.

## Mechanical properties

Following the ASTM D4648/D4648M-16, the laboratory vane shear test was used at a constant shear rate of 18°/min, to measure the undrained cohesion of soft clays, given by (Eq. (3)):

$$C_u = \frac{6 M}{\pi D^2 (3H + D)} \quad (3)$$

Cu is the undrained cohesion (kPa), M the force couple (kN.m). D and H correspond to blade diameter and height (m), respectively.

The blade has a diameter and height of 1.3 cm to limit edge effects on
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Behavior of treated MICP K100 specimen with 18 % water content obtained from the UCS test (a) Failure plane i) before the UCS test; ii) during the UCS test; iii) after the UCS test (b) Stress-strain curve.</figcaption></figure>

the sample. UCS tests, according to ASTM D2166/D2166M-16, are carried out on low water content specimens with 50 mm diameter and 100 mm height. The UCS tests are performed at a constant strain rate of 0.1 mm/min, fast enough to maintain undrained conditions.

The combination of these two laboratory experiments allows a wide range of water contents to be prospected. The vane shear test was used for high water contents corresponding to a liquidity index (LI) range between 0.7 and 1.5 LL, while the UCS test was used for water contents below 0.7 LL.

### Calcium carbonate content measurement

Calcium carbonate content measurement is carried out on soils after mechanical tests. Three specimens were taken from the top, middle, and bottom of each sample.

The CaCO₃ content (CCC) of samples is measured using Dietrich-Frühling calcimeter test according to AFNOR standard NF P94-048.

The “precipitation efficiency” parameter analyses the effectiveness of the MICP treatment for different water contents and MICP solution concentrations. Precipitation efficiency (E<sub>FF</sub>) is defined as the ratio between the experimental precipitated content of CaCO₃ and the theoretical precipitated content of CaCO₃ (Eq. (4)). The experimental precipitated content of CaCO₃ corresponds to the difference between the

CaCO₃ content of the MICP-treated sample and that of the untreated sample. The theoretical precipitated CaCO₃ content is the maximum amount of CaCO₃ that can be precipitated by MICP process, considering the total reaction of urea and calcium sources.

$$E_{ff} (\%) = \frac{\text{Experimental precipitated content of CaCO}^3 (\%)}{\text{Theoretical precipitated content of CaCO}^3 (\%)} * 100 \quad (4)$$

Three repetition measurements were carried out for each specimen.

### Composition analysis

For the composition analysis, specimens' selection was considered the same as that for the calcium carbonate content measurement.

The analytical techniques of X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were used to characterize the mineralogy of treated and untreated specimens with 40 % preparation water content. Crystal structure was determined by XRD on dry powder samples using the Xpert-Pro Powder diffractometer (PANalyticalB.V., Almelo, Nederland), in normal Bragg-Brentano geometry at a voltage of 40 kV and an intensity of 40 mA. The radiation source was a cobalt anode. XRD analysis was performed on an air-dried deposition, with data recorded in 0.013° steps between 5° and 80°.

FTIR analysis was conducted on wet pastes or dry powders using a
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Mechanical results obtained from the UCS test of treated MICP and untreated K100 specimens for several water contents (a) Stress–strain curves (b) UCS value and E₅₀.</figcaption></figure>

Shimadzu Spirit FTIR spectrometer system equipped with a single-reflection ATR measurement accessory. For each sample 32 scans were made with an accuracy of 4 cm⁻¹ within the wavelength range from 4000 to 400 cm⁻¹. Background signal was subtracted to the sample spectrum and the samples spectra were normalized using the K100 peaks 960–1040 cm⁻¹ range of (ν1) Si-O* symmetric stretching.

## Results and Discussions

The mechanical strength of the material was studied by a vane shear test and an UCS test. The two results were then correlated. After shearing, the calcimeter was used to measure the calcium carbonate content of the material. Finally, the crystalline and chemical properties of the material were analyzed using ATR-FTIR and XRD methods.

### Laboratory vane shear test

Fig. 2(a) illustrates the undrained cohesion-time curves of K100 specimens treated with MICP from calcium acetate, obtained from laboratory vane shear tests. The specimens had water contents of 40 % and 60 % corresponding to 1.0 LL and 1.5 LL respectively. The curve of the latter exhibits a high liquidity index, causing just a minor increase in cohesion over time. This slight increase occurs between 12 and 24 h, and then the curve stabilizes, whereas the 1.0 LL curve, starting in a state between liquid and plastic, leads to a greater increase in Cu compared to that of the 1.5 LL curve. Moreover, this curve reaches a stable state after 12 h. In these tests, MICP influences the mechanical properties during the first 24 h. This would be the time considered for calcium carbonate precipitation in this clay. Based on this, a safety time factor was
<figure><img src="image_9.png" /><figcaption>Fig. 6. Mohr's Coulomb circle of a treated MICP and untreated K100 specimen with 18 % water content under UU conditions obtained from the UCS test</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 7. MICP cohesion of treated MICP K100 specimens as a function of water content and liquidity index.</figcaption></figure>

considered, and all proceeding tests were sheared with a curing period of 3 days.

Fig. 2(b) shows that the undrained cohesion of the untreated samples was 1.5 kPa. Plus, the cementation and bacterial solution had no influence on undrained cohesion with respect to that of the untreated ones. However, MICP treatment, for K100 mixed with any of the three different calcium sources considered, resulted in an increase in Cu; compared to that of the untreated samples as shown in Fig. 2(b). MICP-

Nitrate yielded a Cu of 7 kPa, while MICP-Chloride exhibited a Cu of 27 kPa. MICP-Acetate demonstrated the highest Cu of 48 kPa. Hence, in order to improve mechanical strength, MICP treatment with calcium acetate was used for further experimentation of this study.

Fig. 3 presents undrained cohesion-water content for untreated and treated MICP K100 specimens subjected to various equimolar urea-calcium acetate concentrations. The increase in undrained cohesion with decreasing water content was due to the reduction of water content
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Effect of water content and MICP concentration on (a) Precipitation efficiency in specimens from the laboratory vane shear test (b) Calcium carbonate content in specimens from the laboratory vane shear test (c) Precipitation efficiency (d) Calcium carbonate content.</figcaption></figure>

in a saturated medium (Sr = 100 %), which leads to an increase in dry density in untreated samples. As a result, the spacing between soil particles was reduced. An increase in Cu was observed when the concentration of the MICP solution is increased for the same water content. The maximum MICP concentration at 1.4 M corresponds to the solubility limit of calcium acetate in the cementation solution, close to 1.9 M. At this concentration limit, optimum undrained cohesion was achieved in K100. The highest Cu value obtained for treated K100 samples was 66 kPa, with 28 % water content and 1.4 M MICP solution. This Cu value represents 650 % of the undrained cohesion of untreated K100. Therefore, the following tests used exclusively a cement concentration of 1.4 M to achieve better mechanical improvements.

### Unconfined Compressive Strength test

For lower water contents UCS test is considered. The material's homogeneity is demonstrated by the clear existence and continuity of the failure plane. Under unconsolidated undrained (UU) conditions, the theoretical failure plane of soil exhibits a 45° angle. In Fig. 4(a), the

treated K100 specimen with a 18 % water content shows a failure plane according to the theoretical expectations.

Fig. 4(b) presents the stress-strain curves of the UCS test for the K100-treated sample with 18 % water content. These curves are used to determine the maximum compressive strength value (UCS value) and the deformation modulus $E_{50}$. The UCS value represents the peak of the curve, where it is twice the value of Cu under UU conditions. $E_{50}$ modulus demonstrates the stiffness of clayey soils in the high plastic deformation range and is defined as the ratio of half the UCS value over the corresponding strain.

Fig. 5(a) presents the stress-strain curves of treated and untreated K100 specimens subjected to various water content levels. The nomenclature “Treated MICP (w= %)” and “Untreated (w= %)” corresponds to treated and untreated samples with the respective percentage water content. These curves reveal a consistent pattern for all samples where axial stress increases to a peak (UCS value), then decreases significantly. These curves' profiles demonstrate the mechanical behavior of the samples. The reduction in water content made the sample more brittle than ductile. For the same water content, treated and untreated samples
<figure><img src="image_12.png" /><figcaption>Fig. 8. (continued).</figcaption></figure>

exhibit comparable behavior. This implies that water content mainly affects mechanical behavior at low water content ranges, while calcium carbonate precipitation acts as a secondary factor. Furthermore, all K100 samples treated with MICP have higher UCS values than untreated samples, highlighting the positive impact of MICP on clay.

Based on the maximum stress and deformation modulus obtained, Fig. 5(b) provides an analysis of the results. It reveals an increase in the strength of the K100 samples as the water content decreases and/or as they are treated with MICP. When the water content decreases with a fixed dry density, the void ratio remains constant, but suction increases. As a result, mechanical strength increases. Similarly, treatment with MICP increases the value of UCS. MICP induces a gain of 150 kPa at a water content of 18 %. In contrast to the vane test results, the highest strength provided by MICP in the UCS test corresponds to the sample with the highest water content. Like the UCS behavior, the E$_{50}$ modulus increases as water content decreases and/or when treated with MICP. On one hand, this indicates that the material becomes less saturated, less plastic, and stiffer. On the other hand, the improvement of E$_{50}$ value through MICP treatment is significant. MICP induces an improvement of 46 % of E$_{50}$ value, reaches 40.8 MPa at a water content of 18 %.

Relationship between the two mechanical tests

In order to establish a correlation between the vane shear and UCS results and to highlight the cohesion provided by MICP treatment, a parameter called “C$_{MICP}$” was introduced. C$_{MICP}$ is defined as the difference between the total cohesion of a treated sample and the total cohesion of an untreated sample (Eq. (5)). It specifically quantifies the cohesion enhancement due to MICP.

$$C_{MICP} = C_{total} - C_{withoutMICP} \qquad (5)$$

The total cohesion of a material is composed of different types of cohesion. “C$_{without MICP}$” includes all forms of cohesion present in the material other than those induced by MICP (Eq. (6)). This includes “C$_{effective}$”, which represents cohesion generated by compaction and densification of the material. In addition, “C$_{without MICP}$” incorporates “C$_{capillary}$”, which represents cohesion resulting from suction.

$$C_{withoutMICP} = C_{effective} + C_{capillary} \qquad (6)$$

C$_{total}$ represents the overall cohesion of the material treated with MICP.
<figure><img src="image_13.png" /><figcaption>**Fig. 9.** ATR-FTIR spectra of (a) Dry and wet CaCO₃ precipitated by MICP in aqueous solution with calcium acetate (b) Untreated and treated MICP dry K100 (c) Untreated and treated MICP wet K100 (d) Dry K100 with different additives (e) Wet K100 with different additives.</figcaption></figure>

Using Mohr's circles and the Mohr-Coulomb failure criterion, MICP cohesion resulting from the UCS test was defined.

Fig. 6 shows two Mohr circles plotted for both treated and untreated K100 samples at $\gamma_d = 15 \text{ kN.m}^{-3}$ and $w = 18 \%$. They are obtained through the UCS test under UU conditions. The total cohesion corresponds to the maximum shear stress. The friction angle is considered to be zero due to the undrained conditions. For the untreated K100 sample, the total measured cohesion is equal to the cohesion without MICP. Similar to the laboratory vane shear test, MICP cohesion is determined as the difference between the maximum shear stress of a MICP-treated

sample and an untreated sample. In Fig. 6, $C_{\text{total}}$ amounts to 233 kPa, while $C_{\text{without MICP}}$ is 157 kPa. Consequently, $C_{\text{MICP}}$ is approximately 76 kPa.

In Fig. 7, $C_{\text{MICP}}$ values are compiled for all treated K100 samples subjected to a 1.4 M MICP solution (from both vane and UCS tests) into a single curve as a function of water content and Liquidity Index. MICP cohesion increases as water content decreases, with the peak value reached at 18 % water content, i.e., 76 kPa. These findings reveal the consistent profile of the $C_{\text{MICP}}$ – water content curve, which maintains a similar slope along its extension despite the change in mechanical
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>Fig. 9. (continued).</figcaption></figure>

<caption>**Table 3**</caption>

Assignment of dry and wet ATR-FTIR spectra of CaCO3 precipitated by MICP in aqueous solution with calcium acetate.

<table><thead><tr><th>Wavenumber (cm<sup>-1</sup>)</th><th>Assignment</th><th>Reference assignment</th></tr></thead><tbody><tr><td>Dry / Wet</td><td></td><td></td></tr><tr><td>- / 1642</td><td>H-O-H bending</td><td>[78]</td></tr><tr><td>- / 1555</td><td>(v<sub>3</sub>) Asymmetric stretching of C-O</td><td>[67]</td></tr><tr><td>1439 / 1431</td><td>(v<sub>3</sub>) Asymmetric stretching of CO<sub>3</sub><sup>2-</sup></td><td>[65,66] and [79]</td></tr><tr><td>1411 / 1390</td><td>(v<sub>1</sub>) Symmetric stretching of CO<sub>3</sub><sup>2-</sup></td><td></td></tr><tr><td>1086 / 1086</td><td>(v<sub>2</sub>) Out-of-plane bending CO<sub>3</sub><sup>2-</sup></td><td></td></tr><tr><td>875 / 872</td><td>(v<sub>4</sub>) In-plane bending of CO<sub>3</sub><sup>2-</sup></td><td></td></tr><tr><td>744 / 741</td><td></td><td></td></tr></tbody></table>

testing method. However, the curve decreases when it reaches 12 % of water content. The profile of the curve between 12 % and 7.5 % is unknown due to the high uncertainty of the results. The result data underwent statistical analysis to determine the standard deviation and, consequently, the uncertainty of the results, as shown in the figures presenting the results. It is important to note that these uncertainties only consider the dispersion of the results and not the precision of the measurements provided by the experimental devices.

The results of the C<sub>MICP</sub>-Liquidity Index curve shown in Fig. 7 highlight the presence of three distinct behaviors depending on the sample state. As water content decreases, the liquid state initially displays a slight increase in C<sub>MICP</sub>, followed by a significant increase in the plastic state and finally a decrease in the solid state. This behavior can be explained by several factors:

1) In the liquid state, the inter-particle distance is larger than in other states, which explains why it is not possible to maintain a fixed dry density. Consequently, the effect of calcium carbonate cementation in
<table><caption>**Table 4**
Assignment of dry and wet ATR-FTIR spectra of K100 with and without MICP treatment.</caption><thead><tr><th>Sample</th><th>Wavenumber (cm<sup>-1</sup>)<br>Dry / Wet</th><th>Assignment</th><th>Reference assignment</th></tr></thead><tbody><tr><td rowspan="12">K100</td><td>– / 3379</td><td>(v<sub>1</sub> and v<sub>3</sub>) Symmetric and asymmetric stretching of O–H (water)</td><td>[78]</td></tr><tr><td>– / 1639</td><td>H–O–H bending</td><td></td></tr><tr><td>3694 / 3688</td><td>OH<sup>−</sup> stretching</td><td>[80,81] and [68]</td></tr><tr><td>3651 / 3651</td><td></td><td></td></tr><tr><td>3621 / 3620</td><td></td><td></td></tr><tr><td>1114 / 1112</td><td>(v<sub>3</sub>) Asymmetric stretching of Si–O–Si</td><td>[80,82] and [83]</td></tr><tr><td>1031 / 1024</td><td>(v<sub>1</sub>) Symmetric stretching of Si–O</td><td>[80,81] and [82]</td></tr><tr><td>1005 / 992</td><td>(v<sub>1</sub>) Symmetric stretching of Si–O</td><td>[80,81] and [68]</td></tr><tr><td>912 / 908</td><td>Bending of Al–O–H<sup>−</sup></td><td>[80,84] and [82]</td></tr><tr><td>778 / 776</td><td>(v<sub>1</sub>) Symmetric stretching of Si–O–Si</td><td>[80,84] and [82]</td></tr><tr><td>693 / 690</td><td>(v<sub>1</sub>) Symmetric stretching of Si–O<sup>+</sup> (Quartz)</td><td>[80]</td></tr><tr><td>533 / 523</td><td>Deformation of Al–O–Si</td><td>[80,81] and [83]</td></tr><tr><td rowspan="6">K100 with MICP</td><td>1454 / –</td><td>Calcium acetate and urea superposition: Symmetric C–O and asymmetric C–N stretching</td><td>[67] and [85]</td></tr><tr><td>1609 / –</td><td>(v<sub>3</sub>) Asymmetric stretching of C–O</td><td>[67]</td></tr><tr><td>1538 / 1553</td><td>(v<sub>2</sub>) Out-of-plane bending of CH<sub>3</sub></td><td></td></tr><tr><td>– / 1416</td><td>(v<sub>4</sub>) In-plane bending of CH<sub>3</sub></td><td></td></tr><tr><td>– / 1347</td><td>(v<sub>2</sub>) Out-of-plane bending of CO<sub>3</sub><sup>2−</sup></td><td>[69]</td></tr><tr><td>876 / 874</td><td></td><td></td></tr><tr><td rowspan="3">K100 with urea</td><td>1672 / 1668</td><td>Stretch C = O</td><td>[85] and [86]</td></tr><tr><td>1622 / 1635</td><td>Deformation of N–H</td><td></td></tr><tr><td>1470 / 1466</td><td>(v<sub>1</sub>) Asymmetric stretching of C–N</td><td></td></tr><tr><td rowspan="3">K100 with calcium acetate</td><td>1609 / –</td><td>(v<sub>3</sub>) Asymmetric stretching of C–O</td><td>[67]</td></tr><tr><td>1538 / 1553</td><td>(v<sub>1</sub>) Symmetric stretching of C–O</td><td></td></tr><tr><td>1446 / –</td><td></td><td></td></tr></tbody></table>

the soil is less efficient. This results in a slight increase in the C<sub>MICP</sub>.

2) In the plastic state, a decrease in saturation degree occurs within a liquidity index range of 0.6 to -0.1. This reduction may promote the formation of water meniscus between clay particles, improving soil cohesion by calcium carbonate precipitation and considerably improving C<sub>MICP</sub>, as Cheng et al. [62] observed in sandy soils. In this range, the dry density is fixed at 15 kN/m³.

3) As water content continues to decrease in the solid state, there is a reduction in the mass of CaCO₃ for an equivalent level of efficiency. Consequently, the cementation rate becomes less important, leading to a decrease in C<sub>MICP</sub>. In contrast to Cheng et al. [62], who observed that the lower degree of saturation promotes higher strength when the calcium carbonate content is similar. However, it should be noted that in the study of Cheng et al. [62], the calcium carbonate content was similar for each sample, unlike this study. In addition, their samples were treated by injection and not by soil mixing.

These observations are closely linked to the discussions presented in section 3.4 of this study. A dissimilarity trend was observed between the results of the mechanical tests and the calcium carbonate content. This dissimilarity could be explained by localized precipitation of calcium carbonate with decreasing water content, relatively due to preferential localization of water between soil particles, forming menisci. This localized precipitation could strengthen the material more effectively. Furthermore, the amount of water plays a role in the type and shape of the precipitated crystals, which could also impact the mechanical

strength of the treated clay material.

## Calcite precipitation rate

Fig. 8(a) illustrates the assessment of MICP efficiency for each treated K100 specimen employed in the vane test. The results reveal an efficiency E<sub>ff</sub> that appears unaffected by variations in MICP concentration and water content. The average E<sub>ff</sub> level is around 45 %. However, there is an exception in samples treated with a 0.4 M MICP solution. E<sub>ff</sub> is higher with about 65 % across different water content levels. This increase in efficiency matches with [63], who demonstrated that reduced MICP solution concentrations can enhance precipitation efficiency induced by bacterial density of OD600 = 1 in lateritic soil.

Considering the precipitation efficiency is roughly equivalent for different MICP concentrations and water content levels, and given that the MICP solution constitutes the total liquid used in sample preparation, Fig. 8(b) highlights an increase of CCC with rising MICP concentration and water content. A slight presence of natural calcium carbonate, around 0.5 %, was detected in the untreated reference K100 sample. This slight trace of CaCO₃ can be explained by the proximity of the P300 kaolin quarry to limestone areas such as the Monts d'Or in the Rhône basin. In fact, the region's kaolin clays are associated with alluvial and sedimentary formations. The highest CCC was achieved with a MICP concentration of 1.4 M and a water content of 60 %, corresponding to 4 % of CaCO₃. To obtain the optimal amount of calcium carbonate, it is necessary to increase both water content during preparation and the concentration of the MICP solution, while keeping within the solubility limits of the least soluble reagent. However, these results also highlight the need to consider the residual nutrient quantities, which become more significant as cement concentration and water content increase in the MICP process. These factors may potentially impact the behavior of the treated material.

Precipitation efficiency varies between 41 and 56 % for a water content range of 7.5 to 60 %, as shown in Fig. 8(c). Despite uncertainty, the results suggest an average efficiency level of approximately 47 % for both mechanical tests. This similarity in E<sub>ff</sub> for various water content levels implies that the influence of water content on E<sub>ff</sub> may not be highly significant.

Furthermore, the quantity of precipitated calcium carbonate shown in Fig. 8(d), decreases as water content decreases. This loss in the amount of calcium carbonate precipitation may account for the decline in MICP cohesion observed in Fig. 7 between 18 % and 12 % water content levels. The result is a diminution of around 19 % of CCC or, equivalently, of 28 % of the calcite that had precipitated. The linear relationship between CCC and water content might explain the C<sub>MICP</sub>–water content curve. Basically, the MICP cohesion depends on the content of calcite precipitation and the arrangement of calcite crystals within the solid matrix, ultimately affecting the soil cementation or soil densification.

## Composition analysis

Composition analysis is essential to highlight the precipitation of calcium carbonate in clay samples through MICP treatment. Both XRD and FTIR analysis are used in this case.

FTIR spectroscopy using an ATR device revealed the presence of CaCO₃ in the treated samples. Fig. 9(a) shows the spectrum of dry calcium carbonate precipitated by MICP in pure aqueous solution. This spectrum displayed four distinct carbonate peaks (CO₃<sup>2−</sup>) at 744, 875, 1086, and 1411 cm<sup>-1</sup>, representing various vibrational modes, such as in-plane bending (v<sub>4</sub>), out-of-plane bending (v<sub>2</sub>), symmetric stretching (v<sub>1</sub>), and asymmetric stretching (v<sub>3</sub>) [64]. The in-plane bending of the carbonate characterizes the CaCO₃ crystalline phase. Moreover, its wavenumber of 744 cm<sup>-1</sup> corresponds to vaterite [65]. The broad peak partially split around the asymmetric stretch at 1439 and 1411 cm<sup>-1</sup>
<figure><img src="image_16.png" /><figcaption>Fig. 10. X-ray diffractogram of (a) CaCO₃ precipitated by MICP in aqueous solution with calcium acetate (b) Untreated K100 and treated MICP K100 (c) Calcite peak intensity enhancement of untreated K100 and treated MICP K100.</figcaption></figure>

suggests the presence of an amorphous phase of calcium carbonate [66]. The same analysis of this wet sample, shown in Fig. 9(a), exhibits these carbonate vibrational modes, associated with new characteristic hydroxyl groups such as H-O-H bending at 1642 cm⁻¹. However, the interference caused by humidity results in a shift in the characteristic wavenumber of some bonds. In this case, the $v_3$ vibration of the carbonate is attributed to the peak at 1390 cm⁻¹ (previously 1411 cm⁻¹ for the dry material). The shoulder near 1555 cm⁻¹ matches the C-O antisymmetric stretching vibration in calcium acetate [67].

Table 3 summarizes the bonds present in the dry and wet states of precipitated CaCO₃.

Fig. 9(b) superimposes the ATR-FTIR spectra of dry treated and untreated K100 from 40 % of water content before drying. The untreated powder shown specific stretching bands in the 3694–3621 cm⁻¹ range, corresponding to the antisymmetric and symmetric OH modes of water, according to the literature [68]. Symmetric Si-O and asymmetric Si-O-Si stretching bands were observed at 1114, 1031 and 1005 cm⁻¹ respectively. Bands at 935 and 912 cm⁻¹ represent in-plane bending vibrations of Al-OH. Comparison of the spectra of treated and untreated K100 revealed the presence of new molecular functional groups induced by MICP. Specifically, these are the C-O antisymmetric stretching vibrations represented by the peaks at 1609 and 1538 cm⁻¹, indicating the presence of calcium acetate [67]. The peak at 1454 cm⁻¹ corresponds to the overlap between the symmetric C-O stretching bands of calcium acetate and the asymmetric C-N stretching bands of urea. The transmittance band of carbonate vibration in out-of-plane bending, represented by the peak at 876 cm⁻¹, corresponds to calcium carbonate but does not identify its phase [69]. However, the presence of bonds, such as $v_3$ from $(CO_3)^{2-}$, is difficult to discern due to the overlap of absorption bands with close wavelengths, mainly between 1300 and 1600 cm⁻¹.

The appearance of these molecular functional groups confirms the presence of CaCO₃ induced by MICP in the treated K100 sample, as well as residual compounds such as urea and calcium acetate. The precipitation process is not fully efficient, inducing residues from the nutrient solution [70]. ATR-FTIR analysis of wet K100 paste confirms the

presence of CaCO₃ (wavelength peaks at 874 cm⁻¹ and broad band at around 1416 cm⁻¹ corresponding to $v_2$ and $v_3$ vibrations of $CO_3^{2-}$) upon treatment (Fig. 9(c)), despite the residual presence of calcium acetate (symmetric $CH_3$ bending vibrations at 1347 and 1416 cm⁻¹ and asymmetric C-O stretching vibrations at 1553 cm⁻¹). These results agree with [46] carried out on MICP-treated soils. The water content of these K100 samples implies the existence of bonds characteristic of the liquid state with O-H stretching vibrations (broad and intense absorption band between 3200 and 3400 cm⁻¹) and H-O-H bending characterized by an intense peak at a frequency close to 1639 cm⁻¹ [71]. Table 4 summarizes the bonds present in the dry and wet states of K100 with and without MICP treatment.

Given the complexity of identifying chemical bonds in this treated MICP clay, Fig. 9(d) shows the ATR-FTIR spectra of K100 dried with different MICP treatment components. The spectrum can thus be split and the characteristic presence of chemical bonds specific to each chemical component used for MICP or the precipitate components induced by MICP can be recognized. Compared with untreated K100, the addition of urea is marked by the stretching of a C=O carbonyl group (peak at 1672 cm⁻¹), the deformation of N-H (peak at 1622 cm⁻¹) and the asymmetric stretching of C-N (peak at 1470 cm⁻¹). The presence of calcium acetate is indicated by peaks at 1609 and 1538 cm⁻¹ corresponding to asymmetric C-O stretching, as well as a peak at 1446 cm⁻¹ corresponding to symmetric C-O stretching. The equimolar mixture of calcium acetate and urea in K100 thus presents an IR spectrum with covering bands for each of these components, including the peak at 1454 cm⁻¹ corresponding to the overlap of symmetric C-O stretching with asymmetric C-N stretching. The existence of this same peak in the treated K100 leads to the same conclusions. Fig. 9(e) follows the same approach, presenting the ATR-FTIR spectra of wet K100 including different MICP treatment components. The urea containing K100 paste shows peaks similar to those of its dry powder, with a slight band shift caused by water. Carbonyl C=O stretching and N-H deformation are represented by peaks around 1672 and 1622 cm⁻¹, but remain difficult to identify due to the intense H-O-H peak near 1639 cm⁻¹. Asymmetric
<figure><img src="image_17.png" /><figcaption>Fig. 10. (continued).</figcaption></figure>

C-N stretching is most easily observed at 1466 cm⁻¹. K100 paste treated with calcium acetate exhibits asymmetric C-O stretching, out-of-plane and in-plane CH₃ bending, with peaks at 1553, 1416 and 1347 cm⁻¹, respectively. Finally, the wet mixture of K100 treated with urea and calcium acetate shows a spectrum overlapping the vibrational frequencies of the bonds of its chemical components without new bonds, confirming the non-reaction of calcium acetate with urea in the absence of ureolytic bacteria.

Using X-ray diffraction analysis, Fig. 10(a) presents the predominant presence of vaterite when CaCO₃ is precipitated by MICP in aqueous solution, along with calcium acetate residues. The use of calcium acetate under ambient temperature conditions (20-30°C) is known to lead to the precipitation of vaterite crystals, in agreement with ATR-FTIR results and the literature [72].

Fig. 10(b) shows diffractograms of dry K100, untreated and treated

with MICP, samples. It highlights the composition of K100, consisting of kaolinite, quartz and illite. As revealed by calcimeter tests, untreated samples contain low levels of natural CaCO₃ (≈ 0.5 %) and remains below 3.5 % in treated samples. Thus, the intensity of CaCO₃ peaks on diffractograms is low and requires special consideration. Pure clay powder is found to contain a small fraction of calcite. The intensity of these peaks is increased by the MICP treatment. This is explained by an increase in the amount of calcium carbonate in the material. As a result, Fig. 10(c) highlights the variation in intensity for inter-reticular distances specific to the calcite structural arrangement and should not be confused with peaks from other structures. The presence of calcite can be indistinct due to the more intense diffraction of other K100 components. The peak at 2.284 Å, difficult to interpret due to the presence of quartz at 2.283 Å, is an example. No increase in intensity was observed for the structural arrangements typical of other crystalline forms of
calcium carbonate.

Like the ATR-FTIR spectroscopy, component analysis by XRD confirmed the precipitation of CaCO₃ in the treated samples. It also revealed the crystalline form, corresponding to calcite. However, the use of the same MICP process in an aqueous medium favored the precipitation of vaterite. The incorporation of calcium acetate in the MICP process was shown to improve the mechanical properties of clays, such as K100, in contrast to the results reported by [72] in the context of sandy media. These results suggest that the media used has an influence on the CaCO₃ precipitation phase induced by bacterial activity and, consequently, on mechanical behavior.

Finally, component analysis by ATR-FTIR is proving to be a simple and fast way of checking the presence of CaCO₃ in dry or wet clay samples. It also provides an effective identification method for chemical components processed in clay samples without oven-drying them, thus preserving any drying-related changes to their microscopic component. This means that these samples can be analyzed by more time-consuming and costly complementary methods, such as XRD or SEM. In addition, its ability to read atomic bonds lets it detect the phases present, whether crystalline or amorphous. As a complement, XRD can be used to specifically identify the crystalline structure. Further analysis of the diffractograms will determine the size of the CaCO₃ crystals.

## Conclusion

Based on the results of tests conducted on a clay, Kaolin P300, the following conclusions can be drawn:

1) Experimental tests using laboratory vane shear reveal a significant increase in strength due to biocalcification. Undrained cohesion values are compared for treated and untreated kaolin P300. These results highlight the influence of water content and MICP solution concentration on the strength improvement. The findings indicate that the highest undrained cohesion is achieved with a MICP solution concentration of 1.4 M, corresponding to the solubility limit of 1.9 M calcium acetate in the cementation solution.

2) UCS tests provide complementary results to those obtained from the laboratory vane shear tests. These tests explored lower water content levels for kaolin P300, resulting in higher UCS values and higher deformation modulus due to MICP treatment.

3) The outcomes of these two mechanical tests are correlated using the Mohr-Coulomb failure criterion under unconsolidated-undrained conditions. A new mechanical parameter called "C<sub>MICP</sub>" is introduced to explicitly quantify biocalcification's cohesion. C<sub>MICP</sub> demonstrates biocalcification's effective functioning and its positive impact on improving clay's mechanical properties, with an optimal strength observed between the solid and the plastic states of kaolin P300.

4) Beyond the strength enhancement observed in these clay samples through MICP treatment, the biocalcification efficiency of around 45 % is verified by post-treatment using the Dietrich-Fruhling calculator. XRD analysis reveals the precipitation of calcite in K100 and vaterite in aqueous media for the same MICP treatment. ATR-FTIR spectroscopy is used to characterize the molecular functional groups in dry and wet K100 samples, such as CaCO₃ and calcium acetate.

MICP treatment by soil-mixing proved effective in the case of kaolin P300, despite a lower precipitation efficiency than that observed in more permeable treated sands in other studies. There remains scope for further research, including refining sample preparation methods to enhance precipitation efficiency and increase calcite mass, thus reducing the concentrations of the MICP solution.

## CRediT authorship contribution statement

**Olivier Maston:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Tariq Ouahbi:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
**Said Taibi:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.
**Ahmad El Hajjar:** Writing – review & editing, Validation, Investigation, Conceptualization.
**Leslie Sapin:** Writing – review & editing, Validation, Methodology, Conceptualization.
**Annette Esnault-Filet:** Writing – review & editing, Validation, Methodology, Conceptualization.
**Benoit Duchemin:** Writing – review & editing, Validation, Methodology, Formal analysis, Conceptualization.
**Jean-Marie Fleureau:** Writing – review & editing, Validation, Methodology, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgement

The Normandy Region financed this work with RIN EMERGENT BIOFIS project. The author's funding is provided by the university of Le Havre Normandie. Soletanche Bachy industry gives the necessary bacteria.

## Data availability

Data will be made available on request.

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