1. Introduction
The natural flaws found in rocks, such as bedding planes, fissures, and joints, play a significant role in influencing the chemical composition, seepage conditions, stress fields, and shear strength of rock masses [
1,
2]. The stability of a rock mass is mainly controlled by its joints, and failure in this area can have catastrophic consequences [
3]. Therefore, it is essential to focus on the treatment and reinforcement of rock joints to prevent disasters. Current leading prevention techniques include fully grouted rock bolts under aerodynamic effects [
4,
5], inflatable and yielding bolt reinforcement [
6], rock bolts reinforcement [
7], new polymer reinforcement [
8], silicate resin rock reinforcement [
9], organomineral resin injection reinforcement of soft rock [
10], tailing reinforcement by MICP [
11], and gold mine tailing reinforcement [
12].
The abovementioned strengthening techniques can enhance the shear strength parameters of rock masses to a specific level and reduce potential disasters. However, there are also some drawbacks, including poor durability, high energy consumption, high cost, and high pollution emissions [
13]. To address these drawbacks and improve durability while reducing costs, it is crucial to develop and research a novel reinforcement technique for jointed rock masses that is environmentally friendly and economically feasible [
14]. As a result, microbially induced calcium carbonate precipitation (MICP) technology has been utilized for soil and rock reinforcement due to its good injectivity, cost-effectiveness, and environmental friendliness [
13,
15,
16]. MICP technology utilizes a microbial mineralization process to produce inorganic compounds that form cement-like gelling materials. These gelling materials strengthen jointed or broken rock masses [
17]. The principle of the MICP method involves utilizing urease concealed by bacteria to produce carbonate particles in a non-acidic environment, which are then mixed with calcium ions to create crystals of cemented calcium carbonate [
1,
18].
Following in-depth research into the MICP technique, it has made significant contributions in the field of soil and rock engineering, such as rough crack repair [
19], earthquake liquefaction control [
20], fresh river sand and silt reinforcement [
21,
22], erosion protection of sandstone and sand [
23,
24], and the improvement of the disintegration resistance and mechanical properties of residual soil [
25]. Many studies, including papers and patents, have utilized MICP technology to repair concrete cracks of varying widths and shapes [
26,
27,
28,
29,
30,
31,
32,
33]. The impermeability of the repaired cracks has been improved to different extents. The microbial grouting technique has also been used to improve the permeability constant of fractured limestone and phosphogypsum material [
17,
34]. In addition, the MICP technique can be used to repair cracked sandstone and granite [
13,
35]. Deng et al. [
36] selected
Sporosarcina pasteurii to reduce the porosity of fractured yellow sandstone. Zou et al. [
37] investigated the influences of crack inclination angle, bacterial concentration, crack opening, and crack roughness on the fracture permeability of transparent rocks through four seepage experiments. The authors found that the permeability of the fracture filled with microbially induced CaCO
3 rose as the crack inclination, opening, and roughness increased first and then decreased. Song et al. [
38] found that the unconfined compressive strength (UCS) of a low-strength sandstone can be improved significantly with MICP, while this technique cannot effectively improve the mechanical properties of high-strength sandstone. These results provided a solid foundation for the reinforcement of fractured rocks by using the MICP technique. Furthermore, previous studies mostly improved the impermeability constant and unconfined compressive strength and reduced the porosity of different fractured rocks in natural environments. However, the peak shear strength of rock masses is mostly controlled by the shear strength of joints. As such, research on improving the shear strength of rock joints via MICP in a saline–alkaline environment needs to be conducted.
This study proposes a new five-gradient artificial domestication scheme for Sporosarcina pasteurii in a saline–alkaline environment. The direct shear test was then conducted to assess the shear strength of sandstone joints repaired by microbially induced calcium carbonate precipitation (MICP). The subsequent applications of the MICP technique provided valuable insights into increasing the shear strength of rock joints in a saline–alkaline environment.
Over time, rock masses develop joints and discontinuities, which can significantly weaken their shear strength properties, such as cohesion and friction angle. This damage can lead to various consequences, including the dislocation of upper and lower rock masses, collapses, roof arch failures, cracks, and landslides. According to statistical data from the International Consortium on Landslides (ICL) Association, worldwide devastating landslide accidents are caused by the deformation and failure of rock structural planes.
Figure 1 illustrates the global distribution of landslide events prior to 2023, based on data from ICL published over the years.
To address the engineering challenges posed by geological accidents induced by rock mass joints, numerous scholars have proposed various techniques to enhance the shear strength of rock joints since the 1950s. Currently, grouting reinforcement methods employing traditional grouting materials, such as lime, epoxy resin, and cement, are widely utilized [
39,
40]. However, it has been noted that these traditional cementitious materials can impact the pH of the rock mass, leading to environmental erosion and potential harm to groundwater [
41]. Furthermore, chemical grouting materials, like epoxy resin, phenolic resin, and polyurethane, are known to be toxic. The negative environmental impact and high energy consumption associated with traditional grouting reinforcement methods emphasize the crucial need to explore nonpolluting, green, efficient, and cost-effective reinforcement techniques for rock joints. One emerging environmentally friendly geotechnical reinforcement technology is the microbially induced calcium carbonate precipitation (MICP) method. This technique offers several advantages, including low energy consumption, simplicity, and environmental safety [
13,
16,
42]. Since the 2000s, numerous researchers have conducted extensive experimental studies to enhance the permeability and uniaxial compressive strength of rock masses while reducing their porosity through the MICP technique, yielding promising results [
1,
2,
43,
44,
45]. However, previous studies have yet to explore the application of the MICP technique to increase the shear strength of rock joints in order to mitigate catastrophic landslides in saline–alkaline environments, which are prevalent worldwide.
Given that saline–alkaline conditions can diminish the shear strength of rock joints, it is imperative to investigate new reinforcement techniques suitable for such environments in practical engineering. This study introduces a novel approach that involves domesticating
Sporosarcina pasteurii in a saline–alkaline environment. Various methods of bacterial domestication were assessed, leading to the identification of the most effective approach to enhance bacterial activity and urease enzyme activity in saline–alkaline environments, which was then implemented to increase the shear strength of rock joints.
Table 1 displays the worldwide distribution of saline–alkaline land [
46].
In this study, the strains suitable for the saline–alkaline environment were obtained using domestication. The cracks in the saline–alkaline environment were filled by strains to increase the peak shear strength of rocks.
2. Materials and Methodology
2.1. Sporosarcina pasteurii and Medium
Currently, the most commonly used microbial mineralization technology involves urea bacteria, which are widely found in nature.
Sporosarcina pasteurii is a Gram-positive bacterium known for its ability to induce calcite precipitation from biological cementation by the production of urease [
47]. In addition to
Sporosarcina pasteurii, researchers have utilized Microc-occusureae, Proteus, Salmonella, Escherichia coli, and Staphylococcus [
48].
Sporosarcina pasteurii has been proposed for use as an ecologically sound biological construction material. Studies have demonstrated that when combined with plastic and hard minerals,
Sporosarcina pasteurii can form a material stronger than bone [
21,
37,
49]. The bacterium is commonly used for MICP due to its non-pathogenic nature and its ability to produce high amounts of urease, which hydrolyzes urea to carbonate and ammonia [
50].
Sporosarcina pasteurii is preferred due to its classification as a spore-forming bacteria. Spore-forming bacteria can produce round or oval spores within the cell during the later stages of growth and reproduction. These spores have thick walls, low water content, and high-stress resistance, giving the bacteria strong environmental resilience. Additionally, under alkaline conditions, the urease activity of
Sporosarcina pasteurii surpasses that of other bacteria [
51].
Recent studies have focused on the isolation of bacteria in alkaline conditions, with outcomes demonstrating the effective use of laboratory-extracted bacteria for repairing geological materials [
2,
13,
51,
52,
53]. In this context, laboratory-extracted
Sporosarcina pasteurii (from the China General Microbial Culture Preservation Management Center (CGMCC), the number is 1.3687) was utilized to assess the reinforcement effect of microbially produced CaCO
3 on the shear strength of rock joints under a saline–alkaline environment. The purchased strains are in a freeze-dried powder form. The bacterial suspension used in the experiment was an activated bacterial suspension. The culture conditions of bacterial activation are as follows: 30 °C, 150 rpm, for about 5 days. The culture medium contained beef extract (5 g/L), peptone (5 g/L), and NaCl (5 g/L). After observing the turbidity of the bacterial solution, the mother solution was obtained by mixing the bacterial suspension with the neutral medium at a volume ratio of 1:100 for 48 h. The OD600 value was 1.411. The components of the saline–alkaline environment include sodium chloride (NaCl), magnesium phosphate (MgSO
4), potassium chloride (KCl), and peptone (
Table 2). Detailed information regarding the materials used for the domestication of bacteria in the saline–alkaline medium is presented in
Table 2. The pH value of the medium was adjusted by 1 mol/L NaOH solution and 1 mol/L HCl solution. The pH value of the saline–alkaline medium was adjusted to 9, while the neutral medium was adjusted to pH 7. The pH of the solution was measured with a pH detector while adding NaOH or HCl solution until the desired value was reached.
The bacteria were cultured by an incubator, with the incubation process lasting for 48 h at 150 r/min (revolutions per minute) and a temperature of 30 °C. The optimal densities of Sporosarcina pasteurii were measured using an ultraviolet (UV) spectrophotometer. The UV spectrophotometer, comprising components, such as the entrance slit, collimating mirror, monochromator, sample holder, and detector, enabled precise measurements by controlling the alignment of the incident light beam, focusing the light beam, separating different wavelengths of light, holding the solution in place, and measuring the intensity of light reaching it after passing through the sample.
To provide a nitrogen source for the appropriate growth of Sporosarcina pasteurii, a saline–alkaline suspension with a 0.5 M/L concentration of urea and calcium chloride was utilized. The reaction solution was prepared by mixing the bacterial suspension with the gelling solution in a 1:1 volume ratio.
In order to compare the MICP performance of domesticated bacteria, a neutral medium was prepared by removing MgSO
4, KCl, and MgCl
2 from the medium in
Table 2 for undomesticated bacteria. The neutral medium was prepared by removing MgSO
4, KCl, and MgCl
2 from the unacclimated bacteria medium in
Table 2 and reducing the concentration of NaCl to 5 g/L. The test results were used as a reference.
2.2. Sandstone Samples
The study utilized red sandstone samples with uniaxial compressive strength, cohesion, and internal friction angle values of 38.8 MPa, 3.79 Mpa, and 35.81°, respectively, obtained from the field. A standard sandstone sample measuring 100 mm × 100 mm × 50 mm (length × width × thickness) was fabricated using grinding and cutting tools to produce two rock blocks (
Figure 2). The sample’s typical joint surface was displayed in
Figure 2, prepared by an artificial joint surface preparation method resembling the standard rough surface of the 10# joint [
54]. This 10# crack exhibits a rough and irregular surface with a joint roughness coefficient of 18.7. Additionally, an acrylic gasket of 5 mm × 2 mm × 1 mm (length × width × thickness) was placed at the four corner points of the upper and lower parts of the joint to form a 1 mm high cavity for conducting the repair test. The cavity was sealed on three sides with glue, leaving one side unsealed to serve as an inlet for the subsequent infusion of bacterial fluid and nutrients.
2.3. Domestication Scheme
The term “domestication” refers to the breeding of bacteria in a controlled environment to produce specific strains that thrive in artificial settings for various human or industrial needs [
55]. This process helps the microorganisms to adapt to the targeted environment and exhibit improved characteristics and growth rates [
16,
56,
57]. Cheng et al. acclimated the bacteria by adjusting the pH value of the medium every 8 h, resulting in a higher hydrogen production rate for the bacteria [
58]. Similarly, Cheng et al. acclimated bacteria with three cultures for three days each and improved the energy conversion efficiency of hydrogen and methane production in the two-phase fermentation of Spironema arthropods [
59]. In this research, we aim to employ three different domestication approaches, namely direct domestication, three-gradient domestication, and five-gradient domestication of
Sporosarcina pasteurii in saline–alkaline conditions. The domestication medium was created as follows. First, an artificial sterilized medium was designed based on previous studies [
60,
61]. Urea was then added to the sterilized medium at a concentration of 0.5 M/L. The final domesticated medium was prepared by adjusting the pH of the sterilized medium to 9. The complete domestication process under different domestication methods is clearly outlined in
Figure 3. The medium comprises a mixture of a saline–alkaline medium and a neutral medium. The percentages in
Figure 3 indicate the proportion of the saline–alkaline medium.
The particular domestication method is divided into three domestication schemes.
Direct domestication: A total of 1 mL of
Sporosarcina pasteurii was added to bottle “A” containing 100 mL of saline–alkaline sterilized medium (
Figure 3a). The bottle was then placed on a shaker and cultured for 48 h at a temperature of 30 °C. The shaker used has a motor-powered plate that oscillates orbitally with a speed of 150 rpm. After 48 h, the bacteria in bottle “A” were considered to be domesticated bacteria.
Three-gradient domestication: A total of 1 mL of
Sporosarcina pasteurii was added to bottle “B” containing 100 mL of NaCl-diluted saline–alkaline medium (
Figure 3b). From bottle “B” 1 mL of the bacterial suspension was transferred to bottle “C” containing 100 mL of saline–alkaline medium diluted to 1/3, as shown in
Figure 3b. After culturing for 48 h, the bacterial suspension (1 mL) from bottle “C” was transferred to bottle “D” containing 100 mL of domesticated medium. It was cultured for 48 h in bottle “D” to complete the three-gradient domestication stage (
Figure 3b). At this stage, the bacteria in bottle “D” were considered three-gradient domesticated bacteria.
Five-gradient domestication: A volume of 1 mL of bacteria was placed in bottle “E”, which contained a 100 mL medium whose salt–alkaline concentration was diluted to 1/5. Then, bacteria were cultured in bottle “E” for 48 h. Next, 1 mL of the bacterial suspension from bottle “E” was transferred to bottle “F”, which contained 100 mL medium whose salt–alkaline concentration was diluted to 2/5. Then, the bacteria were cultured for 48 h before being moved to bottle “G,” which included 100 mL of saline–alkaline medium diluted to 3/5. After another 48 h of culturing, 1 mL of the bacterial suspension from bottle “G” was then transferred to bottle “H,” which contained 100 mL of a saline–alkaline environment diluted to 4/5, and was left to culture for 48 h. Finally, 1 mL of the bacterial suspension from bottle “H” was moved to bottle “I,” which contained 100 mL of domestication medium, and was also cultured for 48 h. At this stage, the bacteria in a conical bottle “I” were considered to be five-gradient domesticated bacteria.
After each inoculation of bacteria, the culture bottle was placed in a gas bath shaker and cultured at 30 °C and 150 rpm for 48 h. One culture lasts for 48 h, and the measurement results of the experiment determine the number of cultures in each domestication stage. During each domestication process, the concentration of
Sporosarcina pasteurii and unit urease activity were measured at intervals of 6 h. When the difference between the maximum bacterial concentration and the maximum unit urease activity measured by the previous and subsequent cultures was less than 1%, it was considered that domestication had reached a stable stage. The current stage of domestication culture had come to a halt, and the next stage had begun. The specific process is shown in
Figure 4. OD
600 is the absorbance of the solution at 600 nm wavelength [
38], which can indirectly indicate the concentration of bacteria. According to the main composition and content of the saline–alkaline environment, the pH value of the medium is adjusted to 9. In a saline–alkaline environment, the growth curve of
Sporosarcina pasteurii is shown in
Figure 5. As can be seen from
Figure 5, the growth rate of microorganisms is divided into four stages. The first is the bacterial adjustment stage, the second is the rapid reproduction stage, the third is the stable growth stage, and the fourth is the slow decay stage.
After each inoculation of bacteria, the culture bottle was placed in a gas bath shaker and cultured at 30 °C and 150 rpm for 48 h. The concentration of
Sporosarcina pasteurii and unit urease activity were measured at 6 h intervals during each domestication process. The domestication culture was stopped when the maximum concentration of bacteria in the two cultures was close, and the next stage was started. The absorbance of the solution at a 600 nm wavelength [
46] can indirectly indicate the concentration of bacteria and is denoted as OD
600. The pH value of the medium was adjusted to 9 based on the main composition and content of the saline–alkaline environment. In a saline–alkaline environment, the growth curve of
Sporosarcina pasteurii is shown in
Figure 5, illustrating the four stages of the growth rate of microorganisms: the bacterial latent phase, exponential growth phase, stable growth stage, and slow decay stage.
2.4. Microbial Concentration
The turbidimetric method was used to measure the concentration of bacteria in this paper. According to this approach, the number of microorganisms could be ultimately defined by assessing the absorbance of the solution [
62]. The principle of this method is that the turbidity of the bacterial suspension is proportional to the cell concentration. An ultraviolet–visible spectrophotometer was used to determine the absorbance (OD
600) of the cells at a 600 nm wavelength.
2.5. Urease Activity
Based on the characteristics of the strains’ reproduction and growth, urease activity is a critical factor influencing the quantity of CaCO
3 [
63]. The amount of urea is directly related to the change in solution conductivity during hydrolysis. Urease activity can be measured by the change in conductivity per minute [
64]. Urease activity was assessed using the following method. Initially, 5 mL of bacterial suspension was mixed with 55 mL of urea solution at room temperature. Subsequently, a conductivity meter was used to measure the fluctuation of electrical conductivity within 5 min. The average fluctuation amount per minute was then converted to determine the amount of hydrolyzed urea, thus revealing urease activity (mM urea hydrolyzed∙min
−1∙OD
−1, simplifying the equation for the urea decomposition ability of the bacteria. The specific calculation formula is illustrated in Equation (1).
2.6. Production Rate for Calcium Carbonate
The process of calcium carbonate precipitation is a complex biochemical process that requires enough carbonate ions and calcium. The amount needed depends on such factors as pH value, calcium concentration, nucleation sites, and inorganic carbon concentration [
61]. Additionally, temperatures between 10 °C and 37 °C significantly impact the performance of microbially induced calcium carbonate precipitation (MICP) and the growth rate of
Sporosarcina pasteurii [
65,
66]. This study considers two ambient temperatures, namely 10 °C and 30 °C. Various domestication schemes were tested to measure the productivity of CaCO
3 [
20]. The specific process is outlined below.
The bacteria obtained from different domestication schemes were cultured at 150 rpm and removed for use after 48 h. The MICP calcification test was carried out in a tissue culture tube with a breathable film. A 20 mL culture solution of urea and calcium chloride with molar concentrations of 0.5 M/L was prepared. The same volume of suspension and bacterial liquid was mixed for a precipitation reaction. The reaction temperature was controlled at 10 °C and 30 °C, respectively. After 6 days of reaction, the mass of calcium carbonate was weighed, and the productivity was calculated. The measurement method of calcium carbonate productivity was as follows [
67]:
- (1)
The mixed solution after the precipitation reaction was washed and filtered with deionized water three times. The tissue culture tube and filter paper were then dried in the oven at 110 °C for 24 h and weighed to obtain mass M1;
- (2)
Next, the tissue culture tube and filter paper were soaked in a prepared hydrochloric acid solution (0.5 M/L), rinsed with deionized water twice, and dried in a 110 °C oven for 24 h. After drying, they were weighed to obtain mass M2;
- (3)
The actual mass of calcium carbonate produced is calculated by finding the difference between the two masses, M1-M2. The theoretical mass of calcium carbonate is obtained by multiplying the calcium ion concentration (m) used in the gel solution by the volume (V) used in the culture solution;
- (4)
The final calcium carbonate productivity is determined by dividing the mass of calcium carbonate experimentally produced by the mass of calcium carbonate theoretically generated.
2.7. MICP Test of Rough Surface
The MICP test for the rock joint rough surface consists of two simple steps, as follows:
- (1)
Initial infusion of the bacterial suspension: The sealed rock joint specimen was laid flat with the entrance facing upwards. The bacterial suspension was then injected into the cavity using a syringe (see
Figure 6). To ensure the complete saturation of the joint rock wall and sufficient bacterial attachment; the cavity was injected with the bacterial suspension every 6 h, for a total of 4 times. Following the initial perfusion, the joint rock wall appeared to be wet.
- (2)
Injection of bacteria and reaction fluids: The bacterial suspension and reaction solution were mixed in a 1:1 volume ratio and immediately injected into the cavity using a pipette until the mixture almost overflowed the cavity. This process was repeated every 3 h until calcium carbonate crystals completely blocked the cavity, and no more liquid could be injected.
The prepared MICP-cemented rock joint was divided into 3 groups based on curing times of 7, 21, and 28 days, respectively, and then used for subsequent laboratory direct shear tests. In addition, for comparative analysis, uncemented rock specimens with rough joint were also prepared. A total of thirty sample were tested using the direct shear test apparatus.
2.8. Direct Shear Test of Rock Joints
In this section, a servo-hydraulic direct shear machine (DSM-150A) with an accuracy of 10 N was used to determine the shear curve of rock specimens reinforced with MICP (
Figure 7). The samples were divided into two categories: the first category included uncemented sandstone samples, and the second category included MICP-cemented sandstone samples. The cemented sandstone specimens were further categorized into four types; apart from the three types of specimens repaired using strains obtained from three domestication methods, an additional experiment was conducted to compare the repair effect of undomesticated strains in a neutral environment. This experiment involved using a mixture of undomesticated strains and a neutral medium to perform MICP repair on a group of specimens.
The tertiary normal stress (
σn) is used, with values of 0.05
σc, 0.1
σc, and 0.2
σc, corresponding to low, medium, and high stress conditions, respectively. Normal stress is applied using stress control at a loading rate of 0.2 kN/s. Tangential stress is applied using displacement control at a shear rate of 0.2 mm/min. The test stops when the shear displacement reaches 10% of the length of the specimen. The test scheme is detailed in
Table 3.