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Article

Application of Microbially Induced CaCO3 on the Reinforcement of Rock Discontinuity

by
Simiao Zhang
1,
Shuhong Wang
1,*,
Zulkifl Ahmed
1,* and
Fahad Alshawmar
2
1
School of Resource and Civil Engineering, Northeastern University, Shenyang 110819, China
2
Department of Civil Engineering, College of Engineering, Qassim University, Buraydah 51452, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2024, 14(19), 8952; https://doi.org/10.3390/app14198952
Submission received: 6 August 2024 / Revised: 24 September 2024 / Accepted: 26 September 2024 / Published: 4 October 2024
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Abstract

:
Microbially induced calcium carbonate precipitation (MICP) is a technique used in geotechnical engineering to reinforce soil and rock. While it is commonly used for soil reinforcement, its application for rock reinforcement in saline–alkaline environments is limited. In order to improve the reinforcement effect of microbially induced calcium carbonate on rock joints in saline–alkaline environments, experiments were conducted to cultivate Sporosarcina pasteurii. The strengthening effects of MICP on rock joints were evaluated using the direct shear test. Samples of sandstone with rough surfaces were reinforced by MICP. The shear strength characteristics of rock joints reinforced by CaCO3 were then assessed. The results showed that after being domesticated in a saline–alkaline environment, the bacterial concentration reached over 96% of that in a neutral environment. The domesticated Sporosarcina pasteurii performed well at temperatures between 10~30 °C in saline–alkaline conditions. In the saline–alkaline environment, the shear strength of rock joints and the production rate of CaCO3 were higher, and the Sporosarcina pasteurii with domestication showed better joint repair performance. The peak shear strength of rock joints reinforced by MICP increased with curing time, with a quicker strength development in the early stage and a slower increase later on. The peak shear strength of cemented rock joints significantly surpassed that of uncemented rock joints. This research can provide valuable insights for the application of MICP technology in reinforcing rock joints in saline–alkaline environment.

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 CaCO3 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 CaCO3 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 (MgSO4), 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 MgSO4, KCl, and MgCl2 from the medium in Table 2 for undomesticated bacteria. The neutral medium was prepared by removing MgSO4, KCl, and MgCl2 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. OD600 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 OD600. 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 (OD600) 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 CaCO3 [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).
Unit   urease   activity ( mM   urea   hydrolysed · min − 1 OD 600 − 1 ) = Urease   activity   ( mM   urea   hydrolysed · min − 1 ) Bacterial   concentration   ( OD 600 )

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 CaCO3 [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.

3. Results and Discussions

3.1. Microbial Concentration and Urease Activity

In Section 2.1, the turbidimetric method was used to indirectly measure the number of bacteria by analyzing the absorbance of the bacterial suspension. This method is based on the fact that cell concentration is directly related to the turbidity of the bacterial suspension, which in turn is related to the value of absorbance (OD600). In Figure 8, we can see the lowest concentration of bacterial suspension after the first 48 h expanded culture (shown by a red circle), which is called the “initial domestication stage”, and the highest concentration after multiple expanded cultures (shown by a black pentagon) for the three domestication schemes. For comparison, the figure also shows the highest bacterial concentration of undomesticated bacteria after being cultured 48 h in a neutral medium, labeled as “undomesticated”. In Figure 8, “1/3” represents the prolonged growth of bacteria in a solution with a 1/3 salt and alkaline concentration during the three-stage domestication process, and so on.
The key to this experiment is to use urease secreted by Sporosarcina pasteurii to hydrolyze urea to produce carbonate ions, providing raw materials for carbonate precipitation [68,69]. Therefore, bacterial urease activity is an important indicator of MICP efficiency [70,71]. In this study, the maximum urease activity of bacteria was measured by the method discussed in Section 2.5 during the expanded culture of the bacteria, and the results are shown in Figure 9.
In the direct domestication scheme, the maximum concentration of bacterial suspension and the maximum unit urease activity of bacteria after the initial expanded culture was significantly lower, and even after multiple expanded cultures, the increase was limited, but the maximum concentration of bacterial suspension and the maximum unit urease activity of bacteria were still small. This result indicated that a high concentration of saline–alkaline medium had a significant inhibitory effect on the growth and reproduction of Sporosarcina pasteurii, which was the same with the maximum unit urease activity of bacteria.
For the three-gradient and five-gradient domestication schemes, the maximum concentration of the bacterial suspension and the maximum unit urease activity of bacteria after the initial expanded culture were relatively low in the process of each level of concentration domestication. However, after multiple expanded cultures, the concentration of bacterial suspension and the maximum unit urease activity of bacteria increased rapidly and gradually became stable.
In the process of gradient domestication, with the gradual increase in the salt and alkaline concentration in the medium, the maximum concentration of bacterial suspension and the maximum unit urease activity of bacteria at the stable stage of domestication showed a trend of gradual decrease. The above phenomenon is because the osmotic pressure gradually increases with the increase in the salt and alkaline concentration in the medium. With the domestication process, although Sporosarcina pasteurii has a certain adaptability to the increase in osmotic pressure, the high osmotic pressure is bound to have a certain impact on the growth and reproduction of bacteria. Liu et al. [72] cultured Sporosarcina pasteurii in seawater and observed that the growth of Sporosarcina pasteurii was inhibited, consistent with the findings of this paper. Therefore, after the completion of each stage of domestication, the maximum concentration of bacterial suspension and the maximum unit urease activity of bacteria were lower than that of the medium domesticated with a higher saline–alkaline concentration.
After the completion of three-gradient domestication and five-gradient domestication, the maximum concentrations of the bacterial suspension were 1.253 and 1.372, respectively, which could be similar to the maximum concentration of undomesticated bacteria in a neutral environment, indicating that after gradient domestication, bacteria could gradually adapt to the saline–alkaline environment. The maximum concentration of the bacteria and the maximum unit urease activity after five-gradient domestication were slightly higher than that after three-gradient domestication, which were closer to the maximum concentration and the maximum unit urease activity of undomesticated bacteria in a neutral environment, indicating that the effect of five-gradient domestication was better than that of other methods. Ma et al. [71] utilized serial subcultivation to isolate strains that removed specific genes. In this study, the same approach was used to domesticate the strains at each stage in the saline–alkaline environment, acquiring a strain adapted to this environment. The experimental findings demonstrated the feasibility and effectiveness of this method.

3.2. Production Rate for CaCO3

Domestication greatly impacted the productivity of calcium carbonate in Sporosarcina pasteurii. The test results are displayed in Table 4.
(1)
In the direct domestication scheme, the productivity of calcium carbonate was significantly lower than that of undomesticated Sporosarcina pasteurii at both temperatures, and the former was only 13.9% to 14.6% of that of the latter, indicating that Sporosarcina pasteurii had poor adaptability to a strong saline–alkaline environment, and the MICP process was significantly inhibited [72]. First of all, the yield of CaCO3 is related to the activity coefficients of Ca2+ and CO32−, and a high concentration of NaCl will reduce the activity coefficients of Ca2+ and CO32−, thus hindering the crystallization of CaCO3 [73]. Secondly, salt ions decreased the urea hydrolysis rate, leading to a lower calcium carbonate yield [74]. In addition, the high concentration of salt ions caused bacteria to lose water and die, and the alkaline environment caused the activity of urease to be reduced. The reasons mentioned above resulted in a lower yield of CaCO3 in the saline–alkaline environment.
After the three-gradient domestication, the calcium carbonate generation increased by 5.66–7.22% compared with that of the undomesticated strain. After the five-gradient domestication, the calcium carbonate generation increased by 12.26–15.46% compared with that of the undomesticated strain. The experimental results showed that after the gradient domestication, the adaptability of Sporosarcina pasteurii to a saline–alkaline environment was enhanced. In addition, the calcium and magnesium ions in the saline–alkaline medium can provide more calcium and magnesium sources, and the carbonate production is higher than that in a neutral environment to a certain extent.
(2)
The comparison of calcium carbonate production under the two temperature conditions showed that in the neutral environment before domestication, the production of calcium carbonate by bacteria at 30 °C was 9.28% higher than that at 10 °C, which was consistent with the effect of temperature on MICP obtained in previous studies [75]. After three- and five-gradient domestication, the calcium carbonate production of bacteria at 30 °C was 7.69% and 6.25% higher than that at 10 °C, respectively, which gradually reduced. The above results showed that after gradient domestication of bacteria, the influence of temperature on MICP was significantly weakened. In other words, the bacteria, after domestication, can not only adapt to the saline environment but also have better temperature adaptability.

3.3. Shear Displacement–Stress Curve

The shear characteristics of MICP-cemented rock joints were analyzed through direct shear tests in this section. Direct shear tests were performed on five types of rock samples after 28 days of curing. The shear displacement–stress curves for normal stresses of 0.05 σc, 0.1 σc, and 0.2 σc are depicted in Figure 10.
Observation of Figure 10 reveals that all shear stress–strain curves exhibit clear peak points, and the entire shear displacement–stress curve can be segmented into a pre-peak elastic stage, post-peak decline stage, and residual stage. The peak shear stress represents the peak shear strength of the rock joint. The strain obtained by the direct domestication scheme had poor adaptability to the saline–alkaline environment, resulting in a low yield of calcium carbonate, so the reinforcement effect on the cracks was not obvious. Therefore, the peak shear strength of the specimens repaired by the direct domestication strain was only slightly higher than that of the unconsolidated structural plane. The repair ability of the strains obtained by the three-gradient domestication and the five-gradient domestication was significantly enhanced, which was not much different from that of the undomesticated strains in the neutral environment. The repair ability of the strains after the five-gradient domestication was obviously the best, indicating that the strains domesticated by this method could adapt well to the saline–alkaline environment and had a good cementation effect on rock joint specimens. This can increase the peak shear strength of rock joints to a greater extent.
Figure 11 shows the shear displacement–shear strength curves of specimens with a curing time of 7 days, 14 days, and 28 days, respectively, under the third normal stress (0.05 σc, 0.1 σc, and 0.2 σc) at normal temperature. It can be seen from Figure 11 that the peak shear strength of cemented joints increased with the increase in maintenance time.

3.4. Peak Shear Strength

The graph in Figure 12 displays the peak shear strength of sandstone under three different levels of normal stress after reinforcement by varying strains. The specific strength values are provided in Table 5. A comparison of the increase in the peak shear strength of artificial joints by different strains is presented in Table 6 when compared with uncemented specimens.
It is evident that the peak shear strength of strains on joints acquired through the direct domestication method shows a slight increase of between 2.33% and 13.55%. In contrast, both the three-gradient domestication method and five-gradient domestication method effectively led to an increase of between 7.62% and 28.56%. The increase in peak shear strength of the five-gradient domesticated strains in the saline–alkaline environment (14.19% to 28.56%) was comparable to that of undomesticated strains in the neutral environment (16.93% to 32.19%).
To compare the effect of different strains obtained from various domestication schemes on reinforcing microbial-induced calcium carbonate precipitation (MICP) on sandstone joints, and to analyze the influence of curing time on peak shear strength, we plotted the relationship between peak shear strength and curing time under three different normal stresses, as illustrated in Figure 13. The key findings are as follows:
  • The peak shear strength increases rapidly during the early curing period, but the rate of increase slows down gradually with the extension of the curing time. This phenomenon is due to the gradual slowdown in the generation rate of calcium carbonate as urea and nutrients are consumed, and the bacterial population decreases in the absence of a supplemental nutrient solution and bacterial suspension [76,77].
  • The repair ability of the strain obtained by direct domestication is significantly inhibited by the saline–alkaline environment, resulting in only a small increase in peak shear strength after restoration.
  • The peak shear strength of the specimens repaired after three- and five-gradient domestication showed a significant improvement after 28 days of maintenance, indicating that the gradient domestication program can help bacteria gradually adapt to the saline–alkaline environment. Furthermore, the peak shear strength of specimens repaired by bacteria after five-gradient domestication is slightly higher than that of bacteria after three-gradient domestication, and it is close to the repair strength of undomesticated bacteria in a neutral environment. The above results suggest that the five-gradient domestication method is more effective than other methods.
From the mechanical analysis of rock joint reinforcement, it is clear that the key to strengthening rock joints is to use engineering measures to improve their shear strength parameter (c) and internal friction angle (φ) [13]. Therefore, a linear fitting analysis was conducted on the test results of peak shear strength of rock joint specimens repaired by strains of three kinds of domestication schemes under three normal stresses. The results are presented in Figure 14, wherein the slope of the curve represents tanφ, and the intercept represents the c value, respectively. The shear strength parameters (c and φ) of rock joint specimens repaired by different strains are summarized in Table 7.
The data presented in Table 7 reveal that uncemented rock joints have values of c and φ at 0.933 MPa and 50.7°, respectively. The decrease in cell activity and urease productivity of directly domesticated strains caused by high-intensity salinization has no obvious effect on the strengthening of rock joints. The values of c and φ of reinforced joints are 1.001 MPa and 50.9°, which only increase by 7.29% and 0.39%, respectively. However, the use of the three-gradient domestication strain resulted in a 58.95% increase in c value and a 0.79% increase in the φ value of the rock joints. Furthermore, the five-gradient domestication strain yielded a 72.56% increase in c value and a 3.75% increase in φ value, closely resembling the 72.56% and 5.33% increase rates observed in the neutral environment with the undomesticated strain. These results underscore the significant enhancement in the mechanical properties of MICP-cemented rock joints through the multi-gradient domestication method, with the five-gradient domestication approach proving to be the most effective.

3.5. Shear Failure Characteristic

The typical shear failure characteristics of sandstone joint specimens are depicted in Figure 15. From Figure 15, it is evident that the shear failure of the sandstone joint specimen, which has been maintained for 28 days, initially begins from the cementation product of the joint rock wall. The final shear failure of the joint surface involves the crushing of the cement layer and the cutting off of the rough convex of the joint surface. Due to the brittleness of the sandstone [16], local fracturing of the joint rock wall may even occur under high normal stress (refer to Figure 15).

4. Conclusions

In this research, we utilized laboratory-extracted Sporosarcina pasteurii to enhance the shear strength of rock joints. We conducted direct shear tests on the rock samples before and after applying MICP to assess the impact of Sporosarcina pasteurii reinforcement on the shear performance of rock joints. This study’s significance lies in the use of environmentally friendly methods to reinforce rock joints and enhance their shear strength. Our research technique presents a novel approach for employing the MICP technique to improve the shear strength of jointed and fractured rocks under specific environmental conditions. The primary conclusions are as follows:
  • The maximum cell concentration of the five-gradient acclimated strains in the saline–alkaline environment was more than 97% of that of the undomesticated strains in the neutral environment. Additionally, the carbonate yield was significantly higher than that of the directly domesticated strains after interaction with the cement fluid.
  • The domesticated Sporosarcina pasteurii has not only good adaptability to the saline–alkaline environment but also has good temperature adaptability at temperatures from 10 to 30 °C.
  • In the saline–alkaline environment, using Sporosarcina pasteurii obtained from multi-gradient domestication leads to a higher carbonate yield and shear strength of sandstone joints strengthened by MICP. Compared to other domestication methods, the five-gradient domestication strain shows better MICP performance, increasing the peak shear strength by 14.19% to 28.56%. The shear strength parameter “c” and internal friction angle “φ “ of the joint surface can be increased by 72.56% and 3.75%, respectively.
  • The peak shear strength of MICP-cemented rock joint specimens increases as the curing time is extended. However, the strength develops most rapidly in the early stage of curing, with the rate of strength improvement gradually slowing down in the later stage of curing.
This paper uses artificial two-dimensional parallel cracks, while the actual engineering situation mostly involves three-dimensional non-parallel cracks. In future studies, we plan to use rocks collected from the field to investigate natural three-dimensional fractures. We will also consider variations in the opening of natural fissures by constructing non-parallel fissures.

Author Contributions

All the authors contributed to this research, S.Z. conducted the tests and analyzed the experimental data. S.Z. wrote the paper, S.W., Z.A. and F.A. revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by [National Natural Science Foundation of China] grant number [51474050 and U1602232] and [Program for Liaoning Excellent Talents in University] grant number [LN2014006].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support this study are included within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of catastrophic landslide accidents (from ICL).
Figure 1. Distribution of catastrophic landslide accidents (from ICL).
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Figure 2. The used sandstone sample with rough surface.
Figure 2. The used sandstone sample with rough surface.
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Figure 3. Schematic diagram of different gradient domestication schemes. (a) Direct domestication, (b) three gradient-domestication, and (c) five gradient-domestication.
Figure 3. Schematic diagram of different gradient domestication schemes. (a) Direct domestication, (b) three gradient-domestication, and (c) five gradient-domestication.
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Figure 4. Detail of the three-gradient domestication scheme.
Figure 4. Detail of the three-gradient domestication scheme.
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Figure 5. Growth rate of Sporosarcina pasteurii in a saline–alkaline environment.
Figure 5. Growth rate of Sporosarcina pasteurii in a saline–alkaline environment.
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Figure 6. MICP test schematic diagram.
Figure 6. MICP test schematic diagram.
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Figure 7. (a) DSM-150A computerized direct shear equipment and (b) the tested specimen.
Figure 7. (a) DSM-150A computerized direct shear equipment and (b) the tested specimen.
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Figure 8. Concentration of bacteria at different domestication schemes.
Figure 8. Concentration of bacteria at different domestication schemes.
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Figure 9. Bacterial concentration under different domestication schemes.
Figure 9. Bacterial concentration under different domestication schemes.
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Figure 10. Shear displacement–stress curves of MICP-cemented sandstone specimens obtained by different domestication schemes after curing for 28 days: (a) σn = 0.05 σc, (b) σn = 0.1 σc, (c) σn = 0.2 σc.
Figure 10. Shear displacement–stress curves of MICP-cemented sandstone specimens obtained by different domestication schemes after curing for 28 days: (a) σn = 0.05 σc, (b) σn = 0.1 σc, (c) σn = 0.2 σc.
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Figure 11. Displacement curve of MICP-consolidated sandstone joints of five gradient domesticated strains. (a) σn = 0.05 σc, (b) σn = 0.1 σc, and (c) σn = 0.2 σc.
Figure 11. Displacement curve of MICP-consolidated sandstone joints of five gradient domesticated strains. (a) σn = 0.05 σc, (b) σn = 0.1 σc, and (c) σn = 0.2 σc.
Applsci 14 08952 g011
Applsci 14 08952 g012
Figure 12. Comparisons of the peak shear strength of artificial rock joints.
Figure 12. Comparisons of the peak shear strength of artificial rock joints.
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Applsci 14 08952 g013
Figure 13. Variation in peak shear strength of MICP-cemented rock joints with time under different domestication schemes: (a) σn = 0.05 σc, (b) σn = 0.1 σc, and (c) σn = 0.2 σc.
Figure 13. Variation in peak shear strength of MICP-cemented rock joints with time under different domestication schemes: (a) σn = 0.05 σc, (b) σn = 0.1 σc, and (c) σn = 0.2 σc.
Applsci 14 08952 g013
Applsci 14 08952 g014
Figure 14. Fitting curves of shear strength parameters of sandstone joints cemented by different strains.
Figure 14. Fitting curves of shear strength parameters of sandstone joints cemented by different strains.
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Applsci 14 08952 g015
Figure 15. Typical shear failure characteristics of three kinds of artificial rock joint specimens.
Figure 15. Typical shear failure characteristics of three kinds of artificial rock joint specimens.
Applsci 14 08952 g015
Table 1. Distribution of global saline–alkali areas.
Table 1. Distribution of global saline–alkali areas.
DistrictArea
(Thousand Hectares)		Percent
North America1575.51.65
Mexico196.50.21
South America12,916.313.53
Africa8053.88.43
South Asia8760.89.17
East and Central Asia21,168.622.17
Southeast Asia1998.32.09
Australia35,733.037.42
Europe5080.45.32
Totally95,483.2100
Table 2. Components of the saline–alkaline medium.
Table 2. Components of the saline–alkaline medium.
MaterialContent (g/L)
NaCl20
MgSO41.204
KCl1.118
MgCl21.619
Peptone15
Beef extract5
Table 3. Details of uncemented and cemented rock specimens.
Table 3. Details of uncemented and cemented rock specimens.
Specimen TypeCuring Time/DayTest QuantityTotal Quantity
0.05 σc0.1 σc0.2 σc
Uncemented--1113
Undomesticated71113
141113
281113
1/171113
141113
281113
3/371113
141113
281113
5/571113
141113
281113
Note: “Uncemented” refers to an uncemented specimen. “Undomesticated” means an undomesticated strain that is mixed with the neutral medium. “1/1” indicates a directly domesticated strain mixing with the saline–alkaline medium. “3/3” signifies a three-gradient domesticated strain mixing with the saline–alkaline medium. “5/5” denotes a five-gradient domesticated strain mixing with the saline–alkaline medium. The same conventions apply to the subsequent text.
Table 4. Calcium carbonate production rate under different domestication schemes.
Table 4. Calcium carbonate production rate under different domestication schemes.
Domestication Scheme10 °C30 °C
Undomesticated38.8%42.4%
1/15.8%6.2%
3/341.6%44.8%
5/544.8%47.6%
Table 5. Peak shear strength of sandstone specimens repaired by different strains.
Table 5. Peak shear strength of sandstone specimens repaired by different strains.
SpeciesPeak Shear Strength (MPa)
0.05 σc0.1 σc0.2 σc
Uncemented3.2665.15010.310
1/13.3425.84810.530
3/33.8716.31911.096
5/53.9886.62111.773
Undomesticated4.2346.80812.055
Table 6. Percentage increase in peak shear strength of joints strengthened by different strains.
Table 6. Percentage increase in peak shear strength of joints strengthened by different strains.
SpeciesThe Percentage Increase
0.05 σc0.1 σc0.2 σc
1/12.3313.552.13
3/318.5222.707.62
5/522.1128.5614.19
Undomesticated29.6432.1916.93
Table 7. Shear strength parameters of sandstone joints cemented by different strains.
Table 7. Shear strength parameters of sandstone joints cemented by different strains.
Bacterial SpeciesShear Strength Parameters
C (MPa)φ (°)
Uncemented0.93350.7
Undomesticated1.61053.4
1/11.00150.9
3/31.48351.1
5/51.61052.6
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Zhang, S.; Wang, S.; Ahmed, Z.; Alshawmar, F. Application of Microbially Induced CaCO3 on the Reinforcement of Rock Discontinuity. Appl. Sci. 2024, 14, 8952. https://doi.org/10.3390/app14198952

AMA Style

Zhang S, Wang S, Ahmed Z, Alshawmar F. Application of Microbially Induced CaCO3 on the Reinforcement of Rock Discontinuity. Applied Sciences. 2024; 14(19):8952. https://doi.org/10.3390/app14198952

Chicago/Turabian Style

Zhang, Simiao, Shuhong Wang, Zulkifl Ahmed, and Fahad Alshawmar. 2024. "Application of Microbially Induced CaCO3 on the Reinforcement of Rock Discontinuity" Applied Sciences 14, no. 19: 8952. https://doi.org/10.3390/app14198952

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