Unsaturated soil is a type of soil consisting mainly of solid particles, a liquid matrix, and pore air. Due to the shrinkage interface between liquid and air, the matrix suction becomes an important structural stress within saturated soil [1]. The mechanical properties of unsaturated soil closely depend on the humidity characteristics ^[2][3][2,3]. The dependence of mechanical behaviors on matric suction or moisture involved in many engineering problems has been reported on in studies of foundation engineering, subgrade engineering, and slope engineering ^{[4][5][6][7][8]}[4,5,6,7,8]. For example, after the soft soil humidifying, the stiffness of the soil subgrades decreased evidently, resulting in the settlement and collapse of the pavement structure. Thus, unsaturated soils should be improved in practice by physical and chemical methods.

The existing soil treatments mainly include dynamic compaction methods and the cement grouting method, but these methods all have certain shortcomings ^[9][10][9,10]. Yao et al. [11] strengthened the collapsible dam foundation by means of the dynamic compaction method, and found that its availability of treatment is limited by construction and geological conditions. The grouting method is often used for soft foundation treatment, but it uses a large amount of cement and its production would increase the quantity of CO₂ emissions [12]. More and more attention has been placed on reducing CO₂ emissions in the life cycle of engineering practices, with the increasing awareness of people concerned about the environment [13]. These green and low-carbon practices require a novel technology to decouple the dependence of the soil’s treatment by cement. Therefore, the microbial-induced calcite precipitation (MICP) technology has become one of the interest points in geotechnical and geological engineering fields in recent years, since it is an environmentally friendly, noiseless, and low-cost approach for improving the engineering properties of unsaturated soil ^[14][15][14,15].

At present, the cement process of MICP could mainly be classified into four types of bio-mineralization reactions [16], including the urea hydrolysis type [17], ferric reduction type [18], sulfate reduction type [19] and denitrification type [20]. Urea hydrolysis is the most efficient and advantageous way to conduct MICP technology, since it has the advantages of simple operation and is easily controllable [21]. In addition, it can quickly produce calcium carbonate precipitates and has a high microbial survival rate without a special nutrient solution [22]. Studies have been conducted to investigate the improvement of soil mechanical behaviors by urea hydrolysis through macroscopic and microcosmic experiments ^[23][24][23,24]. Martinez et al. [25] applied the MICP grouting method on expansive soil columns and found that the swelling potential and hydrophilicity of expansive soil decreased after the treatment of MICP. Salifu et al. [26] demonstrated that the penetration grouting approach of MICP-solidified fluid can improve the stability of soil slopes, since the generated CaCO₃ can fill the volume of the micro-pore structures within soil by 9.9%. Sharma and R. [27] found that the compressive strength of MICP-treated soil was 1.45 to 2.26 times of that of the untreated soil by laboratory tests. Some researchers have reported the effectiveness of MICP treatment on soil mechanical properties, while less considerations and comparisons have been made on the changes of the soil hydraulic properties induced by MICP treatment ^[28][29][30][28,29,30]. Specially, it is important to investigate the moisture dependence on the stiffness and deformation of unsaturated soil in subgrade construction.

2. Mechanism of Microbial-Induced Calcite Precipitation (MICP)

The MICP is a widely existing bio-mineralization reaction in nature, accompanied by different microbial activities and chemical processes [31]. Different from the mineralization on the geological surface of earth, bio-mineralization refers to the process in which inorganic elements selectively precipitate from the environment to form minerals on a specific organic matrix with the participation of biological cells. This bio driven mineralization reaction mainly occurs in four ways: urea hydrolysis, denitrification, sulfate reduction, and ferric iron reduction [32]. The method of using microorganism-induced denitrification to precipitate calcium carbonate not only has high cultivation cost, but also has low efficiency of generating calcium carbonate [33]. However, it is undeniable that denitrifying bacteria can grow in situ and play a role under anoxic conditions [34]. In the process of microbial-induced sulfate, the sulfate reduction will produce the hydrogen sulfide gas, which is harmful to the environment and human body [35]. In addition, hydrogen sulfide gas also results in accelerating the corrosion of steel bars in structures. For the ferric iron reduction method, the requirements for the oxidation substrate are very high, meaning that it only works when the solubility of the oxidation substrate is low [36]. The urea hydrolysis method is simple and efficient, given that there is no additional reaction condition and no environmental pollution [37]. Because matrixes (urea and CaCl₂) have a high solubility in water solution, the MICP process can generate a lot of CaCO₃ in a short time [38]. Scholars generally believe that MICP using urea hydrolysis has an enormous potential in soil treatment [39]. In addition, among all biochemical reactions, the reaction of urea hydrolysis is a main technological path to produce ammonium ions and carbonate ions, and its reaction process is relatively simple. Therefore, the reaction mechanism of urea hydrolysis MICP was taken as an example in the following section. Chuo et al. [40] found that 17–30% of bacteria collected from Australia can hydrolyze urea rapidly. The Bacillus pasteurii (BP) has a high urease activity within soil and has been widely used in the MICP treatment [41]. During its metabolism, its cell secretes a large amount of urease to produce adenosine triphosphate (ATP), which promotes the catalytic hydrolysis of urea to produce ammonium and carbonate ions. Meanwhile, the PH value in the system increases in this process. Due to the presence of calcium ions, carbonate ions and calcium ions gradually modulate to form CaCO₃ precipitation. The reaction equation of urea hydrolysis is shown in Equations (1)–(5) [42]. The details of the urea hydrolysis of CaCO₃ precipitated by BP are shown in Figure 1. When BP metabolizes to produce urease, it will secrete a metabolite called bail polymer. Due to the existence of the double electric layer structure of the extracellular polymer and its microorganism, the microorganism tends to adsorb on the surface of the sand particles. Because of the negatively charged functional groups such as hydroxyl, amino, amido, and carboxyl, the surface of the microbial cell wall is also negatively charged and constantly attracts calcium ions in the environment, meaning that a large number of calcium ions gather on the cell surface. The carbonate formed after hydrolysis of urea will form CaCO₃ precipitation with these calcium ions and envelop the bacteria [43]. From the whole reaction process, it can be found that bacteria mainly play two roles: the core of which is to provide urease, and the other is to provide crystal nuclei for the formation of CaCO3 crystals [44]. The reaction equation of urease bacteria is displayed in Equations (6)–(10) [45].

3. Influencing Factors of MICP Reaction

The essence of MICP technology is to induce microorganisms to generate CaCO₃ precipitation between the gaps within soil to achieve the role of bio-cementation and treatment of soil. However, MICP technology will be limited and constrained by many factors in actual operation [46]. The factors that influence the treatment effect of MICP technology contain temperature, PH value, bio-cement concentration, calcium ion concentration, nutrient solution (urea) concentration, and soil particle size [47]. Mortensen et al. [48] showed that the influencing factors were in the order from large to small as temperature, concentration of bio-cementing fluid, nutrient solution concentration, PH value, and calcium ion concentration, respectively, through single factor and orthogonal tests. Sotoudehfar et al. [49] used the optimized orthogonal test method to explore the influence of various parameters in the process of MICP on the curing effect. The results showed that the curing time had the greatest influence on the curing effect, and the bacterial cell concentration, molar concentration ratio of nutrient solution, and the liquid injection flow rate had similar influences on the curing effect.

3.1. Temperature and PH Value

Temperature is the key factor in the success of MICP technology on soil treatment [50]. A change in temperature will affect the growth of bacteria, the activity of microbial enzymes, the biodegradation of bacteria, and the process of binding precipitation, thus affecting the final curing effect. Figure 2a shows the changes of urease and growth activities of BP at different temperatures. An absorbance index at the 600 nm wavelength (OD600) was wildly applied to evaluate the density and growth activity of the BP solution. It found that the growth curve of BP was different when the temperature varied from low to high [51]. It is generally believed that the growth of BP is inhibited at low temperatures, while the urease activity of BP decreases at high temperatures ^[52][53][52,53]. Therefore, the BP should be cultivated under a suitable growth environment (temperature). In addition to the temperature, urease activity of BP is an important factor. The decrease in enzyme activity will lead to an insufficient precipitation of CaCO₃. Kim et al. [54] found that the suitable temperature for the growth of BP Sarcina was 30 °C, at which the strain propagated rapidly and produced high urease activity. When the temperature was less than 10 °C, the urease activity was almost lost. Furthermore, since the urease activity is affected by temperature, Xiao et al. [55] demonstrated that CaCO₃ crystals generated in soil samples had a good homogeneity by grouting at low temperature. PH value is one of the important influencing factors on microbial life activities. Its influence is mainly reflected in three aspects: First, it affects the biological activity of macromolecular substances (such as proteins and nucleic acids) by changing their charge. Second, it reduces the absorption and utilization of nutrients by microorganisms by changing the charge of cell membrane. Third, it also reduces the utilization effectiveness of nutrients in the living environment of microorganisms and enhances the toxicity of harmful substances [56]. Figure 2b shows the changes of urease activity with the increasing PH value.

3.2. Bio-Cementing Liquid Nutrient Solution Concentration

Bio-cementitious fluid plays an important role in microbial soil treatment. Bio-cementing liquid shall be used in the reaction of microbial-induced CaCO₃ deposition, which has a direct impact on the treatment effect [57]. From the MICP principle, the increase in calcium ions and urea in the bio-cementing solution will precipitate more CaCO₃. In the current research, the bio-cementing fluid generally includes urea and calcium chloride, but the selection of parameters such as bio-concentration, number of rounds, and ratio are different [58]. Cui et al. [59] believed that the bio-concentration of bio-cementing fluid had a significant impact on the treatment effect. Meanwhile, the low bio-concentration was helpful to obtain higher treatment strength, and the size of generated CaCO₃ crystal was also large [46]. Cheng and Cord-Ruwisch [60] found that a too low bio-concentration of bio-cement solution will lead to insufficient CaCO₃ and will affect the treatment effect.

3.3. Nutrient Solution Concentration

Mujah et al. [61] found that changing the nutrient solution can affect the nucleation rate and the size of CaCO₃ crystals. Meanwhile, the effect of nutrient solution on different environments was also different. Wong [62] added urea to the culture medium to increase the precipitation rate of CaCO₃, which can overcome the inhibition of solidification under low temperature environments. The adding urea does not only increase the urease activity, but also will cause the soil to become alkaline, thus inhibiting the growth of microorganisms [63]. The high urea content causes the uneven distribution of overall precipitation crystals, resulting in the low strength of soil. Zhao [64] found that the concentration of the nutrient solution has an important impact on the shear strength of solidified muddy soil. With the increasing concentration of the nutrient solution, the internal friction angle first increases and then decreases, and the optimal concentration of the corresponding optimal nutrient solution is 1.60 mol/L. Soon et al. [65] found that the bio-cementation effect will reach a peak value with the change in nutrient concentration, according to which the most appropriate nutrient concentration can be obtained. In addition, cells will shrink due to water loss under a high salt environment, thus affecting the physiological and biochemical reaction process of microorganisms. Therefore, the concentration of nutrient solution is very important in the study of microbial solidification of soil. Whiffin et al. [66] found that the activity of bacterial urease decreased significantly, almost linearly, with the increase in calcium ion concentration, indicating that a high concentration of calcium ions have an evident inhibition on urease under this condition. In conclusion, the output of CaCO₃ is positively related to the concentration of nutrient solution within a certain range, but high concentration inhibits the microbial induced CaCO₃ generation. At low concentration, the microbial induced CaCO₃ is smaller and more evenly distributed in the soil.

3.4. Calcium Source and Its Concentration

The BP cells can be regarded as a formation site to produce CaCO₃ precipitation [67]. The MICP reaction process in Figure 1 indicated that the carbonate ions generated by the urea decomposition of BP will be continuously transported from the intracellular to the extracellular and meet with calcium ions in the environment. Therefore, the PH value of calcium sources and the concentration of calcium ions affect the rate, quality, and output of CaCO₃ crystals produced in the MICP process. Achal and Pan [68] compared the effects of four calcium sources on BP-induced CaCO₃ precipitation, and proposed that calcium chloride is a better calcium source in the MICP process, followed by calcium nitrate. Cheng et al. [69] took seawater as the bio-cementing fluid to conduct MICP process, and the results showed that the strength of samples maximized after 200 grouting times, since calcium ion concentration was low in seawater. The Ca²⁺ concentration is very important for the precipitation and precipitation efficiency of CaCO₃ crystals. Okwadha and Li [70] found that high concentrations of urea and CaCl₂ (more than 0.5 mol/L) reduces the deposition efficiency of calcium carbonate, and urea and CaCl₂ can improve the deposition efficiency of calcium carbonate at low concentrations (0.05–0.25 mol/L). From the current test results, a too low or too high calcium ion concentration cannot have the corresponding calcite yield. The calcium ion concentration should be optimized by laboratory tests to effectively bio-cement the test medium together by MICP calcite. Based on the above reviews, the MICP technology results from the metabolic processes of bacteria including urease-producing bacteria, sulfate-reducing bacteria, denitrifying bacteria, and oxidizing bacteria. Although the type of bacteria and the reaction mechanism may be different in the MICP application, both the nutrient solution and the calcium source are necessary to generate CaCO₃ (calcite). Therefore, the MICP is mainly affected by the concentration of calcium ions and urea.

4. Application of MICP in Engineering

The MICP technology comes from the development of microbiology and geotechnical engineering, and is a novel technology with ecologically friendly and sustainable advantages [23]. Now, the technology has achieved good results in engineering applications, including soil treatment, seepage prevention, and cracking repair. Many scholars have proved that MICP plays a significant role in soil treatment through experimental tests ^[71][72][73][71,72,73]. For example, compared with untreated expansive soil samples, it found that the swelling rate of soil samples after MICP mixing treatment was significantly reduced [25]. Sharaky et al. [74] found that the unconfined compression resistance of treated clay samples increased nearly three times through the MICP pressure grouting treatment test of clay. Yasuhara et al. [75] used MICP technology to strengthen the sand and premixed method. The so-called premixing method refers to the use of the mixing method and unconfined compressive strength test to make the reaction liquid better contact with the soil. Compared with other methods, the advantage of this method is that it can make CaCO₃ uniformly distributed in the soil. The CaCO₃ generated by MICP technology can also fill the pores of the solidified soil and further reduce permeability to achieve the purpose of plugging [76]. A plugging test of MICP was conducted on fly ash modified concrete using giant bacillus [28]. The results suggested that the calcite precipitation between the aggregate and the bio-cement mortar was a primary reason for reducing the permeability of samples. Liu et al. [77] also demonstrated that as the CaCO₃ precipitate is induced by MICP, it plays a role in bio-cementing soil samples, since it fills cracks among soil particles. Wiktor and Jonkers [78] used an energy dispersive spectrometer to observe the status of concrete cracks repaired by MICP, and their results showed that a crack of the width of 0.46 mm was completely healed after 100 days of repair, which was much larger than that of 0.18 mm in the control group. Algaifi et al. [79] found that the MICP has an advantage in the self-healing of cracks in bio-cement slurry by the theoretical model and microscopic test. In general, the usage of MICP technology can effectively repair cracks, but it is undeniable that the time period for treatment and repair is long. Subsequent research should be carried out to develop a more efficient and faster MICP technology in the material source and reaction processes.