**3. Bacterial Precipitation of CaCO3 in Soils**

Bacteria can adapt to varying environmental conditions due to their physiology and genetics, which is because they have existed in nature since three and a half billion years ago [46]. Some important features of microorganisms are that they bear cells with a simple structure without an enclosed nucleus. With multiple chromosomes and unique chemical compositions, microbes are characterized and classified by their cell wall, nutrients, RNA, DNA, and type of biochemical changes [47,48]. Bacteria are the most widely found microbes in soils. A bacterial cell has a diameter in the range of 0.5–3.0 μm, with an elongated, spiral, or spherical shape [49]. The bacterial activity in producing calcite for soil treatment involves various bacteria. Burbank et al. [21] studied CaCO3 precipitation through biological mediation using indigenous bacteria and found them effective in increasing the liquefaction resistance of sands. They concluded that using indigenous bacteria is advisable to make soil biomodification more economically feasible. Lee et al. [50] studied the improvement of soil properties using organic materials and found a 1.5–2.5 times increase in soil strength when compared with samples without an organic stabilizer. The stabilizer used was an organic acid material named Con-α, which was developed by Osaki Corp. in Japan. It allows microbe proliferation with aging. The importance of using this organic material is to ensure safety for the environment. pH tests confirmed that the organic acid was eco-friendly. The tests were conducted by preparing samples mixed with 3% and 6% of the organic biostabilizer by weight of the soil, which were tested for different ages. The authors concluded that the pores in the soil were filled with matter produced by the microbes, improving the soil's strength. Although MICP has potential for soil improvement, upscaling this method, optimization and training/educating the technicians on its effective applications are identified as challenges in its implementation [51]. To grade the worth of MICP, the rate of CaCO3 precipitation is said to be 60 kg/m<sup>3</sup> of soil [52]. MICP is carried out by using bacteria such as *Sporosarcina pasteurii* (*S. pasteurii*)/*Bacillus pasteurii* [21–25,50,52–57], *Idiomarina insulisalsae* [24], *Pseudomonas putida* [54], *Bacillus cereus* [58], *Bacillus sphaericus* [59], and indigenous

bacteria [60]. MICP is even used for the improvement of the performance of construction materials [54], sediment stabilization [61], and reduction of coastal erosion [62].

Soon et al. [10] used MICP for the improvement of the engineering properties of soils. The species of bacteria used was *Bacillus megaterium*, combined with other cementing reagents. They found that the CaCO3 precipitates were effective in soil stabilization, capable of improving shear strength, and even useful in reducing the hydraulic conductivity of soil and sand. Proto et al. [63] studied the reduction of the permeability of saturated sand through the formation of biofilms on the surface of the sand grains, biofilms being the accumulation of cells and extracellular polymeric substances in an organic process. The process of bioaugmentation was incited by strains of non-native bacteria injected in the sand, and bacteria already present in the sand contributed to the precipitation. The use of biofilms resulted in a considerable decrease in the permeability of the soil. Bioaugmentation is a commonly used method for removing contaminants from soil mass; this process is initiated by allochtonic or autochtonic microbes against nondegradable organic matter from soil [64]. The transformation of harmful compounds into different forms by using bacteria should be possible, showing bioaugmentation [65]. The use of additives, along with microbes, has also been tested for the improvement of soil performance. Zhao et al. [66] used fiber felt scrap (activated carbon) with the MICP technique to treat sand. They observed that unconfined compressive strength (UCS) and tensile strength improved, showing the possibility of using a discarded scrap waste material along with MICP treatment to improve soil strength.

#### **4. Enzyme Usage and Sources for Soil Treatment**

The most widely studied enzyme source for soil treatment is the jack bean plant, technically termed *Canavalia ensiformis*. This plant is a draught-resistant species classified in the Fabaceae family [34]. Larsen et al. [67] reported that calcite precipitation increases ten times with the use of the jack bean meal instead of pure urease. The urease enzyme was first crystallized in 1926 by James B. Sumner [68–70]. Oliveira et al. [71] conducted a study on the effect of soil type on the precipitation of calcium carbonate by EICP treatment in the soil by jack bean urease for its improvement. They found that the precipitation of CaCO3 increased the strength of the soil by 40–106%, but the test proved ineffective for organic soils. Renjith et al. [5] used a commercially available enzyme-based additive named "Eko soil" for the construction of unpaved roads in Australia. They found that the treatment methods could be used for cost-effective and sustainable unpaved roads. They also surveyed other enzymes that were commercially available or manufactured from fermented matter, which is converted into a chemical, liquid, or organic form. Javadi et al. [72] used urease enzyme extracted from watermelon seeds. They found that the theoretical maximum precipitation of calcite was around 64%, which was considered promising for soil treatment using urease extracted from watermelon seeds. It is also important to note that using enzymes for soil stabilization is expensive; the cost of enzymes is equal to 90% of the total cost of materials used [73,74].

#### *Use of Additives in the Biotreatment*

The use of additives, along with the urease enzyme, in the biotreatment has also been tested to improve the precipitation process for better soil performance. Almajed et al. [75] used non-fat milk powder as an additive to improve the urease activity and obtained surprising results wherein the amount of CaCO3 precipitated and the UCS values were better than those for soils treated with enzyme solutions without non-fat milk powder. It was noted that the amount of calcite precipitated is not regarded as an indicative factor of increase in strength; rather, the precipitation pattern governs the improvement in the geotechnical properties of soils; that is, even low carbonate precipitation with a suitable pattern may lead to higher strength compared to high carbonate precipitation. The enzyme treatment is performed with an aqueous solution consisting of urea, calcium chloride, and urease enzyme in deionized water for mixing/injecting in the soil for calcite

precipitation [76,77]. Hamdan and Kavazanjian [78] also used non-fat milk powder as a stabilizer, along with urease, in the enzyme solution to test its effectiveness in fugitive dust control. They observed that the treatment with the enzyme resulted in resistance to wind erosion. Putra et al. [79] used magnesium chloride as a substitute in the enzyme treatment to precipitate CaCO3. They found that the ratio of precipitation was 90% of the theoretical maximum, which was obtained with the addition of a small quantity of magnesium chloride. The use of magnesium resulted in a lower precipitation rate, resulting in the higher injectivity of the enzyme solution. It changed the shape of CaCO3 precipitates, simultaneously precipitating aragonite along with calcite. The use of additives to improve the efficacy of the enzyme treatment on soils has become an important part of research. Yuan et al. [39] used soybean urease for silt improvement in flooded areas and with urease. Additional materials, like glutinous powder of rice, brown sugar, and skim milk in powdered form were used to reinforce the urease activity. Hommel et al. [80] developed a numerical model for the EICP method to simulate the outcomes for different dosages of the enzyme solutions in any experimental setup. They developed a model that could give qualitative outcomes for the experimental setup modeled in the program. Therefore, EICP and MICP can be tested for their proposed outcomes using a numerical model before conducting the experiments physically.

#### **5. Geotechnical Applications of the Biocementation Technique**

The use of biostabilization methods wherein CaCO3 precipitation helps in improving soils has attracted the interest of geotechnical engineers substantially [81–83]. The biotreatment of soils further needs suitable environmental conditions to achieve the desired outcomes through the precipitation of CaCO3 [54,84]. However, the use of an enzymebased stabilization method depends on factors like type of soil, method of construction, curing, and temperature, which may result in poor outcomes unless a suitable adjustment is not made to control the hindrances as per the type of enzymes [5].

#### *5.1. Biotreatment Techniques*

Mujah et al. [44] reported that MICP is effectuated by the injection, surface percolation, and premixing methods. In the injection method, the treatment solution is injected in the soil. In the premixing method, soils are mixed with the bacterial solution before dumping the soil in its place to serve the intended purpose. In the surface percolation method, the cementation solution is made to be absorbed in the soil from the surface. Wiffin et al. [81] tested the biotreatment through the injection method on a 5 m long column of sandy soil. They observed that the precipitation of CaCO3 was not even along the length of the column. Sotoudehfar et al. [85] studied the factors influencing MICP applied through the injection method. They used a specially designed pump for injecting the cementation fluid into the soil. They found positive outcomes with their injection method of implementing MICP on soils. The injection method of the biotreatment was carried out by two phase injection procedures wherein initial bacterial strains were injected; later, bacterial feed was injected. Stocks-Fischer et al. [25] reported that injecting bacteria and reagents together may result in clogging at the injection site, especially when the flow rates of the fluids are low in the soil. The injection technique may be suitable when the treatment fluid is of low viscosity. In contrast, injection or biogrouting may need substantial pumping energy to achieve the desired soil strength [86]. The inoculation of bacterial strains in soils is also practiced for contaminant removal from soil mass [65].

Almajed et al. [40] studied the EICP treatment through percolation and premixing methods applied on Ottawa 20–30 sand. They interpreted their findings by comparing both methods of treatment and found that premixing was not effective in maintaining the intactness of the sand specimen, whereas the percolation method portrayed better results. An intact specimen, which could be easily tested for its strength and percolation method, also provides good interparticle bonding. Neupane et al. [87] used percolation for EICP on sand. They observed an almost uniform distribution of calcite precipitates in sand

at 5 ◦C, whereas precipitation was reduced to 5% at a temperature of 23.5 ◦C. It can be understood that implementation techniques also play a vital role in the biotreatment of soils, and specific methods of implementation can be devised depending on the soil type and the environmental conditions.

#### *5.2. Effect of Biotreatment on the Unconfined Compressive Strength (UCS) Test*

The biotreatment of soils is well understood for its degree of effectiveness by UCS values [36,88–90]. Ali et al. [91] stated that the UCS test is most trusted to ascertain the effectiveness of soil stabilization methods. Sharma and Ramkrishnan [92] studied soils (fine grained) treated with MICP and observed the improvement in the UCS value of soil. They also inferred that the particle packing plays an important role in improving soil strength and even leads to improved bearing capacity, reduced settlement and permeability, and diminished shrink-swell characteristics of soils. The development of pore pressure can also be stopped with soil treatment. Strength enhancement in soils treated with biocementation methods is mainly achieved because of the adhesion of soil grains due to calcite precipitates in soil voids. Figure 2 shows a comparison of UCS values obtained for the soils before and after the biotreatment.

**Figure 2.** Comparison of unconfined compressive strength (UCS) results for soil samples before and after biotreatment. 1—Sotoudehfar et al. [85], 2—Wani and Mir [93], 3—Wani and Mir [93], 4—Moghal et al. [94], 5—Moghal et al. [94], 6—Xiao et al. [95], 7—Sharma and R [92], 8—Sharma and R [92], 9—Park et al. [96].

Table 1 shows the UCS test results obtained by researchers for different soils treated with MICP/EICP techniques. Yasuhara et al. [31] used free enzyme (urease) supplied by Kishida Chemical to treat sand through CaCO3 precipitation. They found that the experiments showed the effectiveness of the enzyme treatment on UCS samples and permeability. Strength gain in the soils depends on the amount of calcite precipitated. Notably, for the substantial improvement in the stiffness and strength of soils treated with a biostabilization technique, a minimum of 4% of calcite precipitation per mass of treated soils is required [96].


**Table 1.** Unconfined Compressive Strength (USC) results obtained by different researchers after the biotreatment of soils.

#### *5.3. Reduction of Hydraulic Conductivity by Biotreatment*

The use of biotreatment methods for permeability reduction in soils has led to effective results [10,63,90,108–112]. One of the reasons that leads to the reduction in soil permeability is the cementation of soil grains with precipitates of CaCO3, leading to the blockage of connected pores in the soil mass. Although permeability reduction due to biotreatment depends on the size of soil grains, finer soils have miniscule flow paths due to the proper packing of soil grains; even the size of precipitates plays an important role in reducing permeability. Sometimes, suspended precipitates also get accommodated in the voids of the soil mass, thereby contributing further in the reduction of flow paths [108,113]. Cuthbert et al. stated that permeability reduction due to the precipitation of CaCO3 depends on the quantity of precipitates; that is, with more precipitates, permeability reduction will be higher [114]. Ferris et al. [115] studied the use of bacterially precipitated calcite as a plugging material in porous media and proved a permeability reduction in the sand tested. They concluded that a 40% increase in bacteria paved the way for a 70% reduction in sand permeability, which can be attributed to greater precipitates from more bacteria, leading to

a higher percentage of CaCO3 precipitation in the soil mass. Reduced permeability after the soil treatment by calcite precipitates is also due to the reduction of the pore throat at the points of contact between the soil grains where precipitation occurs [82]. Gui et al. [116] used the MICP method in porous media for bioclogging. They found that permeability reduction was greater than 72%. The main reason they identified for the reduction of permeability was the formation of biofilms on the sand grain surfaces. Figure 3 shows the flow paths in a soil mass and the blockage of the flow paths by biofilms on soil grains, plugging, and sealing of pore throats with calcite after the biotreatment.

**Figure 3.** Schematic representation of soil mass: (**a**) Flow paths allowing water to percolate through soil mass. (**b**) Representation of the blockage of pore throats by calcite precipitation and the formation of calcite biofilms on soil grains, leading to narrowing/blockage of flow paths and reducing permeability after biotreatment.

Nemati et al. [109] compared the microbial and enzymatic processes applied for permeability reduction. They found that bacterial precipitation may include a degradable biomass developed as a plugging agent in soil pores, which may dissolve or decompose with time or after exposure to moisture, whereas enzymatically precipitated calcite proves to be a durable plugging agent that contributes to permeability reduction and is, hence, more convenient for geotechnical applications. Rittmann [117] suggested that reduction in permeability is possible because of the formation of biofilms, which reduces pore sizes after coating, clogs flow paths on the units of porous medium, and increases the friction factor of the porous medium after clogging. Chittoori et al. tested the effect of porosity, consolidation, and the unit weight of expansive clays. Their study was important in the wake of soil treatment with microbes because pore size and size of pore throat play important roles in microbial treatment [118]. Figure 4 shows the maximum reduction of permeability in percentage achieved by different researchers in their studies after the biomineralization of soils. It can be inferred from Figure 4 that permeability reduction is achieved through biocementation, which is also promising in applications like lining the base of water bodies and seepage control in water retaining structures.

**Figure 4.** Maximum permeability reductions in percentage achieved by different researchers in their studies after soil biomineralization. 1—Yasuhara et al. [100], 2—Whiffin et al. [81], 3—Soon et al. [119], 4—Nemati and Voordouw [32], 5—Moghal et al. [120], 6—Handley-Sidhu et al. [110], 7—Zamani and Montoya [108], 8—Ferris et al. [115], 9—Gui et al. [116], 10—Proto et al. [63], 11—Ragusa et al. [121], 12—Cunningham et al. [122], 13—Van Paassen [123], 14—Ivanov et al. [124], 15—Al Qabany and Soga [125], 16—Ivanov and Chu [126].

#### *5.4. Liquefaction Control by Biotreatment*

Earthquakes and explosions make soils vulnerable and liquefy them, causing serious damage to the structures. Poorly graded and saturated sands are potential targets to liquefaction [127]. Major threats encountered due to the liquefaction of soils are landslides, damaged underground sewage lines and tunnels, and quicksand effects, although liquefaction helps in preventing seismic waves from reaching the Earth's surface since it produces a damping effect to the waves [128]. Liquefaction control is achieved by various techniques. Densification or compaction of existing soil is also among the methods adopted, but this method poses a threat to adjacent structures [129]. Biocementation has been proven as an effective method in controlling liquefaction in soils since calcite precipitation reduces permeability in soil [115]. Burbunk et al. [20,21] quoted that MICP improved resistance to liquefaction. Water in the voids of soils develops pressure transmitted through flow paths and tends to detach the soil grains apart or facilitate in the possible space in the vicinity. This pore water pressure contributes to the factors leading to the liquefaction of soils. Soil grains, when gelled together after biocementation, are less prone to liquefaction because of the disruption of flow paths and, later, due to permeability reduction and reduced pore water pressure [130]. Zamani and Montoya [131] tested the use of MICP on the permeability and shear of sand with silt and found a reduction in permeability depending on the amount of fines in the sample. Figure 5 shows the development of pore pressure in soil voids due to pore water and the development of resistance to the pore pressure through precipitates of calcite.

**Figure 5.** Representation of pore pressure in soil void: (**a**) Pore pressure developed by pore water pushing soil grains away from each other, leading to the loosening of the soil mass. (**b**) Cementation of soil grains by bioprecipitated calcite offering resistance to pore pressure.

#### **6. Biotreatment of Soils for Geoenvironmental Applications**

Bacterially precipitated CaCO3 has also been used for capturing heavy metal contaminants to reduce their hazardous effects by converting the heavy metal traces into carbonates [12,132–135]. Bioremediation through urease in soil is effective for contaminant remediation [136]. Although natural calcite is used as an adsorbent for removing ions of heavy metal from contaminated water [137,138], the use of calcium for remediating heavy metal contaminants in soil is also practiced by researchers [139–141]. Natural calcite used as an adsorbent is very rare, and even the quality of naturally available calcite is not feasible for adsorption. Therefore, calcite precipitated by microbes was tested for the purpose of adsorption [142]. Kulczycki et al. [143] used bacterial ferrihydrite for the sorption of cadmium and lead and found that the precipitates of ferrihydrite were effective in providing sites for the heavy metal ions to sorb. Pan et al. [144] studied the microbial strategy for lead remediation. They inferred from their study that use of microbes was cost-efficient and environmentally-friendly as a lead remediation method. Velmurugan et al. [145] studied the kinetics of lead absorption by *Penicillium* sp. MRF-1 in a contaminated mining site in South Korea. Their study covered the use of this metal-resistant fungus stain for the remediation of Pb(II) within the dimensions of time of exposure, pH, and temperature. They concluded that *Penicillium* sp. MRF-1 was an inexpensive and conveniently cultivable fungus for the removal of Pb from contaminated solutions. Moghal et al. [94,120] used the enzyme treatment for adsorption and desorption studies for cadmium, nickel, and lead contaminants and found that the urease enzyme was effective in precipitating the carbonates of cadmium, nickel, and lead. They also obtained encouraging results in the level of desorption of heavy metals even after washing the contaminated soils with harsh extractants like ethylene diamine tetra-acetic acid (EDTA) and citric acid. The sorption studies, on the other hand, depicted better results in providing the sites on the soil grains to sorb. Sorption and desorption studies were conducted for individuals and for cocktail solutions of contaminants. These studies obtained appreciable results, encouraging the application of these techniques in situ. Nathan et al. [146] used the EICP method for heavy

metal remediation in paper pulp deinking. They found that the enzymatic bioremediation is effective in reducing the hazardous effects of heavy metals. It also established that the urease enzyme is a nickel-based enzyme [147] and suggested that calcite precipitates by urease enzyme are active in showing affinity to the nickel contaminant, encapsulating them in the precipitates, and converting them into nickel carbonates [94].

Lauchnor et al. [148] studied the co-precipitation of strontium (Sr) in the porous media along with calcite precipitated by the MICP technique. They inferred that Sr precipitation was effective, thereby indicating the effective implementation of this method on site for the remediation of Sr. Mitchell and Ferris [149] tested the use of calcite precipitated with the MICP method to coprecipitate Sr in contaminated water and found that calcite precipitated by the MICP technique was exceptionally effective in the remediation of groundwater. Sr was also remediated by the formation of SrCO3 in microenvironments of soil mass, leading to the reduced effect of radio nucleoids [150]. Wang et al. [151] studied the effect CaCO3 on immobilizing heavy metals and observed that CaCO3 was successful in serving the purpose. Precipitates of calcite in the soil mass contributing to heavy metal immobilization can also be attributed to the number of heavy metal ions in the soil mass. If there are fewer heavy metal ions, then the sites for the ions to settle down will be sufficient, leading to better results in terms of the immobilization of heavy metals. Varenya et al. [152] studied lead retention using the MICP method and found that the precipitates of CaCO3 could be effective in the remediation of lead. They concluded that the MICP method has the potential to be applied in arid areas where phytoremediation cannot be used to remove heavy metals. The MICP method can be effective in reducing the hazardous effects caused by heavy metals like arsenic, cadmium, chromium, copper, and lead [153]. Therefore, the effectiveness of the biomineralization method can be well understood. The bioremediation of contaminants is a promising technique to reduce adverse effects caused by heavy metals. Table 2 shows a brief list of heavy metals remediated by the biostabilization method.


**Table 2.** List of contaminants remediated by the biostabilization method.
