E ﬃ cacy of Enzymatically Induced Calcium Carbonate Precipitation in the Retention of Heavy Metal Ions

: This study evaluated the e ﬃ cacy of enzyme induced calcite precipitation (EICP) in restricting the mobility of heavy metals in soils. EICP is an environmentally friendly method that has wide ranging applications in the sustainable development of civil infrastructure. The study examined the desorption of three heavy metals from treated and untreated soils using ethylene diamine tetra-acetic acid (EDTA) and citric soil by forming a CaCO 3 shield on the heavy metals, thus decreasing their mobility. An approximate improvement of 30% in the retention of heavy metal ions was observed in soils treated with ESs when compared to untreated soil samples. Therefore, the study suggests that ESs can be an e ﬀ ective alternative in the remediation of soils contaminated with heavy metal ions.


Introduction
Increase in population and attempts to satisfy the ever-growing demands of the same have led to industrialization, widespread construction activity, and extensive mining. Industrial effluents contaminate land and thereby pose a threat to the environment [1,2]. The number of contaminated sites identified in the United States of America alone until 2004 was 294,000 [3], which indicates an alarming need to implement remediation and decontamination methods to maintain a balance in nature. Remediation of the sites is costly because conventional remediation methods, such as excavation and dumping on unused land, are outdated, and contaminant removal by physical methods is difficult. As a result, contaminated soils arrive in engineered landfills. Engineered landfills, which are identified as the most viable means of landfilling solid municipal wastes, have also become sources of leachates rich with high levels of toxicity, fluorides, nitrates, and heavy metals [4][5][6]. Hence, attempts to mitigate the adverse effects of hazardous wastes present in landfills has attracted significant research attention. Urbanization also poses a worldwide challenge for landfill management. Developing an environmentally friendly method to decontaminate soils is a priority research topic.
One of the methods adopted to decrease landfill hazards involves adding liners to landfills that act as barriers relative to leachate infiltration from the landfills to the soil beneath [7][8][9]. A liner is an essential part of an engineered landfill and should be durable and properly designed for the safety of the circumferential environment [10]. The use of locally available, fine, and cohesive soil as liner material is an easy and convenient option [11]. However, not all locally available material is suitable for such uses. In such cases, suitable material is imported, which adds to the overall costs of the project. Another method of decontaminating soils is by washing the soils with suitable chemicals to neutralize the contaminants. In general, these methods for decontaminating soils are costly owing to difficulties involved with physical separation or soil washing. Furthermore, due to the heterogeneous soil texture, removal of soil contaminants by soil washing solutions becomes difficult [3].
Another method to strengthen the landfill liner is to stabilize the soil using lime or cement. Stabilization of soil by cement and lime have been vastly used [12][13][14][15] in landfill liners, and these methods contribute to the emission of greenhouse gases, leading to large scale global warming. Production of cement and lime result in about 800-900 and 600-700 kg CO 2 per ton, respectively [16]. The cement industry contributes about 5% of CO 2 emissions globally [17,18]. With such an alarming situation, researchers have searched for sustainable stabilization methods for soil, wherein partial or total replacement of the cement or lime binders are tested for their reliability. These stabilizers include palm oil fuel ash [19], flyash [20], rice husk ash [21,22], residue of calcium carbide [23], alkali-activated agro-waste [24], and so forth.
As an attempt to contribute to the field of soil stabilization, bio cementation by CaCO 3 is also employed as a sustainable method of soil stabilization [25][26][27][28][29], which includes improvement of geotechnical properties of soil as well as contaminant remediation. This study evaluates the use of one such method, known as enzyme induced calcite precipitation (EICP), to immobilize heavy metals as a method of decontaminating soil. The main objective is to study the ability of the EICP method to retain heavy metal contaminants such as nickel, cadmium, and lead in soil and to determine the dosage of Enzyme Solution (ES) that leads to the maximum retention of heavy metals. Another aim is to identify the percentage of heavy metal contaminants retained in the soil as a result of EICP.

Background
The dumping of contaminated soils into landfills has made landfill management a major challenge, as these sites become major sources of leachates rich in heavy metals, fluorides, and other contaminations. Furthermore, the intrusion of leachates from landfills has been found to introduce large quantities of heavy metals, fluorides, and nitrates to ground water used for irrigation, industrial purposes, and drinking [30]. High concentrations of metals in a low-pH soil increase soil acidity, thereby making the soil vulnerable to contamination [6]. The presence of less than 1000 mg/kg of heavy metals in soil is rarely toxic, although human activities involving the disposal of them into the soil above these levels Sustainability 2020, 12, 7019 3 of 17 poses a threat to the flora and fauna. Heavy metals that are naturally present in the soil cause less damage than those that accumulate due to human activity [31]. Specifically, Cd is naturally available in earth's crust with a concentration of 0.1-0.5 ppm associated with zinc, copper, and lead ores [32]. Industries play an important role in increasing heavy metal concentrations in surface soils above permissible levels by releasing toxic fumes into the atmosphere, because traces of fumes find their way to the soil surface [33,34].
Soil in its natural form exhibits different components, such as phyllosilicates, humic substances, and carbonates, which contribute to the sorption process of heavy metals [35]. Concentrations of metals in the soil are governed by anthropogenic effects, pedogenic processes, and parent material [36]. Immobilization [37], phytoremediation [38,39], and soil washing [40] methods are adopted to achieve remediation of heavy metals in soils [7,41,42]. Remediation of contaminated soils can be performed using in situ or ex situ methods based on site conditions [4,43]. Landfill management continues to constitute a complex issue and should be examined further [39].
Industries also emit toxic elements into water bodies. These elements are then absorbed by aquatic plants, making them unsafe for consumption. Sahu et al. [44] examined concentrations of different heavy metals in macrophytes and observed an average of 13 mg/kg of Pb in seven aquatic plants from the Kharun River in India. Metal contaminants also pose a threat to terrestrial plants because of the increase in anthropogenic activities, which eventually leads to their intrusion in the food chain [45]. Heavy metals that are most harmful to human health include Pb, Cd, As, Zn, Cu, Hg, Cr, and Ni. Heavy metals happen to be the most commonly found carcinogens among the pollutants; for example, Hg leads to mutations and genetic damage, and Cu and Pb can affect the brain and bones [46]. Heavy metals are generally removed from the soil via precipitation-dissolution, oxidation-reduction, and adsorption-desorption processes, among which adsorption-desorption is observed as the most effective geochemical process for contaminant remediation [47]. The removal of metals can also be performed by chemical precipitation, bio-precipitation, ion exchange, adsorption, biosorption, physical separation, electrochemical separation, solvent extraction, flotation, and cementation [48,49]. Another technique adopted to decrease the hazardous effects of heavy metals involves retaining contaminants in soil via encapsulation. Methods adopted for retaining heavy metals by encapsulation decrease the mobility of heavy metal ions in the soil. The use of nano calcium silicate in retaining Cd, Ni, and Pb was observed as an effective [50,51] approach towards contaminant remediation. However, the costs associated with production of nano compounds on a macro scale is not economically feasible.
Among the approaches to encapsulate heavy metals in soils, the use of microorganisms is also adopted by researchers. This method, popularly known as bioremediation, involves processes such as microbial induced calcite precipitation (MICP) to encapsulate heavy metals inside the precipitated calcium carbonate. The MICP technique fosters metabolic activity in certain types of soil bacteria (Sporosarcina pasteurii), which results in the formation of inorganic compounds (such as CaCO 3 ) outside the cellular structure; these compounds can bind soil particles together and stabilize the soil. In addition, it is possible to encapsulate heavy metals present in the soil inside the CaCO 3 crystals. Another branch of bioremediation, known as enzyme induced calcite precipitation (EICP), uses plant-based urease enzymes to precipitate calcium carbonate. The use of enzymes is advantageous, because they are non-toxic and ecofriendly [52].
Nathan et al. [53] found the enzyme treatment effective in reducing the heavy metals in paper pulp. EICP is a biologically inspired soil improvement process designed to initiate urea hydrolysis using urease enzyme extracted from jack beans [25,[54][55][56][57] or watermelon seeds [58]. The precipitation of CaCO 3 through enzyme activity is obtained by mixing urea, calcium chloride, and urease enzyme with deionized water. This solution can then be mixed with soil to prepare soil samples for testing [59]. Additionally, MICP is a process in which CaCO 3 is precipitated by the activity of bacteria, such as Sporosarcina pasteurii, to improve the geotechnical properties of soil [60]. Calcium carbonate precipitates are observed to fill the voids between soil grains, thereby reducing the permeability of soil and improving unconfined compressive strength (UCS) [61,62] and shear strength of the soil [63]. Furthermore, the precipitates mitigate liquefaction below the existing structures, stabilize roadways, control the flow of groundwater, immobilize contaminants [37], and remediate hazardous trace metals in soils [64]. The urease enzyme used in the study was crystallized from jack beans (Canavalia ensiformis) and formed the main source of urea hydrolysis [65]. Plant-based urease is optimal if obtained from jack bean and is identified as the first crystallized enzyme as well as the first nickel metalloenzyme [66]. Concerning remediation of soil contaminants, CaCO 3 precipitates are relatively easy to implement, useful in retaining the contaminants, and economic. They are readily accepted in society, based on an approximate scoring proposed by Dejong et al. [67].

Soil
The present study was performed on two soils, namely black cotton soil collected from Yadgir district (16 • 45 20.556 N, 77 • 9 4.5072 E) and red soil from Bangalore district (13 • 2 14.1396 N, 77 • 37 11.928 E) in Karnataka, India. The dominant mineral in black cotton soil was identified as montmorillonite (henceforth referred to as 'Soil M') and kaolinite in red soil (referred to as 'Soil K'). Basic tests of these soils were conducted and are listed in Table 1. Soil M was classified as clay with high plasticity (CH), whereas Soil K was classified as clay with low plasticity (CL) according to the Unified Soil Classification System.

Heavy Metal Contaminants
Three heavy metals, namely cadmium (Cd), nickel (Ni), and lead (Pb), were spiked in the soils by preparing a stock solution with predetermined load ratios to obtain the target concentrations of contaminants in the soil as described by Mohammed and Moghal [2].
Analytical reagent grade (AR) nitrates of nickel (Ni(NO 3 ) 2 ), cadmium (Cd(NO 3 ) 2 ), and lead (Pb(NO 3 ) 2 ) were used to prepare the stock solutions. The target load ratio was identified as 50 and 100 mg/kg of heavy metal contaminants. Salt solutions were prepared in containers of borosilicate glass to maintain a sufficiently wet consistency to spike soils with contaminants. Oven-dried soil samples of a predetermined quantity were placed in glassware and properly washed with deionized double-distilled water (DI), and the heavy metal stock solution was added to the soil samples and mixed thoroughly to achieve the target Ni, Cd, and Pb load ratios [68]. Soil containers were covered with thin sheets of aluminum with minute perforations to ensure free airflow and to avoid dust intrusion and cross-contamination [51]. Subsequently, the samples were placed on a flat dry platform in a temperature-and humidity-controlled room where the temperature was maintained at 23-27 • C with proper ventilation and average relative humidity of 45%. The soil samples were left to cure for 40 days such that the probability of contaminants desorbing from the soil in the initial stages was minimized. After the soil samples were cured for 40 days, acid digestion was conducted, and the Sustainability 2020, 12, 7019 5 of 17 actual amount of heavy metal concentration in the soils was established using an atomic absorption spectrophotometer (AAS) (PerkinElmer Model A-Analyst 400). The results are tabulated in Table 2. The same procedure was repeated for the soils treated with ESs, and the concentrations of the contaminants in the soils treated with the ESs were determined for comparison with those of untreated soils.

Enzyme Solutions
Three types of enzyme solutions were prepared to treat the soil using Analytical Reagent (AR) grade materials. Specifically, the following materials were used to prepare the ESs: Urease enzyme (Canavalia ensiformis (jack bean) Type III, powder, 15,000-50,000 units/g solid) • Non-fat milk powder The chemicals were procured from Winlab Chemical, Market Harborough, United Kingdom, and the urease enzyme was procured from Sigma-Aldrich, St. Louis, MO, USA. The composition of each of the ESs is presented in Table 3. It can be noted here that ES1 and ES2 have similar compositions, except for the use of non-fat milk powder, which aided in developing nucleation sites in the soil masses, thereby leading to the precipitation of CaCO 3 at the contact points [25]. Similarly, ES2 and ES3 differ by the concentration of the urease enzyme, used as per Almajed et al. [25].

EICP Treatments
Enzyme-treated soil samples were prepared by compacting soil passing through a 425-µ sieve and mixed with an ES to obtain a moisture content equal to the optimum moisture content of the soil by weight. The soil was mixed with ES in a 5-cm diameter mold with a depth of 10 cm in three layers to ensure maximum density and minimal voids. Subsequently, the soil samples were cured in a desiccator for 21 days after they were sealed in an airtight seal pack to ensure proper calcite precipitation.  [69] to gain a better understanding of the effect of ESs on the strength properties of soils. Figure 1 shows the UCS results obtained for soil samples using ESs cured for 7, 14, and 21 days. It was observed that the UCS values of the soil samples increased to the maximum level when the samples were treated with ES2 and cured for 21 days. Non-fat milk powder in ES2 is the main strength booster, because it creates sites of nucleation in the soil, enhancing the precipitation of CaCO 3 . However, non-fat milk powder is also used in ES3, and the strength gain is less than that of the ES2, because the amount of urease enzyme used in ES3 is 0.85 g/L, which is less than that of ES2 [25]. The same reason potentially holds in terms of understanding the amount of CaCO 3 precipitated in the soil when ES3 is used, which constitutes a reduction in CaCO 3 precipitated when compared to precipitation given the use of ES2.
UCS values of the soil samples increased to the maximum level when the samples were treated with ES2 and cured for 21 days. Non-fat milk powder in ES2 is the main strength booster, because it creates sites of nucleation in the soil, enhancing the precipitation of CaCO3. However, non-fat milk powder is also used in ES3, and the strength gain is less than that of the ES2, because the amount of urease enzyme used in ES3 is 0.85 g/L, which is less than that of ES2 [25]. The same reason potentially holds in terms of understanding the amount of CaCO3 precipitated in the soil when ES3 is used, which constitutes a reduction in CaCO3 precipitated when compared to precipitation given the use of ES2.

Measurement of Calcium Carbonate Precipitation
The ES-treated soil samples were kept in a desiccator after they were sealed in air-tight packs and were tested to identify the amount of CaCO3 precipitated by gravimetric acid digestion. 40 to 50 g of soil specimens was soaked in 1 M hydrochloric acid for an hour until the disappearance of the effervescence was observed after soaking (due to the dissolution of CaCO3 in the soil). Subsequently, the samples were rinsed and placed in an oven for drying at a temperature of 105 °C. The difference in the masses of the soil samples before and after soaking in 1.0 M hydrochloric acid was determined to calculate the percentage of CaCO3 precipitated in soil. The soil samples were tested for the amount of CaCO3 precipitated over 7, 14, and 21 days to understand the precipitation trend. These results are presented in Table 4.

Measurement of Calcium Carbonate Precipitation
The ES-treated soil samples were kept in a desiccator after they were sealed in air-tight packs and were tested to identify the amount of CaCO 3 precipitated by gravimetric acid digestion. 40 to 50 g of soil Sustainability 2020, 12, 7019 7 of 17 specimens was soaked in 1 M hydrochloric acid for an hour until the disappearance of the effervescence was observed after soaking (due to the dissolution of CaCO 3 in the soil). Subsequently, the samples were rinsed and placed in an oven for drying at a temperature of 105 • C. The difference in the masses of the soil samples before and after soaking in 1.0 M hydrochloric acid was determined to calculate the percentage of CaCO 3 precipitated in soil. The soil samples were tested for the amount of CaCO 3 precipitated over 7, 14, and 21 days to understand the precipitation trend. These results are presented in Table 4.

Extractants
The extractants used for the desorption tests included ethylene diamine tetra-acetic acid (EDTA) and citric acid in three molar concentrations (i.e., 0.1, 0.25, and 0.5 M) for the removal of heavy metals retained in the soil by acid digestion. A solid-to-liquid ratio of 1:20 was maintained. Both EDTA [70] and citric acid [71] were proven to be effective in extracting heavy metals from soil surfaces. Gu and Yeung [71] noted that citric acid dominant industrial wastewater is effective in desorbing Cd from soil surfaces in the pH range of 4 to 8.

Desorption Tests
A desorption test was conducted on the soil samples spiked with contaminants after a curing period of 40 days. Five grams of contaminated soil samples was placed in 0.1, 0.25, and 0.5 M extractants in 50-mL polytetrafluoroethylene bottles and shaken as per ASTM D3987 [72] in a mechanical shaker at 30 rpm for 24 h to ensure that the soil particles were uniformly distributed in the solution, and thereby releasing heavy metal contaminants. Subsequently, the slurry was filtered through filter papers (Whatman No. 42) to obtain solutions that were tested in AAS to identify their metal contamination levels. Each set of tests was performed with three samples, and an average of the three results was considered as the final value. The pH values of the solutions tested in AAS were determined, and their removal efficiencies were calculated as follows [73].

Results and Discussions
The data obtained from the desorption experiments were plotted (Figures 2-4) with the molar concentrations of the extractants on the abscissa and heavy metal removal efficiencies (%) on the ordinate. A lower removal efficiency percentage is favorable, as it means that heavy metal contaminants are immobilized in the soil and cannot be extracted from it. Among the three heavy metals, it was observed that the removal efficiency of Cd was minimal when compared to that of Ni or Pb.
The formation of CaCO 3 was initiated in the soil by the reaction between urea and calcium chloride; this occurs when the calcium ions and carbonate ions combine [74]. The process of precipitation of calcium carbonate is accelerated by the urease enzyme [75]. The recipe of the ESs adopted provides an environment for efficient utilization of enzyme for CaCO 3 precipitation [76]. CaCO 3 is precipitated along the nucleation sites with ES2 due to the use of non-fat milk powder [25]. The main process that leads to the formation of calcite is hydrolysis of urea by the urease enzyme into CO 2 and NH 3 , and the speciation of NH 3 leads to the development of NH 4+ ions, which creates a suitable environment for  [26]. It is also proposed that the heavy metal ions with ion radii close to that of Ca 2+ (e.g., Pb 2+ , Cu 2+ , Cd 2+ , and Sr 2+ ) are incorporated in CaCO 3 crystal lattice by replacing Ca 2+ ions or by creating defects on the calcium carbonate crystals, or even by penetrating the CaCO 3 interstice. This indicates that the EICP technique can be used for the remediation of heavy metals [77]. The formation of carbonates of heavy metals occurs in the microenvironment of the mineral carbonates, and the process is even applicable for radionuclides such as strontium forming strontium carbonate (SrCO 3 ) [74]. Metal ions in soil tend to cluster with carbonates that are already present in the soil, and heavy metal retention is expected due to the precipitation of heavy metal carbonates [78,79]. Enzymatic mechanism of bioremediation is found to be effective in issues concerning heavy metal bioaccumulation in paper pulp [53].
are incorporated in CaCO3 crystal lattice by replacing Ca 2+ ions or by creating defects on the calcium carbonate crystals, or even by penetrating the CaCO3 interstice. This indicates that the EICP technique can be used for the remediation of heavy metals [77]. The formation of carbonates of heavy metals occurs in the microenvironment of the mineral carbonates, and the process is even applicable for radionuclides such as strontium forming strontium carbonate (SrCO3) [74]. Metal ions in soil tend to cluster with carbonates that are already present in the soil, and heavy metal retention is expected due to the precipitation of heavy metal carbonates [78,79]. Enzymatic mechanism of bioremediation is found to be effective in issues concerning heavy metal bioaccumulation in paper pulp [53].

Cd Retention in Soils
The highest removal efficiency of Cd was observed as 50.39% for untreated Soil M and 46.37% for untreated Soil K (see Figure 2). The lowest removal efficiency for Cd when extracted by 0.5 M citric acid was observed as 3.97% for Soil M and 5.39% for Soil K treated with ES2 (see Figure 2). An increase in clay or silt particles in the soil decreases the release of Cd due to strong deposits of heavy metal traces that occur in the finer fractions of clay [80]. Hence, it was observed that higher silt or clay content expanded soil capacity for heavy metal retention [81,82]. Our results are in line with these observations. The Cd removal efficiency was also low due to the precipitation of a comparatively large amount of CaCO3 in soil treated with ES2 (see Table 4). Wang et al. [83] found that the presence of CaCO3 played a major role in immobilizing heavy metals, as observed in their study, when treating soils with CaCO3. The EICP method used in this study for the retention of heavy metals is comparatively more effective in immobilizing Cd. Therefore, the EICP treatment can be termed an adsorbent selective to Cd contamination.
The role of pH is also important in the retention of Cd in the soil, and the desorption of Cd is observed to increase when pH decreases. Additionally, increase in the pH of soil results in immobilization of Cd ions [32]. Citric acid is observed to desorb Cd to a lesser extent at lower molar concentrations and to a greater extent at higher molar concentrations (see Figure 2). This can be attributed to the formation of Cd-citric acid complexes in an aqueous state, which detach from soil surfaces. EDTA is observed to retain greater amounts of Cd, even at a pH of 5.05 [84]. The range of pH was between 5-8 with citric acid as chelant with 0.1 M for extraction of Cd. This pH reduced (within range of 3-6) at 0.5 M molarity, leading to comparatively higher removal of Cd. The removal efficiencies and associated standard deviation values are provided in the supplementary material  The maximum range of Ni desorption was observed as approximately 72.36-57.2% for soils M and K, respectively, and the minimum amounts of Ni desorption were 34.48% for Soil M and 20.26% for Soil K when extracted by 0.5 M EDTA. Nickel is one of the most commonly found contaminants in the brownfields, and nickel contamination originates from the discharge of industries involving metal plating, nickel refinement, and mining sites. Nickel precipitates into a stable compound in the form of nickel hydroxide [Ni(OH)2] in slightly alkaline and neutral solutions. Nickel can be effectively removed by citric acid and EDTA chelating agents [70], and nickel retention can be understood from the relationship between the sorptive surface and ion concentration that decreases metal ion removal.
When metal ion concentrations are low, the number of binding sites for heavy metals is initially high and results in the immobilization of heavy metals [85]. The mechanism of Ni retention can also be expressed as a mechanism in which metal binds to a carboxylic group, given the competition between protons, metals, and ion exchange interactions in the solution, and leads to the immobility of Ni ions [86]. Precipitation of heavy metals occurs when a solution is saturated with a specific element through a homogeneous or heterogeneous aggregation processes. The former aggregation process is an outcome of nucleation of the supersaturated phase in the soil solution, whereas the latter process involves precipitation formed by the nucleation of other materials (i.e., soil particles), which hold metals as sorbents on the surface [87]. The urease enzyme used in the study is reportedly active and stable when the EDTA solution exhibits a neutral pH. The enzymatic activity plays a major role in already present nickel ions in the urease enzyme. It is also established that nickel ions are active in strengthening the enzyme activity completely; thus, attempts to remove nickel from the urease enzyme were not successful [66]. Thus, it can be assumed that preexisting nickel in urease and added

Cd Retention in Soils
The highest removal efficiency of Cd was observed as 50.39% for untreated Soil M and 46.37% for untreated Soil K (see Figure 2). The lowest removal efficiency for Cd when extracted by 0.5 M citric acid was observed as 3.97% for Soil M and 5.39% for Soil K treated with ES2 (see Figure 2). An increase in clay or silt particles in the soil decreases the release of Cd due to strong deposits of heavy metal traces that occur in the finer fractions of clay [80]. Hence, it was observed that higher silt or clay content expanded soil capacity for heavy metal retention [81,82]. Our results are in line with these observations. The Cd removal efficiency was also low due to the precipitation of a comparatively large amount of CaCO 3 in soil treated with ES2 (see Table 4). Wang et al. [83] found that the presence of CaCO 3 played a major role in immobilizing heavy metals, as observed in their study, when treating soils with CaCO 3 . The EICP method used in this study for the retention of heavy metals is comparatively more effective in immobilizing Cd. Therefore, the EICP treatment can be termed an adsorbent selective to Cd contamination.
The role of pH is also important in the retention of Cd in the soil, and the desorption of Cd is observed to increase when pH decreases. Additionally, increase in the pH of soil results in immobilization of Cd ions [32]. Citric acid is observed to desorb Cd to a lesser extent at lower molar concentrations and to a greater extent at higher molar concentrations (see Figure 2). This can be attributed to the formation of Cd-citric acid complexes in an aqueous state, which detach from soil surfaces. EDTA is observed to retain greater amounts of Cd, even at a pH of 5.05 [84]. The range of pH was between 5-8 with citric acid as chelant with 0.1 M for extraction of Cd. This pH reduced (within range of 3-6) at 0.5 M molarity, leading to comparatively higher removal of Cd. The removal efficiencies and associated standard deviation values are provided in the Supplementary Materials (Tables S1 and S2 respectively).
using reagent-grade stabilizers, namely Ca(H2PO4)2 and CaCO3 in a dumpsite in Taiwan.
The heavy metal desorption process is affected by the pH of the pore fluid solution [42]. Even waste soils with neutral pH values exhibit significant levels of heavy metal sorption [92]. Harter [93] examined the effects of soil pH on heavy metal adsorption and stated that the degree to which metal ions hydrolyze at a specific extractant pH (leading to their release from the host soil) is unknown. In a previous study, Harter [93] suggested that retention of univalent Pb hydroxides (when compared to divalent ions) can increase by 60% when pH increases from 6 to 8 and forms precipitates of metals at high pH levels. The same study revealed that Ni was retained due to a reaction that formed precipitates at high pH levels and concluded that the validity of pH, as a deciding factor in ascertaining the quantity of heavy metal that can be safely retained in the soil, is uncertain, although the pH of the extractants used for Pb retention was within the range 3-5. Gupta and Lataye [94] indicated that the pH of a solution affects the charge and process of ionization of the sorbent in the solution. Therefore, it is presumed that pH increases the duration for which metal contaminants are released by extractants. The process of heavy metal removal was performed in the study by conducting separate desorption tests on soils spiked with individual contaminants, because the simultaneous removal of heavy metals using a single extraction method is difficult [95].

Conclusions
The aim of this study was to examine the efficacy of EICP in fixing Cd, Ni, and Pb that were spiked in Soil M and Soil K in addition to improving their strength characteristics. The following conclusions were made from the study:


Soil grains adhered to each other due to CaCO3 precipitation initiated by the urease enzyme, and there exists a high probability that metal ions are encapsulated between the soil grains and CaCO3 precipitates. This leads to the effective retention of heavy metals in the soil matrix.  Heavy metal retention in the soil occurred in the following order: Cd > Ni > Pb. The EICP-treated soil retained the maximum quantity of Cd among all the heavy metals. Additionally, Cd retention exceeded Ni or Pb retention even after treatment with chelants EDTA and citric acid. This was potentially due to the formation of CdCO3 in the soil matrix.  EICP treatment using ES2 was observed to be better in terms of retaining heavy metals in the  Figure 3 shows the plots for Ni extracted from both soils using EDTA and citric acid. Figure 3a,b shows the removal efficiency for load ratios of 50 mg/kg. Figure 3c,d shows the removal efficiency for load ratios of 100 mg/kg. The plots indicate that the encapsulation of Ni in the soils with ES2 exceeded that in the soils with ES1 and ES3. The difference in removal efficiency was not significant between the two load ratios (50 and 100 mg/kg). Both load ratios yielded removal efficiency values of approximately 15-20%.

Ni Retention in Soils
The maximum range of Ni desorption was observed as approximately 72.36-57.2% for soils M and K, respectively, and the minimum amounts of Ni desorption were 34.48% for Soil M and 20.26% for Soil K when extracted by 0.5 M EDTA. Nickel is one of the most commonly found contaminants in the brownfields, and nickel contamination originates from the discharge of industries involving metal plating, nickel refinement, and mining sites. Nickel precipitates into a stable compound in the form of nickel hydroxide [Ni(OH) 2 ] in slightly alkaline and neutral solutions. Nickel can be effectively removed by citric acid and EDTA chelating agents [70], and nickel retention can be understood from the relationship between the sorptive surface and ion concentration that decreases metal ion removal.
When metal ion concentrations are low, the number of binding sites for heavy metals is initially high and results in the immobilization of heavy metals [85]. The mechanism of Ni retention can also be expressed as a mechanism in which metal binds to a carboxylic group, given the competition between protons, metals, and ion exchange interactions in the solution, and leads to the immobility of Ni ions [86]. Precipitation of heavy metals occurs when a solution is saturated with a specific element through a homogeneous or heterogeneous aggregation processes. The former aggregation process is an outcome of nucleation of the supersaturated phase in the soil solution, whereas the latter process involves precipitation formed by the nucleation of other materials (i.e., soil particles), which hold metals as sorbents on the surface [87]. The urease enzyme used in the study is reportedly active and stable when the EDTA solution exhibits a neutral pH. The enzymatic activity plays a major role in already present nickel ions in the urease enzyme. It is also established that nickel ions are active in strengthening the enzyme activity completely; thus, attempts to remove nickel from the urease enzyme were not successful [66]. Thus, it can be assumed that preexisting nickel in urease and added nickel potentially cluster and this behavior leads to the retention of Ni in the soil. This can explain the results shown in Figure 3. Figure 4 shows the Pb removal efficiency. As shown in the figure, Pb retention was minimal when compared to Ni and Cd retention. Raw soil K contaminated with Pb demonstrated very high removal efficiency, which ranges from 99.96% to 97.52% for soil treated with 0.5 M EDTA extractant and with load ratios of 50 and 100 mg/kg, respectively. The removal efficiencies after acid digestion by citric acid decreased to 11.2% and 26.26% for the same soil with 50 and 100 mg/kg load ratios, respectively, when Soil K was treated with ES2.

Pb Retention in Soils
Pb and Cd get adsorbed on CaCO 3 precipitates, limiting their mobility. The retention of Pb and Cd was observed as predominant by polydopamine CaCO 3 when compared to natural CaCO 3 [88]. Thus, it was inferred that decreases in Cd and Pb removal efficiency observed in the present study occurred due to the presence of CaCO 3 in the soil. Pb retention can also occur via diffusion of solid-state and precipitation reactions resulting in PbSO 4 and PbCO 3 precipitates when the metal contamination levels exceed solubility levels of the carbonates and hydroxides at a given pH [89,90]. Kumpiene et al. [91] performed Pb immobilization studies via phosphorus-containing chemicals, such as apatites and hydroxyapatites, in synthetic and natural forms. This is because precipitation and ionic exchange of pyromorphite-type minerals decrease Pb mobility, and because compounds of Ca are generally efficient in performing Pb immobilization as soil pH increases within the range 8-9, thereby leading to the retention of Pb. Almost 99% of Pb retention was obtained by Wang et al. [83] using reagent-grade stabilizers, namely Ca(H 2 PO 4 ) 2 and CaCO 3 in a dumpsite in Taiwan.
The heavy metal desorption process is affected by the pH of the pore fluid solution [42]. Even waste soils with neutral pH values exhibit significant levels of heavy metal sorption [92]. Harter [93] examined the effects of soil pH on heavy metal adsorption and stated that the degree to which metal ions hydrolyze at a specific extractant pH (leading to their release from the host soil) is unknown. In a previous study, Harter [93] suggested that retention of univalent Pb hydroxides (when compared to divalent ions) can increase by 60% when pH increases from 6 to 8 and forms precipitates of metals at high pH levels. The same study revealed that Ni was retained due to a reaction that formed precipitates at high pH levels and concluded that the validity of pH, as a deciding factor in ascertaining the quantity of heavy metal that can be safely retained in the soil, is uncertain, although the pH of the extractants used for Pb retention was within the range 3-5.
Gupta and Lataye [94] indicated that the pH of a solution affects the charge and process of ionization of the sorbent in the solution. Therefore, it is presumed that pH increases the duration for which metal contaminants are released by extractants. The process of heavy metal removal was performed in the study by conducting separate desorption tests on soils spiked with individual contaminants, because the simultaneous removal of heavy metals using a single extraction method is difficult [95].

Conclusions
The aim of this study was to examine the efficacy of EICP in fixing Cd, Ni, and Pb that were spiked in Soil M and Soil K in addition to improving their strength characteristics. The following conclusions were made from the study:

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Soil grains adhered to each other due to CaCO 3 precipitation initiated by the urease enzyme, and there exists a high probability that metal ions are encapsulated between the soil grains and CaCO 3 precipitates. This leads to the effective retention of heavy metals in the soil matrix.

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Heavy metal retention in the soil occurred in the following order: Cd > Ni > Pb. The EICP-treated soil retained the maximum quantity of Cd among all the heavy metals. Additionally, Cd retention exceeded Ni or Pb retention even after treatment with chelants EDTA and citric acid. This was potentially due to the formation of CdCO 3 in the soil matrix. • EICP treatment using ES2 was observed to be better in terms of retaining heavy metals in the soil when compared to ES1 and ES3.

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The use of non-fat milk powder in the preparation of ES2 played a major role in boosting the UCS strength of the soil and also in retaining heavy metals in the soil due to the precipitation of CaCO 3 .
Overall, the effects of CaCO 3 precipitation due to the ESs consisted of improvements in heavy metal retention and UCS strength when compared to those of raw soils. Hence, it was concluded that the precipitates of CaCO 3 hold heavy metals and improve UCS strength irrespective of whether the quantity of precipitation varies for different ESs.

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Based on these results, it is clear that EICP has sufficient ability to immobilize heavy metals in contaminated soil. This study portrayed appreciable outcomes for Cd when compared to Ni and Pb; this technique can be employed to immobilize specific contaminants by identifying its effectiveness on other heavy metals also. However, further testing in the field is necessary before drawing this conclusion.