Bio-Mediated Method for Immobilizing Copper Tailings Sand Contaminated with Multiple Heavy Metals

: Microbial induced carbonate precipitation (MICP) is a natural bio-mediated process that has been investigated for soil stabilization and heavy metal immobilization in soil and groundwater. This study analyzed the effect and mechanism of MICP for the solidiﬁcation/stabilization of tailings sand with multi-heavy metals. When the concentration of cementation solution (CS) is 1.0 mol/L and the optical density(OD 600 ) is 1.6, the unconﬁned compressive strength of tailings sand treated by MICP is the largest, and the solidiﬁcation efﬁciency of heavy metals in tailings sand is also the highest. The macroscopic and microscopic observations reveal that the mechanism of MICP solidiﬁcation of tailings is bacterial outer oxide, hydroxide, alkaline carbonate, and carbonate precipitation.


Introduction
Copper tailings are powdery or sandy solid wastes produced by copper ore beneficiation. According to the analysis report of China's solid waste treatment industry in 2021, the annual emission of copper tailings slag in China can reach 335 million tons. Immense quantities of tailings accumulate in the form of large hills or water ponds, which may cause severe problems. Sometimes, large tailing dumps are unstable and suffer from slope instability effecting by water content changes, which may cause geo-hazards such as landslides. Furthermore, as reservoirs of heavy metal-containing wastes such as Cu, Pb, Zn, Cd, and Cr, copper mine tailings pose significantly substantial risk to the ecosystem and human health due to their toxicity [1,2]. Small particles of tailings may be carried by wind to nearby communities and cause air pollution. Water and soil pollution may occur when rain falls on the tailings and leaches the metals [3]. Therefore, it is of great importance to find low energy consumption, fast, efficient, and environmentally friendly way to manage these tailings and reduce risks.
Microbial induced carbonate precipitation (MICP) has been proven to be economical, effective, and environmentally friendly [4][5][6][7][8]. One of the most widely adopted pathways to achieve MICP is hydrolysis of urea by urease enzyme, which has the advantages of low cost, high efficiency in triggering cementation, controllable reaction process, and straightforward isolation and harvest process [9]. Among them, Sporosarcina pasteurii is the most effective urease-producing bacterial species. The fundamental principle of MICP is that ureaseproducing bacteria are able to produce urease to decompose urea into ammonium (NH 4 + ) and carbonate (CO 3 2− ), then NH 4 + releases ammonia (NH 3 ) and increases the pH value of the medium until NH 4 + /NH 3 and HCO 3 − /CO 3 2− reach a balance. After these reactions, the pH is close to 9.3 whereby CaCO 3 precipitation occurs via a combination of Ca 2+ and CO 3 2− [10]. The precipitated CaCO 3 fills the soil pores and cements the soil particles, which improves the soil strength and reduces the permeability. Meanwhile, the heavy metal ions are co-precipitated and immobilized, which can significantly reduce the concentration of exchangeable heavy metal ions.
In the MICP process, by replacing the calcium with other divalent elements, strong absorption of heavy metals on the surface and inside the lattice of calcite occurs [11]. However, more than five million contaminated sites have been reported globally, in particular mine tailing sites, covering 20 million hectares of land, in which the soils are contaminated by the heavy metals, such as Cu, Pb, Cd, As, Cr, Co, Hg, Ni, Zn, and Se, and which have soil concentrations above background values or regulatory levels [12][13][14][15][16][17]. The effect and mechanism of ureolytic-driven MICP for immobilizing multi-heavy-metal contaminated soils has not been explained clearly. This mechanism can be crucial in applications, as polluted soil like tailing sand invariably contains multiple metal elements, which may have a significant impact on the effect of immobilization. The presence of heavy metals in the soil will undoubtedly affect the curing efficiency of microorganisms.
Using MICP (including the ureolytic-driven MICP) method, this study chooses tailings sand polluted by various heavy metals for study, and explores the followings: (1) the unconfirmed compressive strength (UCS) and durability of tailings sand under different concentrations; (2) the removal rate of different ions; and (3) the microstructure of solidified tailings sand by XRD and SEM. The solidification mechanism and the hypothesis of multilayer precipitation structure by MICP of various heavy metals is proposed for the first time in this study.

Bacteria and Chemicals
Sporosarcina pasteurii (S. pasteurii, ATCC 6452), a non-pathogenic bacterium with high urease activity, was used in this study for MICP [18,19]. The bacterial solution was prepared by inoculating bacterial colonies into the NH 4 -YE solution medium (20 g/L yeast extract, 10 g/L (NH 4 ) 2 SO 4 , and 0.13 M Tris-base) and was then subjected to shaking incubation at 30 • C for approximately 24 h until the optical density at 600 nm (OD 600 ) reached the designated value. The OD 600 value was measured via a UV-Visible spectrometer (Shanghai Analytical Instruments Co Ltd., Shanghai, China). The prepared bacterial solution was then stored at 4 • C. Prior to its use in the experiment, it was firstly re-harvested in fresh growth medium. When bacteria cells reached the exponential growth stage during resuscitation, they were inoculated into the solution for the formal test. NH 4 -YE medium, CaCl 2 , and urea solutions were sterilized by autoclaving at 121 • C for 20 min.
The actual number of cells can be converted by Equation (1) [20].
where Y was cell concentration (cells ML −1 ) and Z is OD 600

Tailings Sand
Copper mine tailing samples were collected from the Shuimucong tailing pond (117 • 51 2 E, 30 • 55 42 N), which is located in Tongling city, a typical mining city in Southeast China. According to the test results, the average particle size (D 50 ) was 0.28 mm, the nonuniformity coefficient (C u ) was 4.62, and the curvature coefficient (C C ) was 1.58. The tailings were medium-fine sand with poor grading and uniform distribution. The natural moisture content was 9.46%, while the loose bulk density was 1.32 g/cm 3 , and the specific gravity was 2.86. Some physicochemical properties and the background levels of toxic metals in the soil are shown in Table 1. The XRF results showed that the tailings contained more Al and Ca compounds. The results of the electron microprobe show that it contains dolomite, potassium feldspar, plagioclase, muscovite, clinopyroxene, garnet, and pyrite. The heavy metal elements in the tailings sand were determined by the toxicity characteristic leaching procedure (TCLP), and the calcium carbonate content was measured according to the excessive acid dissolution method (other carbonates are converted to calcium carbonate). The results showed that Cu, Zn, Mn, and Pb significantly exceeded the standard (GB36600-2018). As shown in Table 1 by X-ray fluorescence spectrosecopy (XRF) technique to determine the chemical oxides in the soil are mainly silica, aluminum oxide, calcium oxide, iron trioxide, etc. The results of the electron microprobe show that it contains dolomite, potassium feldspar, plagioclase, muscovite, clinopyroxene, dolomite, garnet, and pyrite. The heavy metal elements in the tailings sand were determined by the toxicity characteristic leaching procedure (TCLP), and the calcium carbonate content was measured according to the excessive acid dissolution method (other carbonates are converted to calcium carbonate). The results showed that Cu, Zn, Mn, and Pb significantly exceeded the standard (GB36600-2018).

Sample Concentration
The objective of the mine tailing immobilization test was to determine the heavy metal immobilization efficiency via MICP at different initial bacterial concentrations and calcium sources. Three experimental settings were designed to examine the effects of calcium source and initial bacteria concentration on Cu, Zn, Mn, and Pb the immobilization efficiency via MICP. The test scheme and sample label is shown in Table 2. The concentration of bacteria was chosen as OD 600 = 0.8, 1.0, 1.2, 1.4, and 1.6, and the concentration of the cementation solution (CS) was 0.5 (Group A), 1.0 (Group B) and 1.5 (Group C) mol/L, respectively. The step-by-step grouting method was used, and the bacterial liquid or cementitious liquid was slowly injected into the sample by a peristaltic pump eight times ( Figure 1). The steps of injections are as follows:(a) 50 mL bacterial suspension was injected from the top to bottom with an inflow rate of about 2 mL/min; (b) 5 mL fixation solution was flushed with the same inflow rate as the bacterial suspension; and (c) 400 mL CS was injected from the top to bottom with an inflow rate of about 0.2 mL/min.

(1) UCS test
In accordance with the standard test method for UCS of the immobilization mine tailings (ASTM D2166/D2166M-16 2016), the as-prepared specimen was assembled in a strain-controlled YHS-2 UCS testing apparatus. The UCS test was then performed at a vertical strain rate of 1%/min. The stress and strain of the specimen were recorded at intervals of 5 s until the specimen failed.
(2) Leaching experiment of simulated atmospheric rainfall To measure the immobilization rate of different ions in tailings, this study verified the heavy metal leaching of samples under atmospheric rainfall. A set of self-made devices was used to simulate the rainfall leaching test [21]. The leachates were collected, and the heavy metal content was measured by atomic absorption spectrometry. Then the removal rate of heavy metal can be calculated by Equation (2).
where Cm is the leaching concentration of heavy metals after solidification and C0 is the leaching concentration of heavy metals in the unsolidified sample.

(1) UCS test
In accordance with the standard test method for UCS of the immobilization mine tailings (ASTM D2166/D2166M-16 2016), the as-prepared specimen was assembled in a strain-controlled YHS-2 UCS testing apparatus. The UCS test was then performed at a vertical strain rate of 1%/min. The stress and strain of the specimen were recorded at intervals of 5 s until the specimen failed.
(2) Leaching experiment of simulated atmospheric rainfall To measure the immobilization rate of different ions in tailings, this study verified the heavy metal leaching of samples under atmospheric rainfall. A set of self-made devices was used to simulate the rainfall leaching test [21]. The leachates were collected, and the heavy metal content was measured by atomic absorption spectrometry. Then the removal rate of heavy metal can be calculated by Equation (2).
where C m is the leaching concentration of heavy metals after solidification and C 0 is the leaching concentration of heavy metals in the unsolidified sample. (

3) XRD and SEM analysis
The control and treated soil samples were analyzed by XRD (Hefei, China) and SEM (Hefei, China) to confirm metal bioimmobilization. XRD patterns were obtained using a Rigaku D/Max-2005 V diffractometer with Cu-Kα scanning from 5 to 80 • 2θ. After natural drying, the samples were crushed and ground using a mortar and pestle before being The UCS increasing rate of group B was the highest at high bacterial concentration, which was much higher than that of groups A and C at the same bacterial concentration. As the CS content provided by group A was relatively low, increasing the bacterial concentration did not significantly improve the curing effect. The high concentration of CS in group C could inhibit bacteria growth. The increasing rate of UCS of the three groups showed a trend of "fast followed by slow" with the increase of bacterial concentration, which demonstrated that the effect of increasing bacterial concentration on improving the curing effect was significant at low CS concentrations, and gradually decreased at high concentrations. Therefore, the curing effect of group B was better than that of groups A and C at high bacterial concentrations. The unconfined compressive strength of the column sample of biocured tailings obtained in this test is 238 kpa higher than that of the sample made by Oliveira et al. (2021) [22].

Calcium Carbonate Content
The output of the carbonate in tailings after solidification was used to measure the strength effect of solidification.
where M 1 and M 2 are the qualities of the sand sample before and after the hydrochloric acid reaction, respectively. The carbonate contents of the tailings sand solidified by different bacterial concentrations and CS are shown in Figure 2b. As can be seen from Figure 2b, the content of calcium carbonate is positively correlated with the concentration of bacterial. When the bacterial concentration was low (OD600 = 0.8), the calcium carbonate content was 18.48-20.5%, and the calcium carbonate increment was between 2.66% and 4.68%. At high bacterial concentration (OD600 = 1.6), the calcium carbonate content reached 23.16-27.27%, and the increment of calcium carbonate reached 7.34-11.45%. With the increase in bacterial concentration, the calcium carbonate content increased gradually, showing first fast and then slow trend. The calcium carbonate content of three groups indicated that CS concentration 1 M was the optimal concentration for solidifying copper tailings. The produced rate of calcium carbonate in groups A and C was slower than that in group B.

Leaching Characteristics
The leaching concentration of heavy metals after solidification of tailings sand is an important index for the evaluation of solidification effect. This test compares the variation characteristics of copper, lead, zinc, and manganese in tailings before and after solidification.

Leaching Characteristics
The leaching concentration of heavy metals after solidification of tailings sand is an important index for the evaluation of solidification effect. This test compares the variation characteristics of copper, lead, zinc, and manganese in tailings before and after solidification.

Copper
The results in Figure 3a illustrated that the solidification effect of Cu was remarkable, and a higher bacterial concentration may promote Cu precipitation. The highest removal rate of copper ion can reach 99.6%. Specifically, when the bacterial concentration increased from 0.8 to 1.0, the solidification curve had a maximal slope. When the bacterial concentration OD 600 was 0.8, the solidification effect of group A (0.5 M) was better than that of group B (1.0 M) and group C (1.5 M). With the increase in bacterial concentration, the leaching concentration of Cu in groups A and B was close to 0 mg/kg, while the effect of group C was higher than 0.5 mg/kg. The results in Figure 3a illustrated that the solidification effect of Cu was remarkable, and a higher bacterial concentration may promote Cu precipitation. The highest removal rate of copper ion can reach 99.6%. Specifically, when the bacterial concentration increased from 0.8 to 1.0, the solidification curve had a maximal slope. When the bacterial concentration OD600 was 0.8, the solidification effect of group A (0.5 M) was better than that of group B (1.0 M) and group C (1.5 M). With the increase in bacterial concentration, the leaching concentration of Cu in groups A and B was close to 0 mg/kg, while the effect of group C was higher than 0.5 mg/kg.

Zinc
It can be seen from Figure 3b that the curing efficiency of zinc ions varies greatly under the conditions of different bacterial solution concentrations and different cement solution concentrations. The highest curing efficiency of group A and group C was 29.8%

Zinc
It can be seen from Figure 3b that the curing efficiency of zinc ions varies greatly under the conditions of different bacterial solution concentrations and different cement solution concentrations. The highest curing efficiency of group A and group C was 29.8% and 40.4%, while that of group B was 95.6%. Previously, the curing efficiency using the same strain was only 68.86% [11].

Manganese
As Figure 3c shows, when the concentration of bacterial solution was 0.8, the curing efficiency of manganese ion in group A was only 14.7%, while that in group C was 81.5%. When the concentration of bacterial solution increased, the solidification efficiency of manganese ion in group B increased the fastest. When the bacterial concentration was relatively high (OD 600 > 1.4), the manganese ion solidification effect of all three groups showed good efficacy. The amount of urea and Ca 2+ provided by 0.5 mol/L was slight, and the coprecipitation effect was weak, so the curing effect was poor. However, high concentration of 1.5 mol/L CS can inhibit bacteria, and the curing effect is not as effective as 1.0 mol/L.

Lead
The concentration of Pb 2+ in group C (1.5 M) was essentially the same as that of the control group at low bacterial concentration (OD 600 = 0.8), while the concentration of Pb 2+ of group A (0.5 M) and group B (1.0 M) was significantly lower, indicating that Pb had a certain solidification effect when the bacteria concentration was low and the CS Crystals 2022, 12, 522 8 of 13 concentration was not excessively high. With the increase in the bacterial concentration, the curing effect improved, especially in group C. At high bacterial concentration (OD 600 = 1.6), the concentration of Pb 2+ of groups A, B, and C was at the same level, indicating that the effect of the Pb 2+ concentration was significantly reduced at high bacterial concentrations.
Three possible reasons were deduced. First, when the concentration of the bacteria was low, the CO 3 2− produced by urease decomposing urea was also low. Therefore, the coprecipitation and adsorption effects were weak, making solidification unsatisfactory. When the concentration was high, the reverse applied. Second, when the bacterial concentration was low, the amount of vaterite produced was also lower. A small amount of vaterite cannot adsorb more Mn 2+ , therefore, increasing the bacterial concentration can increase the production of CaCO 3 , so the removal rate of Mn 2+ obviously improved. Third, the concentration of the CS mainly affects the content of urea and Ca 2+ .

XRD Results
The solidified sample was dried and ground into a powder, and then put into the shl-2 X-ray diffractometer instrument for XRD testing. Three groups of samples were marked as A5, B5 and C5, and the original tailings were denoted as A0.
The wave peaks of A5, B5, and C5 among the original tailings and the solidified tailings are shown in Figure 4, which indicates that new minerals and compounds were formed in the tailings by MICP. Aragonite was found in both A5 and C5, where there was no vaterite in the undisturbed tailings. This proved that vaterite was a newly formed mineral of MICP. Comparing the XRD results of undisturbed tailings with the solidified tailings, it was found that vaterite, zinc carbonate, lead carbonate, manganese dioxide, and basic copper carbonate are newly formed in tailings during the MICP process. These results illustrate that MICP produces calcium carbonate precipitation and precipitates Cu, Zn, Mn, Pb, and other heavy metals.

SEM Results
The SEM results of the A5, B5, and C5 of the tailings after solidification are shown in Figure 5. Figure 5a shows the A5 sample of solidified tailings enlarged by 2000 times. Spear, columnar, and massive minerals adhesions were bonded around the tailings, which could be identified as aragonite and calcite according to the XRD results. Figure 5b illustrates the A5 image sample of solidified tailings enlarged by 5000 times, in which the rod-shaped objects are approximately 2~3 μm in length and 0.5~1.5 μm in diameter at the top, which

SEM Results
The SEM results of the A5, B5, and C5 of the tailings after solidification are shown in Figure 5.

Discussion
The above test results show that MICP technology can effectively improve the strength and stability of tailings and solidify heavy metal ions in tailings. Combined with the macro and micro test results, the mechanism of strength enhancement of tailings and the biochemical reaction mechanism of heavy metal ions in tailings are analyzed in detail.

The Mechanism of MICP Improve Strength
The average particle size (D50) of tailings sand is 0.28 mm, which can ensure the unimpeded transmission of microorganisms and nutrients [23,24]. This test adopts the method of injecting bacterial liquid first and then cementing liquid for grouting, with a  Figure 5a shows the A5 sample of solidified tailings enlarged by 2000 times. Spear, columnar, and massive minerals adhesions were bonded around the tailings, which could be identified as aragonite and calcite according to the XRD results. Figure 5b illustrates the A5 image sample of solidified tailings enlarged by 5000 times, in which the rod-shaped objects are approximately 2~3 µm in length and 0.5~1.5 µm in diameter at the top, which is similar to the shape of Sarcocystis bassiana. Figure 5c,d represent the B5 sample of solidified tailings enlarged by 3000 and 4000 times, respectively. Like Figure 5a,b, many spherical minerals and a small amount of massive minerals were cemented on the surface of the tailings sand particles. Figure 5e illustrates the A5 sample image of solidified tailings enlarged by 3000 times, in which more spherical and flocculent minerals are stacked and bonded together. Figure 5f clearly illustrates that numerous spherical and massive minerals are cemented with tailings.

Discussion
The above test results show that MICP technology can effectively improve the strength and stability of tailings and solidify heavy metal ions in tailings. Combined with the macro and micro test results, the mechanism of strength enhancement of tailings and the biochemical reaction mechanism of heavy metal ions in tailings are analyzed in detail.

The Mechanism of MICP Improve Strength
The average particle size (D50) of tailings sand is 0.28 mm, which can ensure the unimpeded transmission of microorganisms and nutrients [23,24]. This test adopts the method of injecting bacterial liquid first and then cementing liquid for grouting, with a total of eight times. In the process of MICP reaction, a large amount of CaCO 3 (aragonite/vaterite) was newly formed. Vaterite fills the gap of tailing sand and cements the loose tailing sand particles together, so as to improve the strength of tailing sand [4,[25][26][27]. The effect improved with increasing bacterial concentration because: (1) the content of CO 3 2− produced by urease increased with increasing bacterial concentration; and (2) when the bacterial concentration was low, there was not enough vaterite to cement the loose tailings. The CS could provide urea and Ca 2+ , but an excessively high concentration of the CS possibly inhibited the crystal structure of CaCO 3 , which formed more calcite or aragonite and had side effects.

The Biochemical Reaction Mechanism of MICP Solidifying Heavy Metal Ions
From XRD and SEM results, it can be concluded that Cu, Zn, Mn, and Pb in the metal tailings were mainly fixed by MICP through coprecipitation and biosorption by bacteria. Based on the research on the solidification of a single heavy metal (Pb) by MICP, Jiang et al., (2019) [17] and Castro-Alonso et al., (2019) [28] explained the solidification mechanism both abiotically and biologically. The abiotic mechanism refers to the chemical reaction between the heavy metals and some elements in the bacterial or cementation solution to form precipitates, while the biological mechanism refers to the formation of cell-centered precipitates by urea hydrolysis. Based on the solidification mechanism, the hypothesis of multi-layer precipitation structure of various heavy metals was postulated and discussed. The bacterial solution was injected when metal tailings were solidified by MICP. As the pH of the bacterial solution was 9, the free Cu 2+ , Zn 2+ , Mn 2+ , and Pb 2+ in the tailings could form initial abiotic precipitation such as Cu(OH) 2 , Zn(OH) 2 , Mn(OH) 2 , and Pb(OH) 2 under alkaline conditions, which is conducive to acid-base balance. When the cementation solution was added, urea began to hydrolyze, and the initial non-biological precipitation began to transform into biological precipitation, accompanied by other new precipitation. With an increase in the grouting time, Cu(OH) 2 , Zn (OH) 2 , Mn (OH) 2 , and Pb(OH) 2 could be transformed into CuCO 3 , ZnCO 3 , MnCO 3 , and PbCO 3 , respectively. According to the XRD results, Cu 2 (OH) 2 CO 3 and MnO 2 were formed instead of CuCO 3 and MnCO 3 . In accordance with the previous experimental and the XRD results, we deduced that with the increase in bacterial concentration, the main precipitates were: As shown in Figure 6, bacteria are negatively charged and can attract positively charged heavy metal ions. The heavy metal ions were precipitated by MICP and wrapped in the outer layer of the bacteria, forming a biological multi-layer precipitation structure. According to the hypothesis diagrams, the innermost two layers were Cu 2 (OH) 2 CO 3 and MnO 2 , and the outer two layers were ZnCO 3 and PbCO 3 . Cu 2 (OH) 2 CO 3 and MnO 2 were formed before the urea hydrolysis, while ZnCO 3 and PbCO 3 were continuously transformed with the urea hydrolysis. The reason that Cu 2 (OH) 2 CO 3 and MnO 2 did not transform into CuCO 3 and MnCO 3 could be the envelopment of ZnCO 3 and PbCO 3 in the outer layer, which prevented the transformation. Generally, CaCO 3 was first formed and wrapped in the outer layer of biological precipitation, and the outer layer of CaCO 3 could protect the inner layer of heavy metal precipitation from washing or acid dissolution. From the SEM images, we can see that there was considerable calcium carbonate precipitation in the surface layer, and there may be calcium carbonate in the inner layer of multilayer precipitation structure. both abiotically and biologically. The abiotic mechanism refers to the chemical reaction between the heavy metals and some elements in the bacterial or cementation solution to form precipitates, while the biological mechanism refers to the formation of cell-centered precipitates by urea hydrolysis. Based on the solidification mechanism, the hypothesis of multi-layer precipitation structure of various heavy metals was postulated and discussed. The bacterial solution was injected when metal tailings were solidified by MICP. As the pH of the bacterial solution was 9, the free Cu 2+ , Zn 2+ , Mn 2+ , and Pb 2+ in the tailings could form initial abiotic precipitation such as Cu(OH)2, Zn(OH)2, Mn(OH)2, and Pb(OH)2 under alkaline conditions, which is conducive to acid-base balance. When the cementation solution was added, urea began to hydrolyze, and the initial non-biological precipitation began to transform into biological precipitation, accompanied by other new precipitation.
With an increase in the grouting time, Cu(OH)2, Zn (OH)2, Mn (OH)2, and Pb(OH)2 could be transformed into CuCO3, ZnCO3, MnCO3, and PbCO3, respectively. According to the XRD results, Cu2(OH)2CO3 and MnO2 were formed instead of CuCO3 and MnCO3. In accordance with the previous experimental and the XRD results, we deduced that with the increase in bacterial concentration, the main precipitates were: As shown in Figure 6, bacteria are negatively charged and can attract positively charged heavy metal ions. The heavy metal ions were precipitated by MICP and wrapped in the outer layer of the bacteria, forming a biological multi-layer precipitation structure. According to the hypothesis diagrams, the innermost two layers were Cu2(OH)2CO3 and MnO2, and the outer two layers were ZnCO3 and PbCO3. Cu2 (OH)2CO3 and MnO2 were formed before the urea hydrolysis, while ZnCO3 and PbCO3 were continuously transformed with the urea hydrolysis. The reason that Cu2(OH)2CO3 and MnO2 did not transform into CuCO3 and MnCO3 could be the envelopment of ZnCO3 and PbCO3 in the outer layer, which prevented the transformation. Generally, CaCO3 was first formed and wrapped in the outer layer of biological precipitation, and the outer layer of CaCO3 could protect the inner layer of heavy metal precipitation from washing or acid dissolution. From the SEM images, we can see that there was considerable calcium carbonate precipitation in the surface layer, and there may be calcium carbonate in the inner layer of multilayer precipitation structure.

Conclusions
(1) The bacterial and CS concentration were the two key factors of MICP. Within a certain range, there was a positive correlation between bacterial concentration and UCS. For CS, an excessively low concentration could not provide enough urea and calcium, while a high concentration could inhibit bacteria activity. Under experimental conditions, the optimal bacterial concentration OD600 was 1.6, and the optimal concentration of cementation solution was 1.0 mol/L. The corresponding compressive strength was 778 kPa; (2) The concentrations of Cu 2+ , Zn 2+ , Mn 2+ , and Pb 2+ in the solidified tailings decreased during simulated leaching. Both bacterial concentration and cementation solution concentrations could affect the removal rate; (3) The micro mechanism of MICP could be explained by abiotic and biological mechanisms. The hypothesis of the multi-layer precipitation structure of various heavy metals was first postulated and discussed in this study.

Conclusions
(1) The bacterial and CS concentration were the two key factors of MICP. Within a certain range, there was a positive correlation between bacterial concentration and UCS. For CS, an excessively low concentration could not provide enough urea and calcium, while a high concentration could inhibit bacteria activity. Under experimental conditions, the optimal bacterial concentration OD600 was 1.6, and the optimal con-centration of cementation solution was 1.0 mol/L. The corresponding compressive strength was 778 kPa; (2) The concentrations of Cu 2+ , Zn 2+ , Mn 2+ , and Pb 2+ in the solidified tailings decreased during simulated leaching. Both bacterial concentration and cementation solution concentrations could affect the removal rate; (3) The micro mechanism of MICP could be explained by abiotic and biological mechanisms. The hypothesis of the multi-layer precipitation structure of various heavy metals was first postulated and discussed in this study.