# Experimental Study on the Shear Strength of Loess Modified by Microbial Precipitation Technology

Authors: Bing-Xia Wang; Li-Jun Shi; Jian-Hua Dong; Lu Wang; Wen-Tong Tian
Journal: Advances in Materials Science and Engineering
 (2022)

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## Abstract

The shear strength (vertical pressure 50, 100, 200, and 300 kPa) and soil structure of the remolded loess with different proportions of the microbial cementing solution were measured and observed by the direct shear test and scanning electron microscopy (SEM).The results showed: (1) the increase in the microbial cementation solution led to the transformation of the stress-shear displacement curve from strain hardening to strain softening, with the transition interval of 26%∼34%. (2) With the increase in the microbial cementation solution, the change of cohesion showed a trend of “M,” the inflection point of the microbial cementation solution was 14%, 22%, and 34%, while the internal friction angle showed a law of “W,” where the inflection points of the internal friction angle were about 14%, 30%, 34%, and 38%, respectively. In the range of 18%∼30%, the cohesion and internal friction angle increase with an increase in the microbial cementation solution.(3) The change of cohesion is affected by the microbial mineralization saturation and the joint action of the occurrence state of Ca2+ and HCO3- in the microbial cementation solution. The change of internal friction angle is affected by the soil particle contact, the existing form of calcium carbonate formed by microbial precipitation, pore morphology, yield, and other forces. (4) By means of SEM, the distribution morphology of the soil particles and the contact microscopic images of the loess samples modified by microorganisms with different proportions were further verified to further verify the macroscopic changes of the shear strength of the modified loess samples cemented by microorganisms with different proportions. It provides a theoretical basis for the improvement of loess soil structure characteristics by microorganism calcium carbonate precipitation technology.


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## Body

## 1. Introduction

Loess is easily eroded by water and its strength decreases greatly, resulting in subsidence. Therefore, loess cannot meet the engineering requirements generally, and it must be improved before it can be used better. In engineering, soil is often improved by adding materials, such as fiber, composite cement, SH curing agent, calcium lignosulfonate, etc., which improve the strength and stability of the soil to a certain extent. However, the above-mentioned materials to a large extent lead to environmental pollution, waste of natural resources, high energy consumption, and even safety risks [1–3].The modification of the soil structure by the microbial precipitation of calcium carbonate is favored for its ecological, environmental, and economic advantages. The principle is that some products of microbial metabolism react with ions or mixtures in the external environment, resulting in subsequent mineral deposition of byproducts of metabolism, and the microbial mineralization process with carbonate as the product. It is called microbially induced carbonate precipitation (MICP).The research of Harkes et al. [4] shows that the compressive strength of the sand column is closely related to the content of calcite, and there is a linear increasing relationship. The study of Whiffin et al. [5] shows that calcium carbonate deposition induced by microorganism MICP leads to different soil properties. The results of Cui Mingjuan et al. [6] show that the combination of high/low concentration of chemical treatment can make the calcium carbonate produced by microorganisms more evenly distributed in the sample. The specimens with higher unconfined compressive strength and secant modulus of elasticity can be obtained under the condition of less grouting times. The research results of Qian Chunxiang et al. [7] show that the compressive strength, calcite content, permeability coefficient, ultrasonic velocity, and microstructure obtained at the same position of the microbial cement-based materials prepared by the preparation process in the same direction are better than those prepared by the preparation process in different directions. However, the uniformity of the microbial cement-based material prepared by the preparation process in the same direction is worse than that of the microbial cement-based material prepared by the preparation process in different directions [8]. The results of Liang Shihua et al. [9,10] show that for sand with good grain size distribution, the precipitation amount and the uniformity of calcium carbonate are better, and the porosity and permeability coefficient are smaller, which increased the unconfined compressive strength of the sample. Microbial-induced calcite precipitation could effectively improve the shear strength of granite residual soil as well as its internal friction angle and cohesion, Besides, the increase of cohesion was larger than that of the internal friction angle. Studies [11] have also shown that the shear strength of the sand body cemented by microorganisms is significantly improved. The research results of YUE Jian-wei et al. [12] show that MICP by Sporosarcina pasteurii bacteria liquid was used to strengthen silt soil; its cohesive force increased by about 30% and the internal friction angle did not change much; MICP by glutinous rice pulp was used to reinforce silt soil, and the cohesive force increased linearly with an increase in the concentration of glutinous rice pulp, and the internal friction angle firstly increased and then decreased. The results of Wang et al. [13] show that under the same reaction conditions (same time, volume), the shear strength increases at first and then decreases with an increase in nutrient concentration. When the nutrient salt concentration reaches 0.5 mol/L, the shear strength reaches the largest value. At this time, the cohesive force and the internal friction angle of the sample are 15.5 kPa and18.83°, respectively. The content of calcium carbonate increases with nutrient concentration. When the concentration of the nutrient salt reaches 0.7 mol/L, the average calcium carbonate content of the sample increases less. The uniformity of calcium carbonate crystal distribution changes in a convex shape with an increase in the nutrient salt concentration from low to high. The strength of the cemented sample depends on the amount of CaCO3; crystals formed and their distribution. The produced calcite-type calcium carbonate crystals are mainly deposited at the contact position of the particles to form accumulated crystals or filled in the pores to form a “bonding bridge,” which produces a cementation effect and enhances the mechanical properties of the sample. The research results of Tang Chaosheng et al.. [14] show that the MICP treatment by spraying the prepared liquid and the cemented liquid on the surface of the soil sample in turn can significantly improve the structural strength of the loess, and form a layer of high-strength hardening crust on the loess’ surface. At present, the microbial improvement of soil structure is mainly limited to coarse-grained soil such as sand, but the performance characteristics of fine-grained soil, especially modified loess, are rarely reported.Based on the above analysis, this paper takes microbial calcium carbonate-precipitated modified loess samples as the research subject. Through preparing microbial loess remolded samples with different proportions, the direct shear test and scanning electron microscopy (SEM) observations are carried out. The shear strength of the microbial loess samples with different proportions was obtained. By means of SEM images, the relationship between the microstructure and the macroscopic mechanical shear strength of the microbe-modified loess samples is further verified and explained, which provides some theoretical reference for engineering design in modified loess areas.


## 2. Materials and Methods

### 2.1. Physical Properties of Loess

The test soil samples were taken from Jiaziping Mountain, Langongping, Qilihe District, Lanzhou (see Figure1).The depth of the soil was about 5 meters.Figure 1
Loess sampling site pictures. (a) Long-distance site photos of loess sampling. (b) Close-up field photos of loess sampling.
(a)(b)


### 2.2. Sample Preparation

#### 2.2.1. Bacteria and Culture Medium

The bacteria sealed by glycerol need to be expanded. The culture medium is LB broth and urea. The detailed preparation process of bacteria and culture medium is as follows: (1) The bacteria selected in this paper was Sporosarcina pasteurii (ATCC 11859), which was purchased from China Shanghai Conservation Biotechnology Center(SHBCC) (see Figure2(a)). (2)The medium used in the experiment was LB broth and urea, where LB broth was purchased from China Shanghai Bio-way technology Co., Ltd and the formula of the LB broth medium (1L) was tryptone 10g, yeast powder 5g, and sodium chloride 10g (see Figure 2(b)). 3) The triangular flask, stirring rod, and pipette gun head by silver paper had been sterilized at high temperature in the autoclave at 126°C for 30 minutes (see Figure 2(c)). (4) 25g of LB broth was taken out and added to 1000 ml distilled water, and heated until it was all dissolved by stirring (see Figure 2(d)). (5) The dissolved LB was put into the triangular bottle prepared in step (2), wrapped by silver paper, placed in an autoclave, and sterilized at 126°C for 30 minutes (see Figure 2(e)). (6) The dissolved LB broth was cooled to 60°C by sterilization, and 20g filtered sterilized urea was added and stirred to mix it evenly. (7) A certain amount of the Sporosarcina pasteurii sealed with glycerol was sorbed by the pipette and inoculated into the culture medium according to the volume ratio of 1 : 100(see Figure 2(f)). (8) The prepared culture medium and Sporosarcina pasteurii were put into a triangular flask and sealed with perforated plastic film, while they were placed in a constant temperature vibration incubator and shaken horizontally at the frequency of 150r • min−1 in order to expand the culture aerobically for 24 hours at 30°C (see Figure 2(g)). (9) The Sporosarcina pasteurii solution cultured by step (8) was adjusted to OD = 1.5 by adding distilled water. In order to ensure that the experiment is carried out in a sterile environment, the above operations should be carried out on a sterile operation platform or on alcohol as much as possible.Figure 2
Test sample preparation process diagram. (a) Sporosarcina pasteurii sealed with glycerin. (b) LB broth and urea. (c) The instrument sterilized in the autoclave. (d) LB broth dissolved in distilled water. (e) LB broth dissolved in distilled water and sterilized in an autoclave. (f)Sporosarcina pasteurii sealed with glycerol was inoculated into the culture medium. (g) Expanding culture Sporosarcina pasteurii in a constant temperature vibration incubator. (h) The loess samples treated by MICP in an annular 61.8 mm × 20 mm metal mold.
(a)(b)(c)(d)(e)(f)(g)(h)


#### 2.2.2. Nutrient Solution Preparation

The nutrient solution consists of urea, calcium chloride, and LB broth (3g·L−1) to provide urea and calcium ions for the MICP process and sufficient nutrients for the growth and reproduction of bacteria. The ingredients of the LB broth were shown in 1.2.1. Urea (CH4N2O) 60g and calcium chloride anhydrous (CaCl2)110g were added to distilled water 1L for preparing 1 mol/L mixture nutrient solution, while the LB broth 3g was added to the 1L mixture nutrient solution.


#### 2.2.3. Sample Preparation

The preparation process of loess samples modified by the microbial calcium carbonate precipitation technology is as follows:(1)
MICP treatment: Grind the air-dried loess, sieve it through a 2 mm mesh, and mix it evenly. In this test, for loess with low permeability, the mixing process was selected to conduct the MICP treatment. Firstly, equal volumes of Sporosarcina pasteurii solution and the nutrient solution were measured according to the volume ratio of 1 : 1, and their mass was weighed respectively. At the same time, the required loess mass was calculated according to the sum of the mass of the solution containing Sporosarcina pasteurii and the nutrient solution (referred to as the “microbial cementation solution” in the paper), which was 10%, 14%, 18%, 22%, 26%, 30%, 34%, and 38%. Secondly, the Sporosarcina pasteurii solution was stirred and sprayed evenly into the soil, wrapped in a plastic wrap, and stood for 8h, so that the Sporosarcina pasteurii solution could fully infiltrate and colonize the soil. Finally, the soil that had infiltrated the Sporosarcina pasteurii solution was sprayed with the nutrient solution, fully stirred, and the nutrient solution and soil that had the infiltrated Sporosarcina pasteurii solution were fully reacted as far as possible.(2)
Modified loess samples: In order to study the influence of the microbial cementation solution on the shear strength of the modified loess, the loess samples treated by MICP were placed under static pressure in an annular 61.8 mm × 20 mm metal mold (see Figure 2(h)). As is shown in Figure 2(h)), the samples were wrapped in a plastic wrap and left standing for 1d, such that the MICP reaction process was completed as much as possible. [14]show that the MICP treatment by spraying the prepared liquid and the cemented liquid on the surface of the soil sample in turn can significantly improve the structural strength of the loess, and form a layer of high-strength hardening crust on the loess’ surface.Twelve parallel loess samples of the same proportion were assigned to each group, and the average value of the experiment was taken.


### 2.3. Direct Shear Test

The samples of loess modified by the microbial calcium carbonate precipitation technology were subjected to quick shear tests on a strain-controlled shearing apparatus produced in Nanjing Soil Instrument Factory. Vertical pressures were set at 50, 100, 200, and 300 KPa, respectively. The shear rate was set at 0.8 mm/min, and the shear was stopped when the mechanical displacement reached 6 mm. The shear stress and shear displacement are determined according to the Standard for geotechnical testing method [15]:GB/T50123-2019, in which the shear displacement is the difference between the mechanical displacement and the reading of the dynamometer, and the principle for determining the shear strength is as follows: the peak shear stress is taken for the strain-softening curve, and the shear stress corresponding to the shear displacement of 4 mm is taken for the strain-hardening curve.


### 2.4. SEM Microstructure Characteristics of Microbial-Modified Loess Samples

In this paper, the SEM microstructure characteristics of the samples with different proportions were observed using ThermoFisher Apreo electron microscope. After the direct shear test, samples of microorganism-modified loess with different proportions wrapped in a plastic film were broken to a size less than 1 cm3. The relatively smooth fresh surface was gilded with an ion sputtering instrument, and then the SEM observation was carried out immediately.


## 2.1. Physical Properties of Loess

The test soil samples were taken from Jiaziping Mountain, Langongping, Qilihe District, Lanzhou (see Figure1).The depth of the soil was about 5 meters.Figure 1
Loess sampling site pictures. (a) Long-distance site photos of loess sampling. (b) Close-up field photos of loess sampling.
(a)(b)


## 2.2. Sample Preparation

### 2.2.1. Bacteria and Culture Medium

The bacteria sealed by glycerol need to be expanded. The culture medium is LB broth and urea. The detailed preparation process of bacteria and culture medium is as follows: (1) The bacteria selected in this paper was Sporosarcina pasteurii (ATCC 11859), which was purchased from China Shanghai Conservation Biotechnology Center(SHBCC) (see Figure2(a)). (2)The medium used in the experiment was LB broth and urea, where LB broth was purchased from China Shanghai Bio-way technology Co., Ltd and the formula of the LB broth medium (1L) was tryptone 10g, yeast powder 5g, and sodium chloride 10g (see Figure 2(b)). 3) The triangular flask, stirring rod, and pipette gun head by silver paper had been sterilized at high temperature in the autoclave at 126°C for 30 minutes (see Figure 2(c)). (4) 25g of LB broth was taken out and added to 1000 ml distilled water, and heated until it was all dissolved by stirring (see Figure 2(d)). (5) The dissolved LB was put into the triangular bottle prepared in step (2), wrapped by silver paper, placed in an autoclave, and sterilized at 126°C for 30 minutes (see Figure 2(e)). (6) The dissolved LB broth was cooled to 60°C by sterilization, and 20g filtered sterilized urea was added and stirred to mix it evenly. (7) A certain amount of the Sporosarcina pasteurii sealed with glycerol was sorbed by the pipette and inoculated into the culture medium according to the volume ratio of 1 : 100(see Figure 2(f)). (8) The prepared culture medium and Sporosarcina pasteurii were put into a triangular flask and sealed with perforated plastic film, while they were placed in a constant temperature vibration incubator and shaken horizontally at the frequency of 150r • min−1 in order to expand the culture aerobically for 24 hours at 30°C (see Figure 2(g)). (9) The Sporosarcina pasteurii solution cultured by step (8) was adjusted to OD = 1.5 by adding distilled water. In order to ensure that the experiment is carried out in a sterile environment, the above operations should be carried out on a sterile operation platform or on alcohol as much as possible.Figure 2
Test sample preparation process diagram. (a) Sporosarcina pasteurii sealed with glycerin. (b) LB broth and urea. (c) The instrument sterilized in the autoclave. (d) LB broth dissolved in distilled water. (e) LB broth dissolved in distilled water and sterilized in an autoclave. (f)Sporosarcina pasteurii sealed with glycerol was inoculated into the culture medium. (g) Expanding culture Sporosarcina pasteurii in a constant temperature vibration incubator. (h) The loess samples treated by MICP in an annular 61.8 mm × 20 mm metal mold.
(a)(b)(c)(d)(e)(f)(g)(h)


### 2.2.2. Nutrient Solution Preparation

The nutrient solution consists of urea, calcium chloride, and LB broth (3g·L−1) to provide urea and calcium ions for the MICP process and sufficient nutrients for the growth and reproduction of bacteria. The ingredients of the LB broth were shown in 1.2.1. Urea (CH4N2O) 60g and calcium chloride anhydrous (CaCl2)110g were added to distilled water 1L for preparing 1 mol/L mixture nutrient solution, while the LB broth 3g was added to the 1L mixture nutrient solution.


### 2.2.3. Sample Preparation

The preparation process of loess samples modified by the microbial calcium carbonate precipitation technology is as follows:(1)
MICP treatment: Grind the air-dried loess, sieve it through a 2 mm mesh, and mix it evenly. In this test, for loess with low permeability, the mixing process was selected to conduct the MICP treatment. Firstly, equal volumes of Sporosarcina pasteurii solution and the nutrient solution were measured according to the volume ratio of 1 : 1, and their mass was weighed respectively. At the same time, the required loess mass was calculated according to the sum of the mass of the solution containing Sporosarcina pasteurii and the nutrient solution (referred to as the “microbial cementation solution” in the paper), which was 10%, 14%, 18%, 22%, 26%, 30%, 34%, and 38%. Secondly, the Sporosarcina pasteurii solution was stirred and sprayed evenly into the soil, wrapped in a plastic wrap, and stood for 8h, so that the Sporosarcina pasteurii solution could fully infiltrate and colonize the soil. Finally, the soil that had infiltrated the Sporosarcina pasteurii solution was sprayed with the nutrient solution, fully stirred, and the nutrient solution and soil that had the infiltrated Sporosarcina pasteurii solution were fully reacted as far as possible.(2)
Modified loess samples: In order to study the influence of the microbial cementation solution on the shear strength of the modified loess, the loess samples treated by MICP were placed under static pressure in an annular 61.8 mm × 20 mm metal mold (see Figure 2(h)). As is shown in Figure 2(h)), the samples were wrapped in a plastic wrap and left standing for 1d, such that the MICP reaction process was completed as much as possible. [14]show that the MICP treatment by spraying the prepared liquid and the cemented liquid on the surface of the soil sample in turn can significantly improve the structural strength of the loess, and form a layer of high-strength hardening crust on the loess’ surface.Twelve parallel loess samples of the same proportion were assigned to each group, and the average value of the experiment was taken.


## 2.2.1. Bacteria and Culture Medium

The bacteria sealed by glycerol need to be expanded. The culture medium is LB broth and urea. The detailed preparation process of bacteria and culture medium is as follows: (1) The bacteria selected in this paper was Sporosarcina pasteurii (ATCC 11859), which was purchased from China Shanghai Conservation Biotechnology Center(SHBCC) (see Figure2(a)). (2)The medium used in the experiment was LB broth and urea, where LB broth was purchased from China Shanghai Bio-way technology Co., Ltd and the formula of the LB broth medium (1L) was tryptone 10g, yeast powder 5g, and sodium chloride 10g (see Figure 2(b)). 3) The triangular flask, stirring rod, and pipette gun head by silver paper had been sterilized at high temperature in the autoclave at 126°C for 30 minutes (see Figure 2(c)). (4) 25g of LB broth was taken out and added to 1000 ml distilled water, and heated until it was all dissolved by stirring (see Figure 2(d)). (5) The dissolved LB was put into the triangular bottle prepared in step (2), wrapped by silver paper, placed in an autoclave, and sterilized at 126°C for 30 minutes (see Figure 2(e)). (6) The dissolved LB broth was cooled to 60°C by sterilization, and 20g filtered sterilized urea was added and stirred to mix it evenly. (7) A certain amount of the Sporosarcina pasteurii sealed with glycerol was sorbed by the pipette and inoculated into the culture medium according to the volume ratio of 1 : 100(see Figure 2(f)). (8) The prepared culture medium and Sporosarcina pasteurii were put into a triangular flask and sealed with perforated plastic film, while they were placed in a constant temperature vibration incubator and shaken horizontally at the frequency of 150r • min−1 in order to expand the culture aerobically for 24 hours at 30°C (see Figure 2(g)). (9) The Sporosarcina pasteurii solution cultured by step (8) was adjusted to OD = 1.5 by adding distilled water. In order to ensure that the experiment is carried out in a sterile environment, the above operations should be carried out on a sterile operation platform or on alcohol as much as possible.Figure 2
Test sample preparation process diagram. (a) Sporosarcina pasteurii sealed with glycerin. (b) LB broth and urea. (c) The instrument sterilized in the autoclave. (d) LB broth dissolved in distilled water. (e) LB broth dissolved in distilled water and sterilized in an autoclave. (f)Sporosarcina pasteurii sealed with glycerol was inoculated into the culture medium. (g) Expanding culture Sporosarcina pasteurii in a constant temperature vibration incubator. (h) The loess samples treated by MICP in an annular 61.8 mm × 20 mm metal mold.
(a)(b)(c)(d)(e)(f)(g)(h)


## 2.2.2. Nutrient Solution Preparation

The nutrient solution consists of urea, calcium chloride, and LB broth (3g·L−1) to provide urea and calcium ions for the MICP process and sufficient nutrients for the growth and reproduction of bacteria. The ingredients of the LB broth were shown in 1.2.1. Urea (CH4N2O) 60g and calcium chloride anhydrous (CaCl2)110g were added to distilled water 1L for preparing 1 mol/L mixture nutrient solution, while the LB broth 3g was added to the 1L mixture nutrient solution.


## 2.2.3. Sample Preparation

The preparation process of loess samples modified by the microbial calcium carbonate precipitation technology is as follows:(1)
MICP treatment: Grind the air-dried loess, sieve it through a 2 mm mesh, and mix it evenly. In this test, for loess with low permeability, the mixing process was selected to conduct the MICP treatment. Firstly, equal volumes of Sporosarcina pasteurii solution and the nutrient solution were measured according to the volume ratio of 1 : 1, and their mass was weighed respectively. At the same time, the required loess mass was calculated according to the sum of the mass of the solution containing Sporosarcina pasteurii and the nutrient solution (referred to as the “microbial cementation solution” in the paper), which was 10%, 14%, 18%, 22%, 26%, 30%, 34%, and 38%. Secondly, the Sporosarcina pasteurii solution was stirred and sprayed evenly into the soil, wrapped in a plastic wrap, and stood for 8h, so that the Sporosarcina pasteurii solution could fully infiltrate and colonize the soil. Finally, the soil that had infiltrated the Sporosarcina pasteurii solution was sprayed with the nutrient solution, fully stirred, and the nutrient solution and soil that had the infiltrated Sporosarcina pasteurii solution were fully reacted as far as possible.(2)
Modified loess samples: In order to study the influence of the microbial cementation solution on the shear strength of the modified loess, the loess samples treated by MICP were placed under static pressure in an annular 61.8 mm × 20 mm metal mold (see Figure 2(h)). As is shown in Figure 2(h)), the samples were wrapped in a plastic wrap and left standing for 1d, such that the MICP reaction process was completed as much as possible. [14]show that the MICP treatment by spraying the prepared liquid and the cemented liquid on the surface of the soil sample in turn can significantly improve the structural strength of the loess, and form a layer of high-strength hardening crust on the loess’ surface.Twelve parallel loess samples of the same proportion were assigned to each group, and the average value of the experiment was taken.


## 2.3. Direct Shear Test

The samples of loess modified by the microbial calcium carbonate precipitation technology were subjected to quick shear tests on a strain-controlled shearing apparatus produced in Nanjing Soil Instrument Factory. Vertical pressures were set at 50, 100, 200, and 300 KPa, respectively. The shear rate was set at 0.8 mm/min, and the shear was stopped when the mechanical displacement reached 6 mm. The shear stress and shear displacement are determined according to the Standard for geotechnical testing method [15]:GB/T50123-2019, in which the shear displacement is the difference between the mechanical displacement and the reading of the dynamometer, and the principle for determining the shear strength is as follows: the peak shear stress is taken for the strain-softening curve, and the shear stress corresponding to the shear displacement of 4 mm is taken for the strain-hardening curve.


## 2.4. SEM Microstructure Characteristics of Microbial-Modified Loess Samples

In this paper, the SEM microstructure characteristics of the samples with different proportions were observed using ThermoFisher Apreo electron microscope. After the direct shear test, samples of microorganism-modified loess with different proportions wrapped in a plastic film were broken to a size less than 1 cm3. The relatively smooth fresh surface was gilded with an ion sputtering instrument, and then the SEM observation was carried out immediately.


## 3. Test and Result Analysis

### 3.1. Shape of the Stress-Strain Curve

The direct shear test results of microbe-modified loess samples with different ratios are shown in Figure3. The morphological characteristics of shear stress-shear displacement curve are related to both the content of the microbial cementation solution and the vertical pressure. When the amount of the microbial cement is 38% (see Figure 3(h)), the curves show strain-softening characteristics at all levels of pressure. When the microorganism cementation solution is 34%, the curves show strain hardening at higher pressures (200 kPa and 300 kPa), and strain softening at lower pressures (50 kPa and 100 kPa) (see Figure 3(g)). When the microbial cementation solution reaches 30%, the strain-softening characteristic appears only at the pressure of 50 kPa, and the strain-hardening characteristic appears at the pressure of 100 kPa and above (see Figure 3(f)). When the microbial cementation solution reaches 26%, it shows slight strain softening at the pressure of 50 Pa, and strain hardening at other pressures (see Figure 3(e)). After that, when the microbial cementation solution was 22%, 18%, 14%, and 10%, the samples all showed strain-hardening characteristics (see Figures 3(a)∼3(d)). The above studies showed that the failure mode changed from brittle to ductile with the change in the microbial cementation solution from high to low, and the transition zone was 26%–34% (see Figures 3(e)∼3(g)). In order to more clearly describe the transition process, the shear stress-shear displacement curves of microbial cementing loess samples with different ratios at 100 kPa pressure were selected (see Figure 4), which can be seen from Figure 4. At a pressure of 100 kPa, the microbial cementation solution is 38%, and the peak shear stress (15 kPa) appears at a shear displacement of 3.2 mm (see Figure 4). The peak shear stress (47 kPa) appeared at the shear displacement of 2.8 mm when the microbial cementation solution was 34% (see Figure 4). However, when the microbial cementation solution reached 30%, 26%, 22%, 18%, 14%, and 10%, the curve began to show strain hardening, and the shear stress basically increased with an increase in shear displacement (see Figure 4).Figure 3
Shear stress-shear displacement curves of the microbial cementation solution-modified loess under different ratios. (a) 10%. (b) 14%. (c) 18%. (d) 22%. (e) 26%. (f) 30%. (g) 34%. (h) 38%.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 4
Shear stress-shear displacement curves of the microbial cementation solution-modified loess samples with different ratios under vertical pressure of 100 mkPa.


### 3.2. Shear Strength and Its Indexes

The shear strength and the curve-fitting tables of the samples at different ratios are shown in Figure5 and Table 1. According to the results in Figure 5 and Mohr–Coulomb law, the shear strength characteristics of the remolded loess samples with different microbial cementation solution cements were obtained from the shear stress-shear displacement curve, and the cohesion and internal friction angle were calculated. The results are shown in Figures 6(a) ∼ 6(b). According to Figures 6(a) ∼ 6(b), the trend of cohesion and the internal friction angle varies with an increase in the microbial cementation solution. Cohesion and internal friction angle show four stages with obvious inflection points. As far as the test results are concerned, the inflection points of the change trend of cohesion C are about 14%, 22%, and 34%, respectively (see Figure 6(a)) and the inflection points of the change trend of the internal friction angle are about 14%, 30%, and 34%, respectively (see Figure 6(b)). In the range of less than 14%, the increase in the microbial cementation solution increases the cohesion, and its peak value is 23.3643, which increases by three times (7.92⟶ 23.3643 kPa) (see Figure 6(a)), while the internal friction angle decreases to 0.3305, which decreases by 81% (27°⟶0.3305°) (see Figure 6(b)). In the range of 14%∼22%, the cohesion decreases to 17.7306 with the increase in the microbial cementation solution (see Figure 6(a)), and in the range of 22%∼34%, the cohesion increases to 24.0512 with the increase in the microbial cementation solution (see Figure 6(a)). In the range of 14%∼30%, the internal friction angle increases to 19.305 (0.3305⟶19.305) with an increase in the microbial cementation solution (see Figure 6(b)). In the range of 30%∼34%, the internal friction angle decreases sharply with an increase in the microbial cementation solution (see Figure 6(b)). In the range of 34%∼38%, the cohesion force and internal friction angle change opposite with an increase in the microbial cementation solution, i.e., the cohesion decreases (see Figure 6(a)), but the internal friction angle increases (see Figure 6(b)).Figure 5
Variation of shear strength of loess samples with different microbial cementation solution ratios under vertical pressure.Table 1
Linear fitting of vertical pressure and shear strength of loess specimen with different ratios of the microbial cementation solution.
Ratio of microbial cementation solution (%)Linear fittingR correlation coefficient10y = 0.377x+10.034R2 = 0.984814y = 0.432x+0.3305R2 = 0.906318y = 0.4246x+6.3814R2 = 0.99822y = 0.3244x+12.334R2 = 0.983926y = 0.3603x+13.4957R2 = 0.987930y = 0.3797x+19.305R2 = 0.990534y = 0.4463x+3.3559R2 = 0.998938y = 0.0237x+12.875R2 = 0.1064Figure 6
The change of the shear index with the content of the microbial cement solution. (a) The change of cohesion with the content of the microbial cement solution. (b) The change of the internal friction angle with the content of the microbial cement solution.
(a)(b)As shown in Figure6(a), the cohesion changes in a gentle “M” shape with an increase in the microbial cementation solution. In other words, the cohesion C increases with an increase in the microbial cementation solution at 10%∼14% (see Figure 6(a). The process of microbial mineralization of calcium carbonate minerals involves phase change kinetics. If the solution is in the unsaturated state, i.e., S < 1, the solid in the system tends to dissolve. The increasing concentration of the microbial cementation solution fills the soil particles, which increases the cohesion of C. If the solid-liquid phase is in equilibrium, i.e., S = 1, the concentration of microorganism cementation increases while the cohesion C decreases with the microbial cementation solution increasing from 14% to 22% (see Figure 6(a), which is consistent with the results of the literature [11]. The reason is that the increase in the concentration of Ca2+ and HCO3- in the soil has a significant weakening effect on the original and inherent bonding forces of the soil [16]. When the microbial cementation solution was between 22% and 34%, and once the system is in the state of supersaturation, i.e., S > 1, it tends to crystallize and grow. With the increase in the microbial cementation solution, microbial calcium carbonate precipitates between and on the surface of soil particles, consolidating the soil particles together, and the cohesion gradually increases to the peak value (see Figure 6(a)). When the microbial cementation solution is between 34% and 38%, the cohesion drops sharply (see Figure 6(a). Although calcium carbonate is formed in the supersaturated state and the soil particles and the surface form a whole, after the addition of the microbial cementation solution, i.e., the liquid phase is excessive, there are more Ca2+ and HCO3−, and thus the cohesion decreases. When the microbial cement is between 22% and 34%, the change trend of cohesion is consistent with the results of reference [12], but the change trend of the microbial cement in other intervals still needs further study.Figure6(b) shows that the internal friction angle changes in a “W” shape with an increase in the microbial cementation solution. As shown in Figure 6(b), the change of the internal friction angle with the microbial cementation solution can be divided into four stages, namely 10%∼14%, 14%∼30%, 30%∼34%, and 34%∼38%, and the content of the microbial cementation solution at the inflection point is 14%, 30%, and 34%, respectively. The inflection points of 14% and 34% of the microbial cementation solution are the same as the inflection points of the cohesive microorganism cementation solution. Loess is a cohesive soil, and the change mechanism of its internal friction angle is relatively complex. The internal friction angle of loess is mainly derived from two parts, one from the friction between particles, the other from the attraction of cohesion. According to the analysis of the experimental results: When the content of the microbial cementation solution is small, i.e., 10∼14%, in the loess sample, the clay particles of the loess are strongly linked by water, resulting in large soil particles, which are relatively difficult to move or break up between particles when subjected to shear, and the internal friction angle is large (see Figure 6(b)). When a small amount of microbial cementation solution is added, the internal friction angle decreases (see Figure 6(b)). The results of this experiment are related to soil particle microbial cementation, while the factors affecting soil particle cementation include the content and distribution of microbial-induced calcium carbonate precipitation, the formation of calcium carbonate crystals, the type and concentration of bacteria, and the activity of enzymes, calcium sources, and medium components , pH value, temperature, molding process and chemical treatment, and other factors. Therefore, adding a small amount of the microbial cementation solution is not enough for the nucleation and growth of calcium carbonate formation; thus, effectively cementing loess particles increases the internal friction angle. A small amount of microbial cementing solution cannot meet the conditions for the nucleation and growth of calcium carbonate crystals; that is, it is not enough to effectively cement loess particles and increase the internal friction angle. However, a small amount of microbial cementing liquid can play a lubricating role, remove soil particles, and block the bound water between soil particles, resulting in a decrease in the internal friction angle. In addition, although a certain thickness of calcite can be generated around the particles, and the soil particles are connected by calcite carbonate, the connection effect is poor, and the performance improvement is not obvious. When the content is 14%∼30%, with the increase in the microbial cementation solution, the calcium carbonate formed gradually increases (see Figure 6(b)) and distributes at the Adam's apple of the pores, forming a good “bridge”; it then fills the whole pores, further strengthening the fixation effect on soil particles, and increasing the internal friction angle [17]. When the content is 30%∼34%, the soil particles are consolidated into a whole; when more microbial cement solution is added, the nucleation point of the calcium carbonate grains becomes insufficient. Excess microbial cement solution plays a lubrication role on the shear action, and the viscosity resistance decreases, leading to a decrease in the internal friction angle again (see Figure 6(b)); when the microbial cement changes between 14% and 34%, the internal friction first increases and then decreases. This trend of change is similar to that of [13]. At 34%∼38%, calcium carbonate crystals can be formed and grow with a further increase in the microorganism cementation solution and reach dynamic saturation; the internal friction angle also increases (see Figure 6(b)), but the phenomenon needs further study.


## 3.1. Shape of the Stress-Strain Curve

The direct shear test results of microbe-modified loess samples with different ratios are shown in Figure3. The morphological characteristics of shear stress-shear displacement curve are related to both the content of the microbial cementation solution and the vertical pressure. When the amount of the microbial cement is 38% (see Figure 3(h)), the curves show strain-softening characteristics at all levels of pressure. When the microorganism cementation solution is 34%, the curves show strain hardening at higher pressures (200 kPa and 300 kPa), and strain softening at lower pressures (50 kPa and 100 kPa) (see Figure 3(g)). When the microbial cementation solution reaches 30%, the strain-softening characteristic appears only at the pressure of 50 kPa, and the strain-hardening characteristic appears at the pressure of 100 kPa and above (see Figure 3(f)). When the microbial cementation solution reaches 26%, it shows slight strain softening at the pressure of 50 Pa, and strain hardening at other pressures (see Figure 3(e)). After that, when the microbial cementation solution was 22%, 18%, 14%, and 10%, the samples all showed strain-hardening characteristics (see Figures 3(a)∼3(d)). The above studies showed that the failure mode changed from brittle to ductile with the change in the microbial cementation solution from high to low, and the transition zone was 26%–34% (see Figures 3(e)∼3(g)). In order to more clearly describe the transition process, the shear stress-shear displacement curves of microbial cementing loess samples with different ratios at 100 kPa pressure were selected (see Figure 4), which can be seen from Figure 4. At a pressure of 100 kPa, the microbial cementation solution is 38%, and the peak shear stress (15 kPa) appears at a shear displacement of 3.2 mm (see Figure 4). The peak shear stress (47 kPa) appeared at the shear displacement of 2.8 mm when the microbial cementation solution was 34% (see Figure 4). However, when the microbial cementation solution reached 30%, 26%, 22%, 18%, 14%, and 10%, the curve began to show strain hardening, and the shear stress basically increased with an increase in shear displacement (see Figure 4).Figure 3
Shear stress-shear displacement curves of the microbial cementation solution-modified loess under different ratios. (a) 10%. (b) 14%. (c) 18%. (d) 22%. (e) 26%. (f) 30%. (g) 34%. (h) 38%.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 4
Shear stress-shear displacement curves of the microbial cementation solution-modified loess samples with different ratios under vertical pressure of 100 mkPa.


## 3.2. Shear Strength and Its Indexes

The shear strength and the curve-fitting tables of the samples at different ratios are shown in Figure5 and Table 1. According to the results in Figure 5 and Mohr–Coulomb law, the shear strength characteristics of the remolded loess samples with different microbial cementation solution cements were obtained from the shear stress-shear displacement curve, and the cohesion and internal friction angle were calculated. The results are shown in Figures 6(a) ∼ 6(b). According to Figures 6(a) ∼ 6(b), the trend of cohesion and the internal friction angle varies with an increase in the microbial cementation solution. Cohesion and internal friction angle show four stages with obvious inflection points. As far as the test results are concerned, the inflection points of the change trend of cohesion C are about 14%, 22%, and 34%, respectively (see Figure 6(a)) and the inflection points of the change trend of the internal friction angle are about 14%, 30%, and 34%, respectively (see Figure 6(b)). In the range of less than 14%, the increase in the microbial cementation solution increases the cohesion, and its peak value is 23.3643, which increases by three times (7.92⟶ 23.3643 kPa) (see Figure 6(a)), while the internal friction angle decreases to 0.3305, which decreases by 81% (27°⟶0.3305°) (see Figure 6(b)). In the range of 14%∼22%, the cohesion decreases to 17.7306 with the increase in the microbial cementation solution (see Figure 6(a)), and in the range of 22%∼34%, the cohesion increases to 24.0512 with the increase in the microbial cementation solution (see Figure 6(a)). In the range of 14%∼30%, the internal friction angle increases to 19.305 (0.3305⟶19.305) with an increase in the microbial cementation solution (see Figure 6(b)). In the range of 30%∼34%, the internal friction angle decreases sharply with an increase in the microbial cementation solution (see Figure 6(b)). In the range of 34%∼38%, the cohesion force and internal friction angle change opposite with an increase in the microbial cementation solution, i.e., the cohesion decreases (see Figure 6(a)), but the internal friction angle increases (see Figure 6(b)).Figure 5
Variation of shear strength of loess samples with different microbial cementation solution ratios under vertical pressure.Table 1
Linear fitting of vertical pressure and shear strength of loess specimen with different ratios of the microbial cementation solution.
Ratio of microbial cementation solution (%)Linear fittingR correlation coefficient10y = 0.377x+10.034R2 = 0.984814y = 0.432x+0.3305R2 = 0.906318y = 0.4246x+6.3814R2 = 0.99822y = 0.3244x+12.334R2 = 0.983926y = 0.3603x+13.4957R2 = 0.987930y = 0.3797x+19.305R2 = 0.990534y = 0.4463x+3.3559R2 = 0.998938y = 0.0237x+12.875R2 = 0.1064Figure 6
The change of the shear index with the content of the microbial cement solution. (a) The change of cohesion with the content of the microbial cement solution. (b) The change of the internal friction angle with the content of the microbial cement solution.
(a)(b)As shown in Figure6(a), the cohesion changes in a gentle “M” shape with an increase in the microbial cementation solution. In other words, the cohesion C increases with an increase in the microbial cementation solution at 10%∼14% (see Figure 6(a). The process of microbial mineralization of calcium carbonate minerals involves phase change kinetics. If the solution is in the unsaturated state, i.e., S < 1, the solid in the system tends to dissolve. The increasing concentration of the microbial cementation solution fills the soil particles, which increases the cohesion of C. If the solid-liquid phase is in equilibrium, i.e., S = 1, the concentration of microorganism cementation increases while the cohesion C decreases with the microbial cementation solution increasing from 14% to 22% (see Figure 6(a), which is consistent with the results of the literature [11]. The reason is that the increase in the concentration of Ca2+ and HCO3- in the soil has a significant weakening effect on the original and inherent bonding forces of the soil [16]. When the microbial cementation solution was between 22% and 34%, and once the system is in the state of supersaturation, i.e., S > 1, it tends to crystallize and grow. With the increase in the microbial cementation solution, microbial calcium carbonate precipitates between and on the surface of soil particles, consolidating the soil particles together, and the cohesion gradually increases to the peak value (see Figure 6(a)). When the microbial cementation solution is between 34% and 38%, the cohesion drops sharply (see Figure 6(a). Although calcium carbonate is formed in the supersaturated state and the soil particles and the surface form a whole, after the addition of the microbial cementation solution, i.e., the liquid phase is excessive, there are more Ca2+ and HCO3−, and thus the cohesion decreases. When the microbial cement is between 22% and 34%, the change trend of cohesion is consistent with the results of reference [12], but the change trend of the microbial cement in other intervals still needs further study.Figure6(b) shows that the internal friction angle changes in a “W” shape with an increase in the microbial cementation solution. As shown in Figure 6(b), the change of the internal friction angle with the microbial cementation solution can be divided into four stages, namely 10%∼14%, 14%∼30%, 30%∼34%, and 34%∼38%, and the content of the microbial cementation solution at the inflection point is 14%, 30%, and 34%, respectively. The inflection points of 14% and 34% of the microbial cementation solution are the same as the inflection points of the cohesive microorganism cementation solution. Loess is a cohesive soil, and the change mechanism of its internal friction angle is relatively complex. The internal friction angle of loess is mainly derived from two parts, one from the friction between particles, the other from the attraction of cohesion. According to the analysis of the experimental results: When the content of the microbial cementation solution is small, i.e., 10∼14%, in the loess sample, the clay particles of the loess are strongly linked by water, resulting in large soil particles, which are relatively difficult to move or break up between particles when subjected to shear, and the internal friction angle is large (see Figure 6(b)). When a small amount of microbial cementation solution is added, the internal friction angle decreases (see Figure 6(b)). The results of this experiment are related to soil particle microbial cementation, while the factors affecting soil particle cementation include the content and distribution of microbial-induced calcium carbonate precipitation, the formation of calcium carbonate crystals, the type and concentration of bacteria, and the activity of enzymes, calcium sources, and medium components , pH value, temperature, molding process and chemical treatment, and other factors. Therefore, adding a small amount of the microbial cementation solution is not enough for the nucleation and growth of calcium carbonate formation; thus, effectively cementing loess particles increases the internal friction angle. A small amount of microbial cementing solution cannot meet the conditions for the nucleation and growth of calcium carbonate crystals; that is, it is not enough to effectively cement loess particles and increase the internal friction angle. However, a small amount of microbial cementing liquid can play a lubricating role, remove soil particles, and block the bound water between soil particles, resulting in a decrease in the internal friction angle. In addition, although a certain thickness of calcite can be generated around the particles, and the soil particles are connected by calcite carbonate, the connection effect is poor, and the performance improvement is not obvious. When the content is 14%∼30%, with the increase in the microbial cementation solution, the calcium carbonate formed gradually increases (see Figure 6(b)) and distributes at the Adam's apple of the pores, forming a good “bridge”; it then fills the whole pores, further strengthening the fixation effect on soil particles, and increasing the internal friction angle [17]. When the content is 30%∼34%, the soil particles are consolidated into a whole; when more microbial cement solution is added, the nucleation point of the calcium carbonate grains becomes insufficient. Excess microbial cement solution plays a lubrication role on the shear action, and the viscosity resistance decreases, leading to a decrease in the internal friction angle again (see Figure 6(b)); when the microbial cement changes between 14% and 34%, the internal friction first increases and then decreases. This trend of change is similar to that of [13]. At 34%∼38%, calcium carbonate crystals can be formed and grow with a further increase in the microorganism cementation solution and reach dynamic saturation; the internal friction angle also increases (see Figure 6(b)), but the phenomenon needs further study.


## 4. SEM Microstructure Characteristics

In order to further study the shear properties of microbe-modified loess samples with different proportions, the relationship between the microbe-modified loess samples with different proportions and its shear strength was established. In this paper, the basic physical property indexes of loess samples are shown in Table2 and the grain size composition is shown in Table 3; the SEM microstructure characteristics of samples with different proportions were observed using ThermoFisher Apreo electron microscope. The variation of the shear strength was explained by the microstructure of the microbe-modified loess samples with different proportions. The SEM images of each sample with a magnification of 200, 300, 500, 1000, and 1500 were obtained. After comparison, the original image size was 2048 pixels ×2 048 pixels. Due to the limitation of space, typical original loess was selected for study at 1000 times magnification. The structural types, contact conditions, and pore morphology of the soil all changed.Table 2
Physical index of undisturbed loess.
Dry densityρd/g⋅cm−3Moisture contentω/%Liquid limitWL/%Plastic limitWP/%Plasticity indexIPVoid ratioe1.601.4830.8917.1512.870.78Table 3
Grading of undisturbed loess.
Granularity groupingSandPowder particleClay particlePercentage11.571.217.3Figure7 shows that the ratio of the microbial cementation solution has a significant effect on the soil structure of loess samples. Due to space limitations, the SEM photographs of microbial cementation at 18% were omitted. Figure 7(a) shows the original specimen. Figures 7(b)–7(h) show photographs of specimen at 10%, 14%, 22%, 26%, 30%, 34%, and 38%, respectively. When the microbial cementation solution is from 18% to 30%, it forms calcium carbonate to cover the surface and a “bridge” between particles, which is consistent with the phenomenon in [11]. The pores are relatively small, and the internal friction angle gradually increases. This soil structure contributes greatly to its strength (see Figures 7(d) and 7(f)). When the microbial cementation solution is from 34% to 38%, calcium carbonate is formed to wrap the soil, blocking the nutrients needed by microorganisms, and microorganisms gradually die. At the same time, there are many holes in the spherical calcite. Therefore, the internal friction angle decreases [18]. With the further increase of microorganisms, the particle-bound water film is thickened, and the internal friction angle also increases (see Figures 7(g) and 7(h)).Figure 7
7 SEM images of loess samples modified by different proportions of the microbial cementation solution. (a) The original specimen. (b) 10%. (c) 14%. (d) 22%. (e) 26%. (f) 30%. (g) 34%. (h) 38%.
(a)(b)(c)(d)(e)(f)(g)(h)


## 5. Conclusions

(1)
The increase in the microbial cementation solution will lead to the transformation of the shear-stress displacement curve from strain hardening to strain softening, and the transition range is 26% ∼ 34%.(2)
With the increase in the microbial cementation solution, the internal friction angle showed a law of “W,” the inflection points of the internal friction angle were about 14%, 30%, 34%, and 38%, while the change of cohesion showed a trend of “M,” the inflection point of the microbial cementation solution was 14%, 22%, and 34%, respectively. In the range of 18%∼30%, the cohesion and internal friction angle increase with an increase in the microbial cementation solution.(3)
The change of cohesion is affected by the microbial mineralization saturation and the joint action of the occurrence state of Ca2+ and HCO3- in the microbial cement solution. The change of the internal friction angle is affected by the soil particle contact, the form and pore form of calcium carbonate formed by microbial precipitation, the yield, and other forces.(4)
By means of SEM, the distribution and contact microscopic images of the soil particles of the modified loess samples with different proportions of microorganisms were further verified to further verify the macroscopic changes in the shear strength of the modified loess samples with different proportions of microorganisms.



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*Source: 3009071-2022-05-24.xml*