# Enhanced Compressive Strength of the Bentonite-Amended Cement via Bio-Mineralization

Authors: Yongshuai Sun; Anping Lei
Journal: Advances in Materials Science and Engineering
 (2022)

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## Abstract

Bentonite, a supplementary cementitious material for Portland cement, has greatly contributed to environmental sustainability. However, few studies have investigated mortar samples produced by substituting bentonite for cement, and cement strength may be adversely affected when cement is replaced with bentonite in larger proportions. Therefore, this paper investigates and discusses the effect of microbially induced calcium carbonate precipitation (MICP) on improving the strength of bentonite-amended cement. The bio-mineralization process of MICP was characterized by SEM-EDS, while the biominerals formed in bentonite-amended mortar were identified by FIIR and XRD analysis. The results showed that: at bentonite concentrations of 0%, 10%, 20%, 30%, and 40% in cement, the bacterial suspension and reaction solution enhanced the compressive strength of bentonite-amended cement by 17%, 20%, 79%, 78%, and 38%, respectively, after 28 days, compared to control specimens; With the increased bacterial concentration in the presence of the reaction solution, the strength of the bentonite-amended cement (20% bentonite) increased remarkably compared to the control specimen (without bacteria). When the bacterial concentration was OD600 2.0, the compressive strength of bentonite-amended cement (20% bentonite) increased by 80% after 28 days; MICP process has a great effect on improving the strength of bentonite-amended cement. It is a green and economical choice to use MICP to improve the strength of bentonite-amended cement.


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## Body

## 1. Introduction

Population growth and accelerating urbanization have increased the consumption of cement because it is cheap and readily available. However, carbon dioxide emissions from cement production processes and environmental damage caused by the extraction of raw material for cement production have resulted in heavy pressure to reduce the consumption of cement due to global warming [1]. The use of environmentally friendly supplementary materials to partly replace cement could reduce cement consumption [2]. These environmentally friendly supplementary materials could be industrial by-products, industrial waste by-products, or naturally available materials. Furthermore, they require relatively less energy to manufacture. Microbially induced calcium carbonate precipitation (MICP), which is an environmentally friendly supplementary material is an innovative way to improve the strength and durability of cement on the basis of urease-catalyzed urea hydrolysis [3, 4].MICP is defined as biologically induced precipitation in which bacteria create a local microenvironment, under conditions that allow optimal extracellular chemical precipitation of mineral phases [5]. MICP is the product of a series of biochemical reactions. The hydrolysis of urea, which is catalyzed in the presence of microbially produced urease, creates ammonia and carbon dioxide. These products are then converted to carbonate ions and ammonium in an alkaline environment (1) [6]. Negatively charged bacteria are absorbed through positively charged Ca2+. If there are enough Ca2+ ions in the solution, CO32− is formed through urease-catalyzed hydrolysis of urea reacting directly with the Ca2+ cross-linked in the bacteria to precipitate CaCO3 with the bacteria serving as the nucleating site (2) and (3) [6, 7].(1)CONH22+2HO2⟶NH4++CO32−(2)Ca2++cell⟶cell−Ca2+(3)CO32−+cell−Ca2+⟶cell−CaCO3The great prospect of MICP application has been demonstrated in many fields and has been applied in environmental treatment [8–10]; wind erosion [11, 12]; consolidated sand [13, 14]; enhanced oil recovery [15, 16]; cracks and mechanical properties of cement structures [17, 18]; and quality improvement of recycled aggregates [19–21]. Crack repair consists of self-healing of cement cracks [22, 23] and healing of the cement cracks [24–26]. The study of various parameters of cement structures in the MICP process has been mainly concentrated on compressive strength [27, 28], permeability [29], water absorption [30], and chloride ingression [30]. The compressive strength of concrete, which affects its durability, is believed to be the most important characteristic of cement. Therefore, it is very valuable to identify an appropriate technology that ensures the mechanical properties of the structure while partially replacing cement with supplementary materials. Calcium carbonate precipitation induced by bacteria, a recently discovered novel product of bio-mineralization, is widely used to improve the durability of cement [31]. The application of bio-cement not only improves the performance of cement, but also helps to reduce CO2 emissions into the atmosphere. Calcium carbonate precipitation catalyzed through bacteria has been developed as supplementary material to improve the major parameters of cement, such as compressive strength; remediation of cracks; and reduction in permeability and corrosion of cement structures [6, 31–33].The related research combined with MICP shows that MICP is widely used in partially replacing cement with auxiliary materials to improve the compressive strength of cement. Most of the current research focuses on the production of mortar samples by using metakaolin and fly ash instead of cement [1, 3, 34, 35], while the related research on the use of bentonite instead of cement to prepare mortar samples is scarce. Therefore, this paper studies and explores the effect of microbial-induced calcium carbonate precipitation (MICP) on improving the strength of bentonite-modified cement. In this study, MICP technology was used to replace a part of cement with bentonite, and the replacement concentrations were 0%, 10%, 20%, 30%, 40%, and 50%. The compressive strength analysis of cement samples replaced by part of bentonite was carried out, and a control experiment was designed to study the effect of bacterial concentration on bentonite-amended cement. The bio-mineralization process of MICP was characterized by scanning electron microscopy and energy dispersive spectroscopy (direct action and indirect action), and the biominerals formed in bentonite-amended mortar were analyzed and identified by FIIR and XRD.


## 2. Materials and Methods

### 2.1. Microorganisms and Growth Conditions

TheSporosarcina pasteurii (ATCC11859) strain was used to develop bentonite-mended cement in this paper. The bacteria were used because they produce urease and have the ability to crystallize calcium carbonate in the experimental system. The culture medium consisted of mixing soy peptone (5 g/L), casein peptone (15 g/L), urea (20 g/L), and sodium chloride (5 g/L) into 1000 mL of deionized water. The pH value of the medium was about 7.3. The culture medium was sterilized at 121°C for 30 minutes. Bacteria were cultured in a 30°C culture medium at 120 rpm for 48 hours. To pellet the bacteria, the culture containing bacteria was centrifuged at 4°C for 8 min at 5434g. The bacterial pellets were resuspended in 1000 ml of deionized water for future use. The optical densities of bacteria diluted to 600 nm (OD600) were 0.5, 1.0, 1.5, and 2.0, respectively. All chemical reagents, such as calcium chloride, urea, peptone from casein, sodium chloride, and peptone from soymeal were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). All of the reagents used in this study were analytical grade and without further purification.


### 2.2. Materials

The bentonite samples were provided by Yueqing Xibian Automation Co., Ltd. The average particle diameter of the bentonite (D50) was 13.89 µm. Grading size distribution curves of the bentonite were tested by Laser granularity meter (Figure S1(a)). Ordinary Portland cement conforming to IS 12269–1987 was used. Grading size distribution curves of the cement were tested by Laser granularity meter (Figure S1(b)). The average particle diameter of the cement (D50) was 17.04 µm. The chemical composition of the cement and bentonite is shown in Table S1.


### 2.3. The Unconfined Compressive Strength Test

TheSporosarcina pasteurii was used to study the compressive strength of bentonite-amended cement. The reaction solution (0.5 mol/L) consisted of 0.5 mol/L of CaCl2 and 0.5 mol/L of urea. The liquid-solid ratio was 1.5 (by weight). The liquid consisted of 225 mL bacterial suspension (OD600 = 1.0) and 675 mL reaction solution (0.5 mol/L) (bacterial suspension: reaction solution ratio = 1 : 3). Bentonite was used to replace the amount of cement at concentrations of 10%, 20%, 30%, 40%, and 50%. The same experiment was performed by adding deionized water in the place of the bacterial suspension and reaction solution. The cement solidification test was carried out using the bentonite-amended cement (20% bentonite) treatment with 225 mL various bacterial suspension concentrations (OD600 = 0.5, 1.0, 1.5, and 2.0) in the presence of 675 mL reaction solution. The control specimens, 20% bentonite, were prepared in a similar way but without adding reaction solution. The unconfined compressive strength tests were carried out with an automatic compression tester. The experiment of the bentonite-amended cement (20% bentonite) was performed in the presence of deionized water, reaction solution, bacterial suspension, and bacterial suspension and reaction solution, respectively. A 70.6 mm cube mold was used, as per IS 4031–1988. After de-molding, all specimens were placed in a curing room at 25°C until the compressive strength tests were carried on day 3, 7, and 28.


### 2.4. FTIR Spectroscopy and XRD

To determine whether bio-mineralization was involved in the bentonite-amended cement. The spectra of the wavenumber range of 400–4000 cm−1 was recorded by FTIR (FTIR8400, SHIMADZU, Japan). The bentonite-amended cement was ground into mortar. Measurements were carried out by using an automatic XRD instrument (Brooke, Germany). The scanning speed was 0.15 s Step-1, and the range of 2 θ is 10°∼90°.


### 2.5. SEM-EDS-TG

The collected bentonite-amended cement specimens were sprayed on an objective table. The microstructure of the bentonite-amended cement specimens were observed using field emission scanning electron microscopy (FESEM, Zeiss Supra 55, Germany). Examination was performed with 25 kV acceleration voltage. The chemical composition of the bentonite-amended cement was characterized by energy dispersive spectroscopy (EDS, HORIBA, 7593-H) associated with the SEM. The thermogravimetric analysis was carried out with TA instrument SDT Q600 at a temperature rise rate of 10°C/min under nitrogen flow.


## 2.1. Microorganisms and Growth Conditions

TheSporosarcina pasteurii (ATCC11859) strain was used to develop bentonite-mended cement in this paper. The bacteria were used because they produce urease and have the ability to crystallize calcium carbonate in the experimental system. The culture medium consisted of mixing soy peptone (5 g/L), casein peptone (15 g/L), urea (20 g/L), and sodium chloride (5 g/L) into 1000 mL of deionized water. The pH value of the medium was about 7.3. The culture medium was sterilized at 121°C for 30 minutes. Bacteria were cultured in a 30°C culture medium at 120 rpm for 48 hours. To pellet the bacteria, the culture containing bacteria was centrifuged at 4°C for 8 min at 5434g. The bacterial pellets were resuspended in 1000 ml of deionized water for future use. The optical densities of bacteria diluted to 600 nm (OD600) were 0.5, 1.0, 1.5, and 2.0, respectively. All chemical reagents, such as calcium chloride, urea, peptone from casein, sodium chloride, and peptone from soymeal were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). All of the reagents used in this study were analytical grade and without further purification.


## 2.2. Materials

The bentonite samples were provided by Yueqing Xibian Automation Co., Ltd. The average particle diameter of the bentonite (D50) was 13.89 µm. Grading size distribution curves of the bentonite were tested by Laser granularity meter (Figure S1(a)). Ordinary Portland cement conforming to IS 12269–1987 was used. Grading size distribution curves of the cement were tested by Laser granularity meter (Figure S1(b)). The average particle diameter of the cement (D50) was 17.04 µm. The chemical composition of the cement and bentonite is shown in Table S1.


## 2.3. The Unconfined Compressive Strength Test

TheSporosarcina pasteurii was used to study the compressive strength of bentonite-amended cement. The reaction solution (0.5 mol/L) consisted of 0.5 mol/L of CaCl2 and 0.5 mol/L of urea. The liquid-solid ratio was 1.5 (by weight). The liquid consisted of 225 mL bacterial suspension (OD600 = 1.0) and 675 mL reaction solution (0.5 mol/L) (bacterial suspension: reaction solution ratio = 1 : 3). Bentonite was used to replace the amount of cement at concentrations of 10%, 20%, 30%, 40%, and 50%. The same experiment was performed by adding deionized water in the place of the bacterial suspension and reaction solution. The cement solidification test was carried out using the bentonite-amended cement (20% bentonite) treatment with 225 mL various bacterial suspension concentrations (OD600 = 0.5, 1.0, 1.5, and 2.0) in the presence of 675 mL reaction solution. The control specimens, 20% bentonite, were prepared in a similar way but without adding reaction solution. The unconfined compressive strength tests were carried out with an automatic compression tester. The experiment of the bentonite-amended cement (20% bentonite) was performed in the presence of deionized water, reaction solution, bacterial suspension, and bacterial suspension and reaction solution, respectively. A 70.6 mm cube mold was used, as per IS 4031–1988. After de-molding, all specimens were placed in a curing room at 25°C until the compressive strength tests were carried on day 3, 7, and 28.


## 2.4. FTIR Spectroscopy and XRD

To determine whether bio-mineralization was involved in the bentonite-amended cement. The spectra of the wavenumber range of 400–4000 cm−1 was recorded by FTIR (FTIR8400, SHIMADZU, Japan). The bentonite-amended cement was ground into mortar. Measurements were carried out by using an automatic XRD instrument (Brooke, Germany). The scanning speed was 0.15 s Step-1, and the range of 2 θ is 10°∼90°.


## 2.5. SEM-EDS-TG

The collected bentonite-amended cement specimens were sprayed on an objective table. The microstructure of the bentonite-amended cement specimens were observed using field emission scanning electron microscopy (FESEM, Zeiss Supra 55, Germany). Examination was performed with 25 kV acceleration voltage. The chemical composition of the bentonite-amended cement was characterized by energy dispersive spectroscopy (EDS, HORIBA, 7593-H) associated with the SEM. The thermogravimetric analysis was carried out with TA instrument SDT Q600 at a temperature rise rate of 10°C/min under nitrogen flow.


## 3. Results

### 3.1. Compressive Strength

The compressive strength of the bentonite-amended cement was tested to determine the efficiency of improvement by theSporosarcina pasteurii. The compressive strength of the bentonite-amended cement in the presence of the deionized water (A), the reaction solution (B), the bacterial suspension (C), and the bacterial suspension and reaction solution (D) on days 3, 7, and 28 are summarized in Figure 1. The compressive strength significantly increased for the bentonite-amended cement containing the bacterial suspension. However, the compressive strength of the bentonite-amended cement containing bacterial suspension and reaction solution improved the most. After 28 days, the compressive strength of the bentonite-amended cement specimens with a CaCl2 aqueous solution decreased with respect to the control specimen.Figure 1
Effect of liquid type (a) deionized water; (b) reaction solution; (c) bacterial suspension; (d) bacterial suspension and reaction solution on the compressive strength of cement cubes amended with 20% bentonite after 3, 7, and 28 days.A moderate bell-shaped relationship was observed between the compressive strength of the bentonite-amended cement (20% bentonite) and the bacterial concentration (Figure2(a)). When the bacterial concentration was OD600 1.0 without reaction solution, the compressive strength of the bentonite-amended cement had improved 32% at the age 28 days. The compressive strength of bentonite-amended cement (20% bentonite) did not improve in the other bacterial concentrations.Figure 2
Compressive strength analyses of cement formed with bacterial suspensions of different concentrations (a); different concentrations of bacterial suspension in the presence of reaction solution (b); different concentrations of bentonite without bacterial suspension and reaction solution (c); and different concentrations of bentonite with bacterial suspension and reaction solution (d).
(a)(b)(c)(d)Figure2(b) shows that the compressive strength of the bentonite-amended cement (20% bentonite) did not significantly improve in the presence of the bacterial suspension (OD600 = 0.5) and reaction solution (0.5 mol/L). However, improvement in the compressive strength of the bentonite-amended cement (20% bentonite) by the bacterial suspension and reaction solution was 90% and 80%, respectively, after 7 and 28 days compared to the control specimen (without bacteria), when the bacterial concentration was OD600 2.0. When the bacterial concentration was OD600 1.0 and 1.5, the increase in the compressive strength was almost the same, with the bentonite-amended cement enhancing the compressive strength around 46% after 7 days and 59% after 28 days.The higher the concentration of bentonite, the lower the compressive strength for all the cement cubes with or without bacterial suspension and reaction solution Figures2(c) and 2(d). However, the compressive strength significantly improved for the cement cubes containing the bacterial suspension and reaction solution. Mixing of the bacterial suspension and reaction solution in cement cubes (20% bentonite) showed around 87.5% and 79% improvement in the compressive strength after 7 and 28 days, respectively, with respect to the control specimen. Conversely, the improvement to the compressive strength of cement (without bentonite) with the bacterial suspension and reaction solution was 15% and 17% after 7 and 28 days, respectively, compared to the control specimen. With a 10% bentonite concentration in cement, the bacterial suspension and reaction solution enhanced the compressive strength by 14% and 20% after 7 and 28 days, respectively, compared to the control specimen. With the 30% bentonite concentration in cement, the bacterial suspension and reaction solution enhanced the compressive strength by 136% and 78% after 7 and 28 days, respectively, compared to the control specimen. At the same time, there was also a good improvement in the compressive strength of cement (40% bentonite) in the presence of the bacterial suspension and reaction solution, leading to a 163% and 38% improvement in the strength after 7 and 28 days, respectively, with respect to the control specimen. However, the compressive strength of the bentonite-amended cement cubes (50% bentonite) did not improve compared to the control specimens (without the bacterial suspension and reaction solution) after both 7 and 28 days.


### 3.2. SEM-EDS Analysis of Bio-Mineralized Cement

To investigate the effect of MICP, the 7 day bentonite-amended cement specimens with deionized water, bacterial suspension, and bacterial suspension and reaction solution were visualized by SEM (Figure3). When comparing the SEM graphs of control and bio-cement specimens, it was observed that the number of calcium carbonate crystals in the bentonite-amended cement was increasing, as well the degree of crystallization. The hydration of bentonite-amended cement can only be observed in the absence of bacterial suspension and reaction solution (Figure 3(a)). A dense growth of calcium carbonate in the presence of the bacterial suspension and reaction solution was observed in the bentonite-amended cement (Figure 3(c)). Therefore, it can be concluded that bio-mineralization catalyzed by bacteria plays an important role in enhancing the strength of the bentonite-amended cement.Figure 3
SEM image of cement formed in deionized water (a), bacterial suspension (b), and bacterial suspension with reaction solution (c).
(a)(b)(c)The EDS analysis of the bentonite-amended cement (Figure4) showed that the major elemental composition around the precipitate was mostly calcium, carbon, and oxygen in the presence of the bacteria. When deionized water was added to the bentonite-amended cement, no carbon element was found, which shows that no or only a small amount of calcium carbonate was produced. With the addition of the bacterial suspension, or the bacterial suspension and reaction solution, the content of carbon element increased significantly. These results suggest that CO3−2 was produced by the hydrolysis of urea as catalyzed by bacterial urease. This maybe the reason why the content of calcium carbonate increased.Figure 4
EDS analyses of bentonite-amended cement formed in (a) deionized water b; (b) bacterial suspension c; and (c) bacterial suspension and reaction solution (d); atomic percentage a.
(a)(b)(c)(d)


### 3.3. FTIR and XRD Analysis of Bio-Mineralized Cement

The molecule groups were characterized by FTIR (Figure5(a)). The bentonite-amended cement (20% bentonite), irrespective of the type of liquid, exhibited peaks of CO32−around 1422 cm−1 [36]. The bentonite-amended cement (20% bentonite) without bacterial suspension and reaction solution exhibited peaks at around 1422 cm−1 and 872 cm−1. This is because bentonite and cement contain a certain amount of calcium oxide, which forms natural chemical processes within the cement. With the addition of the bacterial suspension, and the bacterial suspension with reaction solution, the peak values around 876 cm−1 and 1422 cm−1 became increasingly remarkable. These results show that more calcium carbonate was produced. The reason for this is the organic mineral calcium carbonate was formed as bacteria act as nucleating sites in the presence of the reaction solution.Figure 5
FTIR (a) and X-ray diffraction (b) of bentonite-amended cement formed in deionized water A, bacterial suspension B, and bacterial suspension and reaction solution C (1: CH-portlandite (Ca(OH)2); 2: calcite (CaCO3); 3: quartz (SiO2); 4: C-S-H-calcium silicate hydrate (Ca5Si6O16(OH)2); 5: C3S-Alite 3CaOSiO3/C2S-Belite (2CaOSiO3)).
(a)(b)The granular and anhydrous phases of cement, such as C3S and C2S were analyzed by XRD (Figure 5(b)). Moreover, there were hydration products portlandite (CH) and calcium silicate hydrate (C-S-H) in the bentonite-amended cement. These results agree with previously published literature [37]. MICP-based bio-cement always forms calcite [38, 39]. It can be seen that calcite was dominant in all bentonite-amended cement (20% bentonite). When there was no bacterial suspension and reaction solution in the bentonite-amended cement, calcite peak was not found. This may be because the chemical reaction produced too little calcium carbonate. With the addition of the bacterial suspension, and the bacterial suspension and reaction solution, calcite peak became more and more remarkable. The result is consistent with those shown in Figure 5(a). Thus, this indicates that more calcite was produced in the presence of the bacterial suspension and reaction solution.


## 3.1. Compressive Strength

The compressive strength of the bentonite-amended cement was tested to determine the efficiency of improvement by theSporosarcina pasteurii. The compressive strength of the bentonite-amended cement in the presence of the deionized water (A), the reaction solution (B), the bacterial suspension (C), and the bacterial suspension and reaction solution (D) on days 3, 7, and 28 are summarized in Figure 1. The compressive strength significantly increased for the bentonite-amended cement containing the bacterial suspension. However, the compressive strength of the bentonite-amended cement containing bacterial suspension and reaction solution improved the most. After 28 days, the compressive strength of the bentonite-amended cement specimens with a CaCl2 aqueous solution decreased with respect to the control specimen.Figure 1
Effect of liquid type (a) deionized water; (b) reaction solution; (c) bacterial suspension; (d) bacterial suspension and reaction solution on the compressive strength of cement cubes amended with 20% bentonite after 3, 7, and 28 days.A moderate bell-shaped relationship was observed between the compressive strength of the bentonite-amended cement (20% bentonite) and the bacterial concentration (Figure2(a)). When the bacterial concentration was OD600 1.0 without reaction solution, the compressive strength of the bentonite-amended cement had improved 32% at the age 28 days. The compressive strength of bentonite-amended cement (20% bentonite) did not improve in the other bacterial concentrations.Figure 2
Compressive strength analyses of cement formed with bacterial suspensions of different concentrations (a); different concentrations of bacterial suspension in the presence of reaction solution (b); different concentrations of bentonite without bacterial suspension and reaction solution (c); and different concentrations of bentonite with bacterial suspension and reaction solution (d).
(a)(b)(c)(d)Figure2(b) shows that the compressive strength of the bentonite-amended cement (20% bentonite) did not significantly improve in the presence of the bacterial suspension (OD600 = 0.5) and reaction solution (0.5 mol/L). However, improvement in the compressive strength of the bentonite-amended cement (20% bentonite) by the bacterial suspension and reaction solution was 90% and 80%, respectively, after 7 and 28 days compared to the control specimen (without bacteria), when the bacterial concentration was OD600 2.0. When the bacterial concentration was OD600 1.0 and 1.5, the increase in the compressive strength was almost the same, with the bentonite-amended cement enhancing the compressive strength around 46% after 7 days and 59% after 28 days.The higher the concentration of bentonite, the lower the compressive strength for all the cement cubes with or without bacterial suspension and reaction solution Figures2(c) and 2(d). However, the compressive strength significantly improved for the cement cubes containing the bacterial suspension and reaction solution. Mixing of the bacterial suspension and reaction solution in cement cubes (20% bentonite) showed around 87.5% and 79% improvement in the compressive strength after 7 and 28 days, respectively, with respect to the control specimen. Conversely, the improvement to the compressive strength of cement (without bentonite) with the bacterial suspension and reaction solution was 15% and 17% after 7 and 28 days, respectively, compared to the control specimen. With a 10% bentonite concentration in cement, the bacterial suspension and reaction solution enhanced the compressive strength by 14% and 20% after 7 and 28 days, respectively, compared to the control specimen. With the 30% bentonite concentration in cement, the bacterial suspension and reaction solution enhanced the compressive strength by 136% and 78% after 7 and 28 days, respectively, compared to the control specimen. At the same time, there was also a good improvement in the compressive strength of cement (40% bentonite) in the presence of the bacterial suspension and reaction solution, leading to a 163% and 38% improvement in the strength after 7 and 28 days, respectively, with respect to the control specimen. However, the compressive strength of the bentonite-amended cement cubes (50% bentonite) did not improve compared to the control specimens (without the bacterial suspension and reaction solution) after both 7 and 28 days.


## 3.2. SEM-EDS Analysis of Bio-Mineralized Cement

To investigate the effect of MICP, the 7 day bentonite-amended cement specimens with deionized water, bacterial suspension, and bacterial suspension and reaction solution were visualized by SEM (Figure3). When comparing the SEM graphs of control and bio-cement specimens, it was observed that the number of calcium carbonate crystals in the bentonite-amended cement was increasing, as well the degree of crystallization. The hydration of bentonite-amended cement can only be observed in the absence of bacterial suspension and reaction solution (Figure 3(a)). A dense growth of calcium carbonate in the presence of the bacterial suspension and reaction solution was observed in the bentonite-amended cement (Figure 3(c)). Therefore, it can be concluded that bio-mineralization catalyzed by bacteria plays an important role in enhancing the strength of the bentonite-amended cement.Figure 3
SEM image of cement formed in deionized water (a), bacterial suspension (b), and bacterial suspension with reaction solution (c).
(a)(b)(c)The EDS analysis of the bentonite-amended cement (Figure4) showed that the major elemental composition around the precipitate was mostly calcium, carbon, and oxygen in the presence of the bacteria. When deionized water was added to the bentonite-amended cement, no carbon element was found, which shows that no or only a small amount of calcium carbonate was produced. With the addition of the bacterial suspension, or the bacterial suspension and reaction solution, the content of carbon element increased significantly. These results suggest that CO3−2 was produced by the hydrolysis of urea as catalyzed by bacterial urease. This maybe the reason why the content of calcium carbonate increased.Figure 4
EDS analyses of bentonite-amended cement formed in (a) deionized water b; (b) bacterial suspension c; and (c) bacterial suspension and reaction solution (d); atomic percentage a.
(a)(b)(c)(d)


## 3.3. FTIR and XRD Analysis of Bio-Mineralized Cement

The molecule groups were characterized by FTIR (Figure5(a)). The bentonite-amended cement (20% bentonite), irrespective of the type of liquid, exhibited peaks of CO32−around 1422 cm−1 [36]. The bentonite-amended cement (20% bentonite) without bacterial suspension and reaction solution exhibited peaks at around 1422 cm−1 and 872 cm−1. This is because bentonite and cement contain a certain amount of calcium oxide, which forms natural chemical processes within the cement. With the addition of the bacterial suspension, and the bacterial suspension with reaction solution, the peak values around 876 cm−1 and 1422 cm−1 became increasingly remarkable. These results show that more calcium carbonate was produced. The reason for this is the organic mineral calcium carbonate was formed as bacteria act as nucleating sites in the presence of the reaction solution.Figure 5
FTIR (a) and X-ray diffraction (b) of bentonite-amended cement formed in deionized water A, bacterial suspension B, and bacterial suspension and reaction solution C (1: CH-portlandite (Ca(OH)2); 2: calcite (CaCO3); 3: quartz (SiO2); 4: C-S-H-calcium silicate hydrate (Ca5Si6O16(OH)2); 5: C3S-Alite 3CaOSiO3/C2S-Belite (2CaOSiO3)).
(a)(b)The granular and anhydrous phases of cement, such as C3S and C2S were analyzed by XRD (Figure 5(b)). Moreover, there were hydration products portlandite (CH) and calcium silicate hydrate (C-S-H) in the bentonite-amended cement. These results agree with previously published literature [37]. MICP-based bio-cement always forms calcite [38, 39]. It can be seen that calcite was dominant in all bentonite-amended cement (20% bentonite). When there was no bacterial suspension and reaction solution in the bentonite-amended cement, calcite peak was not found. This may be because the chemical reaction produced too little calcium carbonate. With the addition of the bacterial suspension, and the bacterial suspension and reaction solution, calcite peak became more and more remarkable. The result is consistent with those shown in Figure 5(a). Thus, this indicates that more calcite was produced in the presence of the bacterial suspension and reaction solution.


## 4. Discussion

The strength of the bentonite-amended cement (20% bentonite) increased with the addition of the reaction solution. When only the bacterial suspension was added, the strength of the bentonite-amended cement (20% bentonite) improved more. The strength of the bentonite-amended cement (20% bentonite) improved the most when the bacterial suspension and reaction solution were added together. This shows that MICP process has a great effect on improving the strength of the bentonite-amended cement.The compressive strength of the bentonite-amended cement (20% bentonite) first increased and then decreased with the increasing of bacterial concentrations. At the same time, bacteria secrete more nonreactive extracellular polymeric substances in the absence of the reaction solution, which is not conducive for improving the strength of the bentonite-amended cement [40–42]. The compressive strength of the bentonite-amended cement (20% bentonite) increased with the increasing bacterial concentration in the presence of the reaction solution. If the reaction solution was sufficient, the production of calcium carbonate increased with the increasing bacterial concentration. At the same time, the improved compressive strength came from the calcium carbonate deposition catalyzed by bacteria within the pores of the bentonite-amended cement, which became plugged in the bentonite-amended cement paste [43]. The higher amount of calcium carbonate resulted in better blockage of the cement, thereby strengthening the bentonite-amended cement.More calcium carbonate was produced because the bacteria reacted with the reaction solution. Therefore, the bentonite-amended cement has a maximum compressive strength in the presence of the bacterial suspension and reaction solution. The above conclusions agree with TG analysis (Figure6(a)). The content of calcium carbonate could be determined using TG analyzer by measuring the weight loss of the burned samples at a high temperature (up to 900°C). The calcium carbonate content can be determined by (4) [44]. Where A is the initial weight and B and C are weights at 500°C and 800°C. By analyzing the TG data, it can be concluded that the contents of calcium carbonate in bentonite-amended cement formed in the deionized water (A), the bacterial suspension (B), and the bacterial suspension and reaction solution (C) were 1.7%, 2.4%, and 3.8%, respectively. The results of Figure 6(b) show that the strength of the bentonite-amended cement was proportional to the content of calcium carbonate in the bentonite-amended cement as determined by Figure 6(a). This confirms that calcium carbonate catalyzed by bacterial urease plays a crucial role in the bentonite-amended cement strength, which is consistent with previous studies.(4)C%=B−CA×2.274.Figure 6
TG (a) and compressive strength (b) analysis of bentonite-amended cement formed in deionized water, bacterial suspension, and bacterial suspension with reaction solution.
(a)(b)The higher the concentration of bentonite, the lower the compressive strength for all the bentonite-amended cement with or without bacteria. However, the compressive strength significantly improved for the bentonite-amended cement that contained the bacterial suspension and reaction solution. The reason for the increased bentonite-amended cement strength may be that the reaction with MICP yielded an additional cementitious C-S-H gel, together with crystalline products such as calcite and calcium aluminate hydrates. Voids in the bentonite-amended cement were filled through additional C-S-H gel, which led to an increased bentonite-amended cement density. At present, it is generally acknowledged that the reaction mechanism of cement can be summarized by the following (5) [45].(5)C3SC2S+CO2+H2O⟶C−S−H+CaCO3.The MICP reaction produces carbon dioxide, which promotes the above positive reaction. The formation of calcium carbonate crystals is promoted when CO2 reacts with cement hydration products and un-hydrated products. Then, calcium carbonate fills the pore structure of the bentonite-amended cement, eventually enhancing the compressive strength of the bentonite-amended cement [33, 45].Li et al. [35]studied metakaolin-modified cement mortar samples by replacing cement with metakaolin. They found that the compressive strength of metakaolin-modified cement mortar samples was significantly improved at various metakaolin concentrations. Achal et al. [46]studied fly ash-amended concrete samples by replacing cement with fly ash. Similarly the compressive strength of fly ash-amended concrete samples has been significantly improved at various fly ash concentrations. After 28 days, the ratio of strength improvement of the amended-cement in different studies is shown in Table 1. Compared with other studies (without sand), the effect of MICP on cement (with sand) was more obvious. However, when compared with other soils, the effect of bentonite in the MICP process with or without sand was more significant (Table 1).Table 1
Contrast of proportional strength increase in different studies.
Soil concentration (%)Mengmeng Li et alVarenyam Achal et alThis paper027.40%21%17%10—19%20%20—14%79%2521.10%——30——78%40—10%38%5026.80%——


## 5. Conclusions

Calcium carbonate precipitation catalyzed by bacterial urease has enormous potential to enhance the strength of the bentonite-amended cement. In this paper, the effect of bio-mineralization on bentonite-amended cement strength (which is the most important cement parameter) was studied. When the proportion of bentonite was less than 40%, the strength of the bentonite-amended cement was significantly improved. At the same time, with the increasing bacterial concentration, the strength of the cement containing 20% bentonite increased remarkably compared to control specimen (without bacteria). The presence of calcium carbonate was confirmed by SEM and EDS. In addition, FTIR, XRD, and TG analysis confirmed that the strength of the bentonite-amended cement was proportional to the content of calcium carbonate produced by bacterial urease catalysis. This bio-mineralization technology has great application value, because it can ensure the reuse of industrial by-products, while reducing the amount of cement. Finally, MICP itself will not produce pollutants.



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*Source: 7220528-2022-09-27.xml*