Experimental study on ecofriendly and sustainable aeolian sand bricks with microbial-induced calcite precipitation technology

Desertification persists as a significant global environmental and economic challenge, with one promising mitigation strategy involving the utilization of desert aeolian sand in engineering applications. This approach not only aids in rehabilitating degraded landscapes but also contributes to reducing CO₂ and dust emissions associated with conventional construction materials. It also investigates the application of microbial-induced calcium carbonate precipitation (MICP) technology to enhance soil consolidation and improve the mechanical properties of sand bricks. Specifically, the NH₄-Ye medium at pH 9 is employed to cultivate Bacillus pasteurei, and a stirring method was adopted for sand solidification. The results demonstrate successful consolidation of desert aeolian sand via MICP. The innovative contribution of this research lies in its potential to improve soil quality and augment the structural integrity of vast desert aeolian sand deposits in China, offering promising prospects for sustainable engineering applications.

1 Introduction

There is a considerable amount of aeolian sand in the desert in Northwest China. However, the application of aeolian sand in engineering is limited due to its fine structural particles, poor grading, low viscosity, and poor water retention. If the desert aeolian sand can be improved by microbial mineralization and sedimentation technology to enhance its engineering application potential, it will create huge social, environmental and economic benefits.

Biomineralization is a biological process in which organisms promote mineral precipitation under the control or induction of their cells and organic products. It is a common phenomenon in nature. Microbial-induced mineralization is the most common form of biomineralization. Some microorganisms can actively create a microenvironment suitable for mineral nucleation and crystallization through metabolism and promote the deposition of calcium magnesium phosphate, calcium magnesium carbonate, and other minerals (Fernández et al., 2018). It provides carbonate ions through microbial metabolism, creates an alkaline environment, and rapidly precipitates calcium carbonate crystals with excellent cementation performance. The technology of soil reinforcement, leakage stoppage, and joint repair based on microbial-induced calcium carbonate precipitation (MICP) is commonly referred to as MICP technology.

The principle of different types of microorganisms participating in MICP is the same. They all provide a favorable environment for calcium carbonate deposition through their metabolism. However, there are some differences in the specific deposition process, which can be divided into four categories: urea hydrolysis (Muynck et al., 2010), denitrification (Whiffin, 2004; Dejong et al., 2006), ferric reduction (Gomez et al., 2019; Cheng et al., 2013) and sulfate reduction (Qabany and Soga, 2013; Frederik, 2002). Among them, MICP initiated by urease hydrolysis of urea is simple in mechanism, easy to control in reaction process, and can efficiently produce a large number of carbonate ions in a short time, with a good reinforcement effect, so it has been the research hotspot of microbial-induced mineralization technology. Take Bacillus pasteurei, a bacterium with high urease activity, as an example. The strain will secrete urease during metabolism and hydrolyze urease, as shown in Figure 1. After urea is hydrolyzed, the pH in the surrounding environment will rise and dissociate ammonium ions and carbonate ions, the reaction equations are given in Equations 1–7. Because the surface of the bacterial cell wall is often attached with carboxyl, phosphoryl, and other negatively charged groups, it will constantly adsorb calcium ions in the surrounding environment and combine with the dissociated carbonate ions. Under the control of microbial metabolite extracellular polymer (EPS), the bacterial cell wall will be used as the nucleus to start crystallization, continue to grow and expand, and finally form biomineralization (Hammes et al., 2003; Achal et al., 2015). In the process of carbonate deposition induced by Bacillus pasteurei, the strain mainly plays two roles: one is to hydrolyze urea, create an alkaline environment, and dissociate carbonate ions; the other is to provide nucleation sites for the deposition of mineralized products.

$CO{\left({NH}_2\right)}_2+H_{2}O\to N{H}_{2}COOH+N{H}_3(1)$

$N{H}_{2}COOH+H_{2}O\to N{H}_3+H_{2}C{O}_3(2)$

$H_{2}C{O}_3\to HC{O}_3^{-}+H^{+}(3)$

$2N{H}_3+2H_{2}O\leftrightarrow 2N{H}_4^{+}+2O{H}^{-}(4)$

$HC{O}_3^{-}+H^{+}+2O{H}^{-}\to C{O}_3^{2-}+2H_{2}O(5)$

$C{a}^{2+}+Cell\to Cell-C{a}^{2+}(6)$

$Cell-C{a}^{2+}+C{O}_3^{2-}\to Cell-CaC{O}_3\downarrow (7)$

Figure 1

Figure 1. Calcium carbonate deposition induced by Bacillus pasteurei.

Whiffin (Whelan and Whelan, 1997) first proposed the idea of using microbial-induced calcium carbonate deposition technology to solidify loose sand. The core of this technology is to use microbial-induced mineralization to generate sticky mineral precipitation between loose particles and make it cemented into a structure with certain strength (Stocks-Fischer et al., 1999; Montoya et al., 2019). Consequently, the more calcium carbonate is deposited between sand particles, the stronger is the bonding force between sand particles, and the higher is the unconfined compressive strength of sand cement. Therefore, the mechanical strength of solidified sand is determined by the deposition efficiency and distribution uniformity of calcium carbonate. In addition, Qabany and Soga (2013) pointed out that the nutrient solution (calcium salt and urea) was transformed into calcium carbonate under the effect of microbial-induced mineralization and deposited between the loose sand particles. Some of the deposited calcium carbonate filled the spaces between the sand particles, but it did not serve as a bridge to bond the sand particles together. Some of the deposited calcium carbonate was ineffective, while another portion effectively bonded the sand particles together at the sand joints. Enhancing the effective deposition ratio is the only way to fully utilize the excellent curing performance of MICP. Based on the existing research progress, it is found that the microbial mineralized sediment solidified sandy soil involves a series of complex biochemical reactions, and the cementation effect is mainly affected by the microbial species, calcium source, nutrient solution concentration, temperature, pH value, grouting method, and sand properties (particle size distribution, porosity).

The optimum temperature, pH value, and activity of urease secreted by different strains are different, and the application field and environment are also different. Bacillus pasteurei is mainly used for soil improvement and removal of heavy metal ions in water. Bacillus megaterium, which can still grow normally in extreme environments, is selected as microbial cement bacteria to enhance the strength and durability of concrete (Keykha et al., 2019; Keykha et al., 2013; Keykha et al., 2014). Sun et al. (2018) measured the proliferation rate and urease activity of Bacillus pasteurei and Bacillus megaterium by controlling temperature and pH and used the two bacteria to cement sand bodies. The results show that Bacillus megaterium can grow rapidly at low temperature, and the impermeability effect after solidification is significantly better than that of the Pasteurella cemented sand body.

In the process of mineralization and deposition of calcium carbonate induced by microorganisms, it will adsorb exogenous calcium ions and provide nucleation sites for the formation of colloidal calcium carbonate. Therefore, the ideal calcium source can improve the effect of MICP technology on sand solidification. Zhang et al. (2015) and Han and Cheng (2015a) conducted microbial mineralization cementation tests on sandy soil using calcium chloride, calcium acetate, and calcium ethanoate as calcium sources. The results showed that calcium ethanoate was the best calcium source, with the cemented sand bricks exhibiting superior mechanical properties and impermeability compared to those solidified with other calcium sources. Based on existing research, different calcium sources significantly influence crystal form, morphology, and distribution of precipitated calcium carbonate, thereby determining the final cementation effect. Although calcium ethanoate demonstrates superiority in multiple mechanical and physical indicators, factors such as its cost, environmental adaptability, or long-term stability may introduce uncertainties in practical engineering applications, leading to some controversy in its evaluation within the academic community. In contrast, due to its wide availability, low cost, high solubility, and good compatibility with microbial metabolism, calcium chloride exhibits more stable cementation effects and process feasibility in most MICP applications, and is therefore widely regarded as an ideal and reliable choice of calcium source.

The method of adding bacteria and nutrient solution (calcium source, urea, etc.) to sand is called the grouting method. Recently, the main grouting methods used in the study of sand solidification using MICP technology can be roughly divided into five categories: injection method, step-by-step grouting method, immersion method, stirring method, and spraying method. In the injection method, a 50 mL syringe or a certain size of Plexiglas tube is often used as the injection cavity (i.e., the main part of MICP), and the injection cavity and peristaltic pump (grouting power system) are connected into a complete grouting system by using a rubber stopper and rubber tube. In this way, bacterial liquid and nutrient solution are injected from the upper part through the peristaltic pump, calcium carbonate is deposited in the sand body, and finally the reaction waste liquid flows out from the bottom (Whiffin, 2004). The step-by-step grouting method effectively solves the problems of short solidification distance and uneven cementation degree of the injection method, generating widespread research interest in the application of MICP technology for soil reinforcement among researchers worldwide (Paassen et al., 2009). The immersion method, also known as the water sedimentation method, immerses the sand model with bacteria in the cementation liquid. With the extension of immersion time, the sand particles are gradually cemented into a compact whole (Sl et al., 2021). The stirring method is similar to the method of preparing concrete mortar. The bacteria solution, cementation solution, and sand particles are mixed and fully stirred to make the bacteria solution and culture solution evenly distributed in the sand. After standing for a certain time, the sand sample is evenly filled into the mold and finally placed in a suitable environment for maintenance (Jiang and Soga, 2016). The spraying method is a grouting method in which the prepared bacteria liquid and cement liquid are added into the spray bottle, respectively, or mixed and evenly sprayed on the surface of the sand body to ensure that the bacteria liquid and cement liquid penetrate into the sand evenly (Liu et al., 2016; Gao et al., 2019).

At present, the effect of microbial induced calcium carbonate deposition depends largely on the microbial species and the biological activity of urease secreted by them (Qian et al., 2014). Among them, Bacillus pasteurei are most often selected as an experimental strain to study microbial mineralization (Elipe and Lopez-Querol, 2014). Bacillus pasteurei is an alkali-tolerant bacterium with high urease activity. The optimal pH is 8.5, and the weight of urease secreted by the bacterium accounts for about 1% of the weight of the cell. It can grow normally and maintain urease activity in extreme environments (high osmotic pressure, high pH value) (Deng and Wang, 2018; Gui et al., 2018). Therefore, we selected Bacillus pasteurei as the experimental strain to carry out the related research.

In this paper, beef extract peptone medium is used to activate Bacillus pasteurei, and then NH₄-YE medium is used to subculture and expand the culture. CaCl₂ and urea solutions with different concentrations and proportions are selected as the cementing solution, and the pH is adjusted to 7.5 through NaOH or HCl solution. The bacteria are separated from the bacterial solution by centrifuge, and the bacteria, cementation solution, desert sand are mixed while the pH value is adjusted by Tris HCl buffer solution. Finally, the consolidated desert sand specimens are obtained. The feasibility of microbial sand fixation is determined by measuring the compressive strength of the sand bricks through a uniaxial compressive strength test. A key advantage of this study is the potential of MICP technology to consolidate and enhance the structural strength of the vast quantities of arid aeolian sand in China for use in engineering construction.

2 Experiment methodology

2.1 Grouting method

Recently, the grouting methods include injection method, step-by-step grouting method, immersion method, stirring method, and spraying method. In this study, the stirring method is utilized to solidify the desert aeolian sand.

The process for testing consists mostly of the following steps. To prepare the mortar, place 100 g of desert aeolian sand in a 250 mL beaker, then add an equal-volume mixture of resuspended bacterial solution and cementation solution. The components are properly mixed to achieve even dispersion of the bacterial strain and cementation solution throughout the mortar. For sample preparation, a mold is placed on a Petri dish lined with three layers of gauze, and the prepared mortar is poured into it in phases. Following each filling stage, the mortar is softly compressed to produce a consistent sample density through repeated processes (Figure 2). During the curing stage, samples created using the stirring method are initially placed in a 30 °C oven for 3 days before being transferred to a 70 °C oven for 2 days of drying.

Figure 2

Figure 2. Schematic diagram of test piece by stirring method.

2.2 Sand brick properties

2.2.1 Uniaxial compression test

The mechanical experiment is conducted by using the hydraulic servo universal testing machine (as shown in Figure 3) manufactured by W+B Company, Switzerland. During the single extraction compressive strength test, the loading rate is set at 5 mm/min until the sand body splits. The necessary data is record to calculate the compressive strength.

Figure 3

Figure 3. Hydraulic servo universal testing machine.

2.2.2 Determination of calcium carbonate production

The compressed sample was completely crushed in a mortar and dropped in ultrapure water to eliminate any remaining soluble salt. Finally, the acid pickling procedure was used to determine the calcium carbonate content in the solidified sand block. The specific operational steps are as follows:

1. Dry the solidified sand sample with internal soluble salt removed, weight its mass and record it as M₁.

2. Pour excessive HCl solution into the solidified sand sample until no obvious bubbles are generated.

3. Weight the mass of the quantitative filter paper with an analytical balance and record it as M₂.

4. Filter the sand sample with a funnel, and then wash the sand sample repeatedly with ultrapure water to remove excess chloride ions in the water. Finally, measure the mass of the sand sample and filter paper after drying, and record it as M₃.

5. Calculate the mass m of calcium carbonate generated during mineralization and the proportion n of calcium carbonate in the mass of the specimen by the following formula.

$M=M_1+M_2-M_3(8)$

$n=\frac{M}{M_1}\times 100\%(9)$

3 Experimental analysis

3.1 Determination of strain

3.1.1 Experimental principle

MICP is a novel method for soil improvement and structural remediation. This method is primarily based on the metabolism of particular microbes to make carbonate ions, which are then mixed with exogenous calcium ions to rapidly generate calcite crystals with cementation and connect the loose particles together to form a structure of a given strength. Compared to standard chemical grouting, microbial-induced calcium carbonate precipitation method is more sustainable and easier to control. Depending on the metabolic pathways of microorganisms, MICP can be categorized into urea hydrolysis, denitrification, trivalent iron reduction, and sulfate reduction. Among these, the MICP mechanism based on urea hydrolysis is the simplest and most straightforward, easy to control, and can rapidly produce a large amount of carbonate ions, making it the most widely used.

The core of this technology involves using microbial-induced mineralization to create sticky mineral precipitation that binds loose particles together, forming a structure with specific strength.

3.1.2 Strain selection

Researchers purified a variety of urease-producing bacteria from soil and rock prone to mineralization, including Bacillus megaterium, Sporosarcina pasteurii, Bacillus thuringiensis, Bacillus sphaericus, Sporosarcina aquimarina, Bacillus basophilus, Clostridium, Bacillus pseudofirmus, and Sporosarcina newyorkensis.

The optimal temperature, optimal pH, and the activity of the urease secreted by different bacterial species vary, and their application fields and suitable environments also differ to some extent. Bacillus pasteurei is mostly used to improve soil quality and remove heavy metal ions from water. Bacillus megaterium was chosen as a microbial cement bacteria to improve the strength and durability of concrete since it can develop normally in harsh conditions.

The effect of microbial-induced calcium carbonate deposition largely depends on the species of microorganisms and the biological activity of urease secreted by them. Especially in the test of soil consolidation by microbial mineralization, microorganisms are often in the geotechnical environment with a high concentration of inorganic salts and a pH value greater than 8.5. Therefore, the microorganism must grow normally in extreme environments and maintain high urease activity at the same time so as to break through the technical bottleneck and improve the effect of soil consolidation by microorganisms. There are many microorganisms in natural soil, most of which can secrete urease to promote the decomposition and utilization of organic matter in soil.

Researchers have isolated various bacteria with high urease activity in soil and rock environments, among which Bacillus pasteurei is most often selected as experimental bacteria to study microbial mineralization (As shown in Figure 4) (Nguyen et al., 2019). Bacillus pasteurei is an alkali-tolerant bacterium with high urease activity. The optimal pH is 8.5, and the weight of urease secreted by the bacterium accounts for about 1% of the cell weight. It can grow normally and maintain urease activity in extreme environments (high osmotic pressure, high pH). Therefore, we selected Bacillus pasteurei as the experimental strain to carry out the related research.

Figure 4

Figure 4. Statistics on the application of bacterial strains in MICP.

3.2 Srain activation and culture

3.2.1 Selection of activation medium and experimental equipment

The experimental apparatuses and the selection of activation media are presented in Tables 1, 2, respectively.

Table 1

Table 1. Instruments and equipment.

Table 2

Table 2. Ratio of activated medium.

3.2.2 Activation culture operation

Sporosarcina pasteurii, also known as octococcus pasteurii is a nonpathogenic Gram-positive bacterium widely distributed in soil. Bacillus pasteurei is an aerobic strain, which can synthesize and secrete a large amount of urease in the process of metabolism, but it cannot synthesize and secrete urease under the condition of strict hypoxia. In this section, the activation, cultivation and storage of Bacillus pasteurei are carried out in strict accordance with the instructions for strain recovery which provide a strain guarantee for subsequent experiments. The activation operation of bacteria is as follows:

1. First, the culture medium required for bacterial activation is prepared and sterilized. The culture medium recommended in the instructions for strain recovery is the mixed solution of CASO and urea. See Table 2 for specific components. Pour the prepared liquid medium and solid medium into the blue cap bottle and sterilize with high-pressure steam at 121 °C for 20 min.

2. After sterilization, put the culture medium into the ultra-clean workbench, which has been sterilized by UV for 30 min in advance to continue sterilization. When the solid medium temperature drops to about 55 °C, pour the plate in a disposable Petri dish with a thickness of about 3 mm, and cover it upside down.

3. In the ultra-clean bench, the method of tube beating and resuscitation was used to activate the bacteria. Take out the freeze-dried mushroom powder stored in the refrigerator, wipe the tube wall with 75% alcohol, and then move it into the ultra-clean workbench. In the ultra-clean workbench, first light the alcohol lamp, place the top of the lyophilized tube on the flame for burning, quickly add sterile water to break the tube wall, and gently tap the broken glass with tweezers. Then 0.5 mL of liquid medium was injected into the lyophilized tube and gently blown with a pipette gun to fully dissolve it into bacterial suspension.

4. Use a pipette gun to suck 200 µL of bacterial suspension, pour it into the plate, evenly coat it, and then put it into a microbial incubator at 30 °C. To ensure the success rate of activation, the remaining bacterial suspension was poured into a conical flask containing 100 mL of liquid culture medium and cultured at 30 °C, 150 rpm. After the freeze-dried powder strain was activated for 36 h, pale yellow colonies grew on the solid medium, and the liquid medium also became significantly turbid, as shown in Figure 5, indicating that the strain was successfully activated.

Figure 5

Figure 5. Preparation of strain cryopreservation tube and observation of strain under microscope.

3.2.3 Inspection and storage of bacteria

In order to ensure the preciseness of the experiment, it is necessary to determine the type of strain after further observation. After centrifuging and suspending the liquid culture medium, it was added to 0.5 mol/L cementation solution (mixed solution of urea and CaCl₂), and flocculent precipitation was rapidly generated in the solution. Then, the diluted bacterial suspension was coated on the glass plate, fixed with glutaraldehyde solution, and then soaked in 50%, 60%, 70%, 80%, 90%, and 100% ethanol solution for gradient dehydration. After the sample was completely dried, the microscopic morphology was observed through the microscope, the bacteria appear blue after Gram staining, as shown in Figure 6, confirming that the strain is Gram-positive., which was consistent with the characteristics of Bacillus pasteurei in previous studies, thus proving that the activated strain was Bacillus pasteurei required by the experiment.

Figure 6

Figure 6. The Gram stain result is blue purple, indicating Gram-positive bacteria.

With the subculture of Bacillus pasteurei, its biological activity will continue to decrease. In order to meet the large demand of high urease activity Bacillus pasteurei in the later sand fixation experiment, the storage of primary strains should be done well. After mixing the initial cultured bacterial suspension with a 30% glycerol solution in equal volume, it is stored in a cryopreservation tube and frozen in a −20 °C refrigerator. The rest of the bacterial suspension is stored in a 4 °C refrigerator for use in recent experiments.

3.3 Optimization preparation of culture medium

3.3.1 Selection of culture medium

The stored Bacillus pasteurei can be utilized as the mother liquor for shaking cultivation for 15 h and then changed the culture conditions of the inoculation mother liquor culture medium to optimize the microbial culture process. The selected culture media are tryptic soy broth (TSB), NH₄-YE, and CASO+Urea (see Table 3 for the composition ratio), and experiments were carried out according to Table 4 to explore the optimal culture conditions of microorganisms.

Table 3

Table 3. Composition and proportion of different media.

Table 4

Table 4. Grouping of microbial culture condition optimization experiment.

By measuring the growth characteristics of microorganisms under different culture conditions, we found that the NH₄-YE liquid medium provides sufficient nutrients for the growth of Bacillus pasteurei, which can be utilized for expanding its culture. When pH was 8 or 9, the proliferation rate of Bacillus pasteurei was the fastest, and urease activity was also very high; the growth of Bacillus pasteurei was better at 30 °C; and a lower inoculation ratio and higher oscillation rate were beneficial to the proliferation and activity maintenance of Bacillus pasteurei. Therefore, we selected NH₄-Ye liquid medium with pH 9 as the optimal medium, and the optimal culture conditions were as follows: temperature 30 °C, inoculation ratio 1:150(V_bacteria:V_liquid), and oscillation rate 180 rpm. Under the optimized culture conditions, the growth and urease activity of Bacillus pasteurei are shown in the Figure 7.

Figure 7

Figure 7. Proliferation and urease activity of Bacillus pasteurei.

3.3.2 Preparation of culture medium and culture effect

Take out the strain and pour it into 300 mL of NH₄-YE liquid culture medium. After shaking the culture for 15 h under optimized conditions (30 °C, 180 rpm), use this as the mother liquid and inoculate several conical flasks containing 150 mL of NH₄-YE liquid culture medium at an inoculation ratio of 1:150. After shaking culture for 27 h under the optimized conditions, centrifuge and resuspend it, and store the resuspended bacteria solution in a 30 °C incubator for standby.

3.4 Specimen fabrication

3.4.1 Mold making

We used eight cylindrical molds to cut the 50 mL centrifugal pipe into a hollow tubular structure with a height of 30.00 mm and an inner diameter of 26.50 mm, as shown in Figure 8.

Figure 8

Figure 8. Specimen mould and constant temperature oven.

After using ultra-pure water to clean the mold in the laboratory, we put a preservative film on the bottom of the mold and tied it tightly with a rubber band, and then applied edible oil evenly on the inner wall of the mold, so that the test piece can be demolded easily after drying.

In the process of mineralization and deposition of calcium carbonate induced by microorganisms, microorganisms will adsorb exogenous calcium ions to provide nucleation sites for the formation of colloidal calcium carbonate. Because the sand sample used by the team is desert aeolian sand, which contains many impurities and calcium carbonate, hydrogen chloride solution is used to remove calcium carbonate from the sand sample before grouting. The aeolian sand used in the experiment and the cement solution are shown in Figure 9.

Figure 9

Figure 9. Aeolian sand used in the experiment and preparation of cementing fluid.

0.1 mol/L hydrogen chloride solution is used to remove calcium carbonate from the sand sample. First, the desert aeolian sand was put into a beaker and then the hydrogen chloride solution was poured into the mortar and stirred until no bubbles were generated. Then the waste water is poured into the waste liquid tank. Next, place the sand sample into the oven to dry.

3.4.2 Strain extraction

In the MICP technology, the selection of calcium sources significantly influences the solidification effect of sandy soil, as microorganisms adsorb exogenous calcium ions and provide nucleation sites for the formation of colloidal calcium carbonate, thereby affecting the mineralization process. The type of calcium source directly influences the cementation effect by affecting the crystal form and precipitation rate of calcium carbonate. Calcium chloride has been widely demonstrated as an ideal calcium source for microbial mineralization (Zhang et al., 2015; Han and Cheng, 2015b; Jing et al., 2016; Akoğuz et al., 2019). Therefore, this study selects calcium chloride as the calcium source.

According to the urea decomposition equation of microbial mineralization process, it can be concluded that a molecule of urea can be decomposed into a molecule of CO₃^2-, while the sediment calcium carbonate can be generated from a molecule of Ca²⁺and a molecule of CO₃^2-, so the combination of urea and calcium chloride at a molar ratio of 1:1 can improve the utilization efficiency of cementation liquid in the process of microbial mineralization. To study the effects of the concentration and pH value of cementation solution on the microbial mineralization rate and sediment morphology, different concentrations of cementation solution and bacterial solution fixation solution were prepared according to the ratio of nutrients shown in Table 5. After the solute was completely dissolved, NaOH solution or HCl solution was added dropwise, the pH of cementation solution was adjusted to 7.5, and then placed in a 30 °C incubator for storage.

Table 5

Table 5. Mixing ratio of cementing fluid in single grouting.

The finished bacterial liquid culture medium often contains metabolic waste from its growth process, which will affect the normal growth of bacteria and urease activity. Generally, it needs centrifugation and resuspension before normal use. The specific steps are as follows:

1. Pour all the bacterial liquid cultured for 27 h under the optimized conditions into a 1L beaker, mix it evenly, and then subpackage it in a conical flask for standby.

2. Tris HCl buffer (0.05 mol/L, 25 °C) with pH values of 7, 7.5, 8, 8.5 and 9 were prepared respectively.

3. After shaking the measured bacterial liquid evenly, pour it into a 50 mL centrifuge tube and put it into a high-speed freezing centrifuge. Centrifuge it for 10 min at 4 °C, 8000 rpm.

4. Remove the supernatant, add Tris HCl buffer of equal volume, and prepare bacterial suspension with different pH values for standby.

After using the above method, we first took out the strain, poured it into 150 mL of NH₄-YE liquid culture medium, and used it as the mother liquid after shaking the culture for 15 h under the optimized conditions (30 °C, 180 rpm), and then inoculated it into several conical flasks containing 150 mL of NH₄-YE liquid culture medium at the inoculation ratio of 1:150. After shaking the culture for 27 h under the optimized conditions, we centrifuged and resuspended it and put the resuspended bacteria solution in a 30 °C incubator for standby.

3.4.3 Grouting

The method of adding bacteria and nutrient solution (calcium source, urea, etc.) to sand is called the grouting method. At present, the main grouting methods used in MICP technology for sand solidification can be roughly divided into five categories: injection method, step-by-step grouting method, immersion method, stirring method, and spraying method.

Each grouting method has its advantages and limitations, the stirring method can significantly reduce the permeability of sand, enhance its impermeability, and achieve effective solidification by optimizing the distribution of calcium carbonate (Ma et al., 2018; Ma, 2018; Jiang and Soga, 2016; Kim et al., 2013), so we selected the stirring method. The stirring method is similar to the method of preparing concrete mortar. The bacteria solution, cementation solution, and sand particles are mixed and fully stirred to make the bacteria solution and culture solution evenly distributed in the sand. After waiting for a certain time, the sand sample is evenly filled into the mold and finally placed in a suitable environment for maintenance. We poured the suspended bacteria solution with the pH adjusted by Tris HCl buffer into the sand sample, added the cementing solution, and stirred it evenly. The mixture shall be gently compacted after each filling in batches, and samples with uniform density shall be made after several operations. Three samples were made for each group.

3.4.4 Specimen curing

Put the sample made by the stirring method into a 30 °C oven for curing for 3 days; then it was dried in an oven at 70 °C for 2 days. The test specimen is shown in Figure 10.

Figure 10

Figure 10. Microbial sand fixation specimen.

3.5 Mechanical property test and data analysis of test piece

Two groups of aeolian sand specimens consolidated by Bacillus pasteurei in the desert, with pH value as the control condition, and numbered, shown in Table 6.

Table 6

Table 6. Grouping of test pieces.

3.5.1 Compressive strength

The test was conducted on a hydraulic servo universal testing machine manufactured by W+B Company, Switzerland. The uniaxial compressive strength test adopted a displacement control mode, with the loading rate 5.0 mm/min. Loading continued until cracks appeared in the specimen and failure occurred, with relevant data recorded to calculate its compressive strength. The experimental phenomena are as follows.

For Group A specimens, no significant cementation traces were observed on the surface before compression, and considerable sand particle shedding was evident. When the compressive deformation reached approximately 1mm, the specimens exhibited slight expansion, followed by rapid and severe overall fragmentation. Large sand masses collapsed, leading to complete failure. After failure, the specimens broke into multiple block-like sand masses. The shape change of the test piece is shown in Figure 11.

Figure 11

Figure 11. Specimen failure process of group A. (a) Initial stage; (b) Expand; (c) Avalanche; (d) Destroy.

For Group B specimens, before compression, observe that the surface of the test piece is rigid, and white cementation products are obviously visible between sand particles. The surface of the test piece is dry, and there are almost no sand flakes. When compressed about 1mm, a ring-shaped bulge is generated in the middle of the test specimen, then the bulge is broken and collapsed, after that the test piece is collapsed in large pieces, and finally completely destroyed. It can be observed that part of the sand brick inside is not cemented firmly and is still in a loose sand state, while the fragmented shell is massive and relatively solid. The specimen-shape variation is presented in Figure 12.

Figure 12

Figure 12. Specimen failure process of group B. (a) Initial stage; (b) Expand; (c) Avalanche; (d) Destroy.

The force displacement curves of test pieces 1, 2, 3 and 4 are shown in Figure 13. The maximum axial force and compressive strength of specimens 1 to 4 are shown in Table 7.

Figure 13

Figure 13. Force-displacement curve. (a) Sample 1; (b) Sample 2; (c) Sample 3; (d) Sample 4.

Table 7

Table 7. Test data record.

3.5.2 Determination of calcium carbonate content

The compressed sample was completely crushed in a mortar and dropped in ultrapure water to eliminate any remaining soluble salt. The acid pickling procedure was used to determine the calcium carbonate content in the solidified sand block. The calcium carbonate content can be calculated according to Equations 8, 9, and is given in Table 8.

Table 8

Table 8. Calcium carbonate content.

3.5.3 Analysis of test results

As shown in Table 8, the efficiency of microbially induced calcium carbonate precipitation (MICP) under pH = 8 conditions is higher than that under pH = 9 conditions. To further investigate the influence of pH on the structure and cementation properties of the precipitates, this study used field-emission scanning electron microscopy (FESEM) to analyze the micromorphology of calcium carbonate deposits formed under different pH conditions (Figure 14). The results indicate that pH significantly regulates the crystal morphology and particle assembly of calcium carbonate. In the pH = 8 system, the deposits mainly consist of rhombohedral, plate-like, and spherical particles interconnected to form chain-like structures. In contrast, in the pH = 9 system, the deposits exhibit a relatively uniform “cauliflower-like” structure composed of nested spherical particles. This morphological difference reflects how different pH environments directly affect the crystallization habits, growth patterns, and aggregation behaviors of calcium carbonate.

Figure 14

Figure 14. Microstructure of sediments at different pH values: (a) pH = 8 (magnified 200 times); (b) pH = 8 (magnified 500 times); (c) pH = 9 (magnified 200 times); (d) pH = 9 (magnified 500 times).

To clarify the impact of micromorphology on macroscopic cementation effectiveness, the internal structure of sand samples treated with a 2 mol/L cementation solution (pH = 9) was observed (Figure 15). Figures 15a,b show that in the outer region of the sand sample, microbially induced calcium carbonate effectively fills the gaps between sand grains, forming evident cementation. Figures 15c,d further reveal that cementation via calcium carbonate deposition at sand grain contacts is also observable in the central region of the sample. Combined with the micromorphological analysis, it is suggested that the uniform “cauliflower-like” nested structure formed under pH = 9 conditions facilitates the construction of continuous and dense cementation interfaces between sand grains, thereby potentially enhancing the mechanical properties of the cementation system. In contrast, the chain-like structure formed under pH = 8 conditions differs in terms of connectivity and spatial distribution, which may lead to relatively lower cementation uniformity and strength.

Figure 15

Figure 15. Microscopic morphology of sand blocks solidified by the stirring method at pH = 9: (a) Lateral sand particles (magnified 500 times); (b) Lateral sand grains (magnified 2000 times); (c) Central sand grain (magnified 500 times); (d) Central sand grain (magnified 2000 times).

Although the efficiency of calcium carbonate cementation by Sporosarcina pasteurii under pH = 9 conditions is relatively lower, the total amount of calcium carbonate formed under this condition does not decrease significantly due to sufficient oxygen diffusion into the specimen and adequate incubation time. The ample oxygen supply promotes significantly better overall cementation uniformity compared to pH = 8 conditions, resulting in a compressive strength 3-5 times higher, with a maximum of 1.183 MPa. Both microscopic and macroscopic observations collectively indicate that the “cauliflower-like” calcium carbonate structure formed under pH = 9 conditions is more conducive to establishing a continuous and uniform cementation network between sand grains, thereby enhancing the overall mechanical performance. In contrast, under pH = 8 conditions, despite a faster external deposition rate, the dense outer layer hinders oxygen diffusion inward, and the internal cementation uniformity is poorer, ultimately leading to lower overall strength.

4 Conclusion

This research primarily investigates the application of microorganisms to stabilize aeolian sand in desert environments to enhance the sand structure or utilize aeolian sand for construction. The optimal strain and culture conditions for consolidation are identified. The stirring method is selected for sand solidification. Following extensive experimentation, sand brick specimens are fabricated and their compressive strength is evaluated, clearly prove the effectiveness and potential of employing Bacillus pasteurei to solidify aeolian sand by utilizing MICP technology in desert environment protection and civil construction. The sufficiency of oxygen, the uniformity of its distribution, and the synchronicity of specimen cementation will significantly influence the consolidation results. The moist environment and water flow significantly undermine the integrity of the microbially cemented sand bricks.

We use desert sand as the primary raw material to replace the river sand widely used in traditional civil engineering. In addition to safeguarding riverbeds and their ecosystems, this initiative helps improve air quality and the environment in deserts by mitigating dust storms. Simultaneously, employing this material can diminish the carbon footprint associated with the production and transportation of conventional construction materials, thereby introducing a novel sustainable methodology to environmentally conscious civil engineering.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

YX: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Supervision, Writing – review and editing. SS: Data curation, Formal Analysis, Visualization, Writing – review and editing. MM: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – original draft. F-YD: Data curation, Formal Analysis, Investigation, Writing – review and editing. Y-QG: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing – review and editing. XH: Conceptualization, Formal Analysis, Investigation, Resources, Validation, Writing – review and editing. TZ: Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – review and editing. KS: Formal Analysis, Methodology, Project administration, Supervision, Writing – review and editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This study was financially supported by the National Natural Science Foundation of China with Grant No. 52278505, 52108443, Jiangsu Province International Cooperation Project with Grant No. SBZ2022000169, the Zhi Shan Scholarship of Southeast University, Innovation and Entrepreneurship Program (Innovation and entrepreneurship Doctor) of Jiangsu Province with Grant No. JSSCBS20210132, and the Fundamental Research Funds for the Central Universities 2242025F10009.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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