# Modelling Microbially Induced Carbonate Precipitation (MICP) in Microfluidic Porous Chips

Authors: Shuang Shu; Haoran Chen; Hao Meng
Journal: Geofluids
 (2022)

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## Abstract

Microfluidics is a new experimental approach that investigates the processes related to fluids flowing through small channels or porous media. Microfluidic chips, the core elements of the microfluidic technique, consist of quasi-2-dimensional pore structures etched and sealed in transparent materials. Fluids are allowed to flow through the chips, and real-time observations can be made about the transport and reactions of fluids at the pore scale. In this study, we test the feasibility of using the microfluidic technique to visualise the microbially induced carbonate precipitation (MICP) process. The experimental results obtained in this study show that the generation, evolution, and morphology of calcium carbonate, the effective product in MICP, can be clearly observed during the entire spatial and temporal range of the tests. Comparative tests with different testing variables, including the concentration of treatment solutions, activity of bacterial suspensions, and particle size, were also carried out. The results show that the influence of these variables on the treatment effects, such as the production rate of the reaction, spatial distribution of calcium carbonate, type and morphology of crystals, amount of ammonia gas produced, and presence/absence of clogging issues, can be visualised in microfluidic chips. The results presented in this paper prove that the microfluidic technique could be a helpful tool for unravelling the mechanisms of the MICP process at the pore scale and understanding the factors influencing the MICP process.


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## Body

## 1. Introduction

Microbially induced carbonate precipitation (MICP) is an alternative soil treatment technique. It involves the microbially-mediated precipitation and crystallisation of carbonate salt. The production of carbonate salt in soil pores can greatly alter the strength, stiffness, and permeability of host soils. The MICP method can potentially be used in a wide range of applications, such as ground improvement, seepage control, and rehabilitation of contaminated soil [1–7]. A notable feature of the MICP method is that the materials used in the soil treatment (bacterial suspensions, and treatment solutions containing urea and calcium) have low viscosity and can be applied to soil simply by percolating them through soil pores. The precipitation and crystallisation of carbonate salt can occur along the flow paths. Soil column tests have been adopted in many experimental studies [8–12]. In these experiments, the outcome of the treatment is dependent on many factors, including grain size, porosity of the soil, amount of bacteria, concentration of the treatment solution, and water head applied to the soil. Although considerable amounts of research effort have been exerted, a clear understanding of the effects of these factors on the treatment of soil is still difficult. Conventional experimental methods, such as strength tests, permeability tests, and SEM analysis, also have limitations for understanding the fundamental mechanisms of the MICP process.The microfluidic technique could address the abovementioned problems. Microfluidic chips, the core elements of the microfluidic technique, are miniature models that allow small amounts fluids to flow through. These models consist of flow channels etched and sealed in transparent materials. The flow channels in the chips are designed to mimic channelled or porous structures (e.g., soil pores, rock fractures, or blood veins) to reproduce the real phenomena that occur in these environments. Meanwhile, these phenomena can be visualised and recorded using optical microscopes. Currently, the microfluidic technique has been used in the fields of chemistry, biology, medicine, and geology not only for research but also practically [13–19].The microfluidic technique has several remarkable advantages for the study of MICP. The reactions can be visualised such that the underlying mechanisms of the MICP process at the pore scale are revealed. Only small amounts of liquid are required for the tests compared with tests in real soil. The experimental time can also be reduced. Because of the reduced cost and time, more parallel, parametric tests can be carried out. However, Wang and Soga [20–22] may be the only preliminary study on this topic until now. In this paper, a series of microfluidic porous chips that represent real soil are fabricated and used for MICP study.


## 2. Materials and Methods

### 2.1. Microfluidic Porous Chips

The design of the microfluidic chip used in the study is shown in Figure1. The range of the porous medium consists of quasi-2-dimensional particles and pores. The shapes and sizes of the particles were determined according to ASTM C778-graded sand. The pores were etched into the chips to a depth of 0.2 mm, which is larger than most of the pore throats between particles. A cover slide was attached to the etched side of the chip to form a complete chip. The material used to fabricate the chips was polydimethylsiloxane (PDMS). A chip used in the experiment is shown in Figure 2. The chips used in this study were designed by the authors and fabricated by Wenhao Chip Technology Co., Ltd, China.Figure 1
Schematic of a microfluidic porous chip.Figure 2
Picture of a microfluidic porous chip.


### 2.2. Bacteria and Treatment Solutions

Microbial ureolytic process, which is mediated by ureolytic bacteria, can produce carbonate and ammonium ions. If dissolved calcium salt is present in the system, calcium carbonate can be precipitated. In this condition, precipitated calcium carbonate is prone to forming crystals. In this study,Sporosarcina pasteurii, a species with the capability to hydrolyse urea, was used. The bacterial growth medium consisted of 20 g/l yeast extract, 10 g/l NH4Cl, 10 mg/l MnSO4 · H2O, and 24 mg/l NiCl · 6H2O, and the pH was adjusted to 9 with the addition of NaOH solution. The growth medium was autoclaved before inoculation. The bacteria were cultivated aseptically and aerobically at 30°C for 1-2 days. The bacteria were then centrifuged and suspended in 0.9% NaCl solution prior to use. Bacterial suspensions with different activities were obtained by diluting the biomass with different volumes of NaCl solution. The urease activity of the bacterial suspensions was measured based on the change of electric conductivity of a urea solution in a certain period of time. The test procedure followed that used by Whiffin et al. [23]. An increase in electric conductivity of 1 mS/min/cm is equivalent to a reduction in the urea concentration of 8.83 mM/min in the test. A bacterial suspension with 1 mS/min/cm activity has 3.1 × 108 CFU per mL. The treatment solutions consisted of equimolar urea and calcium chloride at different concentrations for different tests.


### 2.3. Experimental System

The experimental system is schematically shown in Figure3. A syringe pump (Shenchen SPLab01, China) was used to push liquids into the chip at a constant rate. The MICP process in the microfluidic chip was visualised using an optical microscope (Olympus BX61, Japan). Images could be recorded by a camera integrated within the microscope.Figure 3
Schematic of the test set-up.


### 2.4. Testing Procedure

After assembling all the parts of the test setup, the microfluidic chip should be saturated. The saturation was done by either pumping in or extracting de-ionised water through the chip using the syringe pump. After saturation of the chip, the bacterial suspension was injected into the chip for inoculation of the porous medium, and a 2 hour retention time was provided to facilitate cell attachment. Then, the treatment solutions were injected. During the injection of these fluids, photos were taken at different points on the chip.


## 2.1. Microfluidic Porous Chips

The design of the microfluidic chip used in the study is shown in Figure1. The range of the porous medium consists of quasi-2-dimensional particles and pores. The shapes and sizes of the particles were determined according to ASTM C778-graded sand. The pores were etched into the chips to a depth of 0.2 mm, which is larger than most of the pore throats between particles. A cover slide was attached to the etched side of the chip to form a complete chip. The material used to fabricate the chips was polydimethylsiloxane (PDMS). A chip used in the experiment is shown in Figure 2. The chips used in this study were designed by the authors and fabricated by Wenhao Chip Technology Co., Ltd, China.Figure 1
Schematic of a microfluidic porous chip.Figure 2
Picture of a microfluidic porous chip.


## 2.2. Bacteria and Treatment Solutions

Microbial ureolytic process, which is mediated by ureolytic bacteria, can produce carbonate and ammonium ions. If dissolved calcium salt is present in the system, calcium carbonate can be precipitated. In this condition, precipitated calcium carbonate is prone to forming crystals. In this study,Sporosarcina pasteurii, a species with the capability to hydrolyse urea, was used. The bacterial growth medium consisted of 20 g/l yeast extract, 10 g/l NH4Cl, 10 mg/l MnSO4 · H2O, and 24 mg/l NiCl · 6H2O, and the pH was adjusted to 9 with the addition of NaOH solution. The growth medium was autoclaved before inoculation. The bacteria were cultivated aseptically and aerobically at 30°C for 1-2 days. The bacteria were then centrifuged and suspended in 0.9% NaCl solution prior to use. Bacterial suspensions with different activities were obtained by diluting the biomass with different volumes of NaCl solution. The urease activity of the bacterial suspensions was measured based on the change of electric conductivity of a urea solution in a certain period of time. The test procedure followed that used by Whiffin et al. [23]. An increase in electric conductivity of 1 mS/min/cm is equivalent to a reduction in the urea concentration of 8.83 mM/min in the test. A bacterial suspension with 1 mS/min/cm activity has 3.1 × 108 CFU per mL. The treatment solutions consisted of equimolar urea and calcium chloride at different concentrations for different tests.


## 2.3. Experimental System

The experimental system is schematically shown in Figure3. A syringe pump (Shenchen SPLab01, China) was used to push liquids into the chip at a constant rate. The MICP process in the microfluidic chip was visualised using an optical microscope (Olympus BX61, Japan). Images could be recorded by a camera integrated within the microscope.Figure 3
Schematic of the test set-up.


## 2.4. Testing Procedure

After assembling all the parts of the test setup, the microfluidic chip should be saturated. The saturation was done by either pumping in or extracting de-ionised water through the chip using the syringe pump. After saturation of the chip, the bacterial suspension was injected into the chip for inoculation of the porous medium, and a 2 hour retention time was provided to facilitate cell attachment. Then, the treatment solutions were injected. During the injection of these fluids, photos were taken at different points on the chip.


## 3. Results

Results of five tests with different testing variables are presented and comparatively analysed here. These testing variables include the concentration of treatment solution, bacterial activity, and particle size. Detailed parameters of each test are provided in Table1. Images at three points, A, B, and C, as marked in Figure 1, are presented. These three points are located at the front, middle, and rear locations of each chip along the flow direction.Table 1
Testing parameters.
Test no.Parameters of the microfluidic chipsParameters of the bacterial injectionParameters of the treatment solution injectionMean particle size (m)Void ratio∗Bacterial activity (mS/cm/min)Bacterial injection rate (μl/min)Bacterial injection duration (min)Concentration of urea & calcium chloride (M)Treatment solution injection rate (μl/min)Treatment solution injection duration (hr)10.411.510100.1212020.411.510100.522430.410.210100.522440.211.510100.5212 (clogged thereafter)50.210.210100.5224∗: Because the microfluidic porous chips are quasi-2-dimensional in shape, void ratio is defined as the ratio of the area covered by the pores to the area covered by the particles.Test 1 is used here to portray the MICP process in the microfluidic chip. The results are presented in Figure4. After injection of the bacterial suspension, some biomass accumulated in the pores. With the injection of the treatment solution, crystals of calcium carbonate gradually arose and grew. For the three crystals A, B and C, as circled in Figure 4, the lengths of the crystals gradually and continuously grew with time (Figure 5(a)). Meanwhile, at Locations A, B and C, the total number of crystals remained roughly the same during the treatment course, as seen in Figure 5(b). Most of the crystals formed in this test took on a square or cubic shape. This indicates that the crystals formed in the tested condition are mainly calcite. There were also a few crystals with a circular shape, which could be a vaterite form of calcium carbonate. The shapes of different morphologies of calcium carbonate can be seen in Figure 6 (Figure reprinted from Niedermayr et al. [24]). Calcite and vaterite are seen more often than other morphologies [25].Figure 4
Results of Test 1 portraying the MICP process in the microfluidic porous chips.Figure 5
Growth of crystals in a microfluidic porous chip. (a) Length of Crystals A, B and C (as circled in Figure4) over time; (b) Number of crystals over time at Locations A, B and C (the length of crystals is the length of the projection of the crystals in the horizontal direction).
(a)(b)Figure 6
Polymorphs of CaCO3 (reprinted from Niedermayr et al. [24]).
(a)(b)(c)(d)(e)(f)The results of Tests 1 and 2 are comparatively presented in Figure7. The concentrations of the treatment solutions for Tests 1 and 2 were 0.1 and 0.5 M, respectively. The calcite form of crystals (square or cubic shape) clearly dominated in Test 1, while in Test 2, the crystals took on circular or near-circular shapes, which is likely vaterite. As seen in Figure 7, in Test 2, a large volume of gas bubble was generated in the pore spaces, while in Test 1, the volume of bubble was much less. Gas should be of enough concern since it can clog the porous spaces, alter flow channel of the cementation solution and thus lead to a heterogeneous treatment result. According to previous studies, ammonia gas is an adverse byproduct in the ureolysis-based MICP process and an effective way to reduce the production of ammonia gas was to use treatment solutions of lower concentrations [26]. However, As PDMS is a gas permeable material, the bubbles may also be the air penetrating from the surface of the chip or simply due to inappropriate control of inlet pipes. Future studies are required to explore the causes of generating bubbles and their effect on the process of MICP.Figure 7
Comparative results of the final treatment effects in Tests 1 and 2 showing the influence of the concentration of treatment solution on the MICP process.Bacterial activity is also an important factor in the MICP process. In Figure8, the results of Tests 2 and 3 are presented. Tests 2 and 3 have bacterial activities of 1.5 and 0.2 mS/cm/min, respectively. The crystals in Test 3 were smaller and formed more slowly than those in Test 2. This indicates that bacterial activity strongly influenced the reaction rate of the MICP process. Such results also imply that the use of a bacterial suspension with lower activity may lead a larger range of improvement in the scale of real soil grounds.Figure 8
Comparative results of the final treatment effects in Tests 2 and 3 showing the influence of bacterial activity on the MICP process.The size of soil particles affects the permeability and transport of liquids in soil. In Figure9, three tests, Tests 2, 4, and 5, are presented to illustrate the effect of particle size on the MICP process. Test 2 had a mean particle size of 0.4 mm, and Tests 4 and 5 had a mean particle size of 0.2 mm. When the bacterial activity was 1.5 mS/min/cm, precipitated crystals were relatively uniform in Test 2, while in Test 4 the distribution of crystals was non-uniform, and crystals tended to accumulate at the front range of the porous section. The non-uniform distribution of crystals can cause clogging of the soil pores and impede further MICP treatment. In Test 4, pores were clogged after 12 hours of treatment solution application. The clogging of the chip is apparent because the syringe pump stops operating. However, when the bacterial activity was reduced to 0.2 mS/min/cm, the clogging issue was relieved, as displayed in Test 5 in Figure 9.Figure 9
Comparative results of the final treatment effects in Tests 3, 4, and 5 showing the influence of particle size on the MICP process.


## 4. Conclusions

This paper presents an experimental study of using microfluidic porous chips to visualise and understand the MICP process at the pore scale in real time. The following conclusions can be drawn:(1)
Pore-scale observations of the MICP process in real time show that the size of the crystals gradually increases. However, the total number of crystals remains roughly the same during the treatment course. Both calcite and vaterite forms of calcium carbonate appeared in the treatment(2)
Parametric tests on different testing variables, including the concentration of treatment solutions, activity of the bacterial suspensions, and particle size, show that these parameters have significant effects on the results of the test, such as the production rate of the reaction, the spatial distribution of calcium carbonate, the type and morphology of crystals, the amount of gas produced, and the presence/absence of clogging issues. In turn, these differences can greatly influence the quality of the treatment results. Future studies are required to apply the results obtained in microfluidic chips to mid- and large-scale studies



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