Improvement of the Sand Quality by Applying Microorganism-induced

Calcium Carbonate Precipitation to Reduce Cement Usage

KONGTUNJANPHUK S.1,*, PIANFUENGFOO S.2, SUKONTASUKKUL P.2

1Department of Biotechnology,

King Mongkut’s University of Technology North Bangkok,

1518 Pracharat 1 Rd. Wong Sawang, Bangsue, Bangkok 10800,

THAILAND

2Construction and Building Materials Research Center (CBMRC),

King Mongkut’s University of Technology North Bangkok,

1518 Pracharat 1 Rd. Wong Sawang, Bangsue, Bangkok 10800,

THAILAND

*Corresponding Author

Abstract: - This research determines the Microbially Induced Calcium Carbonate Precipitation (MICP) process

utilized by the bacteria found in Thailand. Many researchers typically use the high-efficiency MICP bacteria to

precipitate calcium carbonate. However, it is only available in some countries, leading to a high import

expense. Therefore, the methodology for using the bacteria capable of producing calcium carbonate in Thailand

was investigated. The five pure bacteria strains are obtained from the Thailand Institute of Scientific and

Technological Research (TISTR), i.e., Proteus mirabilis TISTR 100, Bacillus thuringiensis TISTR 126,

Staphylococcus aureus TISTR 118, Bacillus sp. TISTR 658 and Bacillus megaterium TISTR 067. To screen

urease production, the bacteria were spread on Christensen's Urea Agar (UA) slant surface via a colorimetric

method. All bacteria strains can produce urease enzymes by observing the color changes in the UA. Berthelot's

method was used to determine the urease activity. The result shows the bacteria's urease activity: 2389, 1989,

1589, 789, and 589 U/ml, respectively. These directly lead to calcium carbonate production: 3.430, 3.080,

2.590, 1.985, and 1.615 mg/ml, respectively. Despite the bacteria in this research having a low precipitation

efficiency compared to the strain used in many research studies, they can improve sand stabilization in 7 days.

Proteus mirabilis TISTR 100 was the most stable and effective strain for the MICP process in Thailand. Hence,

this research reveals the ability of the local bacteria to bond with the sand particle. Briefly, the improvement of

the MICP process in sand stabilization can be improved to reduce imported expenses. In addition, the MICP

process can reduce the use of cement in sand stabilization work.

Key-Words: - MICP, Cement consumption, Biofilm, Improvement, Ureolytic bacteria, Cemented sand.

Received: July 21, 2023. Revised: April 19, 2024. Accepted: June 6, 2024. Published: July 3, 2024.

1 Introduction

The current world is facing the issue of global

warming, partly caused by environmental

degradation resulting from the mining of limestone

to produce cement. This process leads to pollution,

such as dust and air pollution, and the destruction of

natural habitats for wildlife and forests. Hence, the

researchers have discovered the significance of the

combination of biotechnology and industrial

construction in developing innovative approaches to

reduce cement usage for environmental

sustainability.

Microbially induced carbonate precipitation

(MICP) may be defined as one environmentally

friendly approach that can improve the properties of

soil and sand, [1], [2]. The MICP method uses the

ureolytic bacteria which can produce the enzyme

urease to accelerate the process of urea hydrolysis,

leading to the formation of calcium carbonate, [3],

[4].

This research selected the pure strain of each

bacteria, mainly within Thailand from the Thailand

Institute of Scientific and Technological Research

(TISTR). The growth conditions of the bacteria

strain were controlled by maintaining temperature

and adjusting the pH value. The optimal conditions

to ensure the production of urease enzymes,

temperature, and pH are the major roles in inducing

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

295

Volume 20, 2024

calcium carbonate precipitation from various

calcium sources, such as calcium lactate, calcium

nitrate, [5], [6] and calcium chloride [7]. Calcium

chloride is typically used in MICP processes due to

the higher rate of calcium carbonate precipitation

than the other sources, [8].

The MICP mechanism starts with the

catalyzation of urea hydrolysis by urease activity.

This reaction generates the carbonate and

ammonium compounds forming, as shown in

Equation 1. Subsequently, the presence of

ammonium increases the pH value of the solution,

promoting the precipitation of calcium carbonate, as

shown in Equation 2, [9].

CO(NH2)2+2H2O→CO3

2-+2NH4

+ (1)

Cell+Ca2++CO3

2-→Cell+CaCO3 (2)

The MICP is mainly focused on calcium

carbonate deposits in soil and sand to enhance their

stability and reduce soil erosion, [10], [11], [12]. It

can be used as a bonding between sand particles to

improve their cohesiveness and formability, which

is superior to untreated sand, [13], [14], [15], [16].

Furthermore, the bacterial biofilm formation

effectively enhances the adhesiveness of the bacteria

to sand surfaces. This biofilm formation also

supports the efficacy of the MICP process, resulting

in higher sand stability, [17], [18], [19], [20], [21].

This research aims to investigate the quality

improvement of sand stabilization by applying the

MICP process to precipitate calcium carbonate as a

binding material between sand particles to enhance

shape stabilization. This study potentially opts for

the bacteria strains that found in Thailand.

Experimental trials are conducted to determine the

optimal concentration of calcium chloride solution

that induces the highest calcium carbonate

precipitation for various bacterial strains. These

selected conditions will be further studied to

improve the bonding characteristics and strength of

the sand, which can be based on sustainable future

developments in real-world construction.

2 Materials and Methods

2.1 Materials Preparation

The sample sand used in this research was obtained

from the Chao Phraya River, Nonthaburi Province,

Thailand. Sand passed through the sieve No. 30 and

was retained on sieve No. 200 in a sieve shaker

(RETSCH®, AS 200 Basic, Germany), which have a

particle size within the range of 75-600 µm using a

sieve (RETSCH®, Woven Wire Mesh Sieves -ⵁ 200

mm., Germany) that complied with ASTM E11

standards, [21]. After that, the sand was washed

thrice with deionized water (DI) and then dried in a

hot air oven (Memmert, Universal oven UN30m,

Germany) at 105°C for 48 hr.

The maximum dry density of the sand was

conducted according to the ASTM D698 (Standard

Efford Compaction), [22] and it was found that the

sand in this research had an optimum moisture

content of 17.5% and a maximum dry density of

1.60 g/cm3.

2.2 Medium Preparation and Cultural

Method

2.2.1 Cultural Medium Preparation

The preparation of cultural medium using Nutrient

Broth containing urea (NBU) began by weighing 13

g of the Nutrient Broth powder (HIMEDIA®, M002,

India) and dissolving it in 1000 ml of distilled water.

Then, 20 g of urea (KEMAUS, Australia) was

added, and the medium was dissolved completely

using a hot plate and magnetic stirrer (IKA®, C-

MAG HS 7, Germany). Next, 100 ml of the

prepared NBU was transferred into a 250-ml

Erlenmeyer flask and then sterilized in an autoclave

(HIRAYAMA, HICLAVE HVA-110, Japan) at a

temperature of 121°C and a steam pressure of 15 psi

for 15 min.

2.2.2 Urease Test

The urease screening by Christensen's Urea Agar

(UA) (HIMEDIA®, M112, India) was prepared by

dissolving 24 g of nutrient powder in 950 ml of

distilled water. The medium was dissolved

completely and sterilized in the autoclave at a

temperature of 115°C and a steam pressure of 10 psi

for 20 min. Afterward, the solution was left cool to

50°C.

The concentrated urea solution of 40 %w/v 50 ml

was aseptically added to the UA. Finally, the UA 5

ml was transferred into the empty sterilized vials,

and the vials were placed at an inclined angle of 30

degrees to allow for proper solidification (agar

slant).

The urease screening by Stuarts' Urea Broth

(UB) (HIMEDIA®, M111, India) by completely

dissolving 18.71 g of the nutrient powder in 950 ml

of distilled water. Subsequently, the solution is

sterilized in the autoclave at a temperature of 121°C

with a steam pressure of 15 psi for 15 min.

Afterward, the solution was left cool to 50°C.

The 50 ml of concentrated urea solution was

aseptically added to the UB solution. Finally, the 5

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

296

Volume 20, 2024

ml of UB solution is divided into empty sterilized

vials.

2.2.3 Biofilm Detection

The preparation of Tryptone Soya Broth (TSB)

(HIMEDIA®, M011, India) for biofilm-forming of

the bacteria. By weighing 30 g of the nutrient

powder and dissolving it in 1000 ml of distilled

water using the hot plate and magnetic stirrer. Then,

100 ml of the TSB solution is transferred into a 250-

ml Erlenmeyer flask and sterilized in the autoclave

at a temperature of 121°C and a steam pressure of

15 psi for 15 min.

2.2.4 Calcium Source Preparation

The calcium source for calcium carbonate

precipitation in the research is a calcium chloride

dihydrate solution (KEMAUS, Australia). It was

dissolved by DI water to adjust the concentrations

range from 100 to 300 mM. The various

concentrations are modified to determine the

optimal conditions for producing calcium carbonate

in each bacteria strain.

2.2.5 Bacteria Cultural Method

The selection of pure bacterial strains used in the

experiments was conducted at the Thailand Institute

of Scientific and Technological Research (TISTR).

Five pure strains were chosen, including P. mirabilis

TISTR 100, B. thuringiensis TISTR 126, S. aureus

TISTR 118, Bacillus sp. TISTR 658, and B.

megaterium TISTR 067. Cell activation was

performed by adding freeze-dried cells in an

ampoule to vials containing Nutrient Broth (NB) as

the culture medium, with a volume of 5 ml

(comprising NB at a concentration of 13 g/L). The

cells were incubated at 37°C for 24 hr. in an

incubator. Subsequently, the bacterial strains were

inoculated into the NBU medium to achieve a cell

density of 1%. The cultures were further incubated

at 37°C for 24 hr.

Following incubation, each bacterial strain was

subjected to a growth curve analysis. The growth

rate was measured at regular intervals using a

visible spectrophotometer (JENWAY®, 7200,

United Kingdom) with a wavelength of 600 nm.

This analysis aimed to determine the maximum

growth rate for each bacterial strain, which would

be utilized in subsequent experimental steps. From

the reading O.D.600, calculate the cell concentration

using Equation 3, [23].

Y = 8.59 × 107 ∙ Z1.3627 (3)

Where, Y is the cells concentration (cells/ml)

Z is the reading O.D.600

2.3 Microbiological Analysis

2.3.1 Gram Staining Method

The glass slides were cleaned using 95% ethanol

and then passed through the flame of a Bunsen

burner using a slide holder. Following this, a loop

sterilized by flame was used to transfer a small

amount of bacterial-suspended solution onto the

glass slide. The slide was allowed to air dry and

then held at one end while passing through the

flame of the Bunsen burner several times with the

smear-side facing up. Afterward, the smear on the

slide was stained with crystal violet for 1 min. The

slide was then gently rinsed with distilled water.

Next, Gram's iodine was applied to the smear for 1

min., followed by elution with 95% ethanol until the

eluted solution ran almost clear. The slide was

rinsed again with distilled water and then stained

with safranin O for 1 min. Subsequently, the slide

was rinsed with distilled water and blot-dried before

being observed under an optical microscope (Nikon,

Eclipse E100, Japan) at 100x magnification.

2.3.2 Biofilm Detection and Screening

To investigate biofilm formation by the Microtiter

plate assay of different bacterial strains' attachment

to calcium carbonate granules, [17]. Each strain of

bacteria was cultured in TSB at 37°C for 24 hr.

Subsequently, the bacterial strains were diluted to a

concentration of 1% in TSB and dispensed into a

96-well Microtiter plate. Incubate the plate at 37°C

for 24 hr. After incubation, the bacteria were

washed twice with phosphate-buffered saline (PBS)

and allowed to air dry for 1 hour. The biofilm

staining is performed by adding 1% crystal violet

solution (200 µl) for 5 min., resulting in a purple

coloration. Subsequently, the stain was rinsed off

with distilled water thrice. Finally, the absorbance

of the wells with 95% ethanol (200 µl) measures at

570 nm using a microplate reader (Metertech,

M965+, Taiwan)—this measurement allowed for the

quantification of light absorption, indicating the

extent of biofilm formation.

2.3.3 Urease Screening

The bacteria were cultured on the UA slant using a

sterile loop to screen bacteria capable of producing

urease for urea hydrolysis reaction between urea and

calcium chloride to form calcium carbonate

precipitate. The slants were then incubated at 37°C

for 24 hr. to test for any color changes in the culture

medium. A pinkish-red color change in the culture

medium indicated a positive result (+).

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

297

Volume 20, 2024

2.3.4 Urease Activity

Using Berthelot's Method, the enzyme samples were

collected from the bacterial culture medium in

Section 2.2. The bacterial culture was transferred

into tubes and then centrifuged at 4000 rpm for 30

min. at 4°C (Eppendorf, 5810R - Benchtop

Centrifuge, Germany). After centrifugation, the

supernatant with 250 µl was collected. From the

collected sample, a 20-fold dilution of the

supernatant is performed with DI water. Potassium

phosphate buffer with a pH of 8 and a concentration

of 100 mM (1 ml) was added. Then, a starting

solution of urea with a concentration of 100 mM

(2.5 ml) was added. The solution was incubated at

37°C for 5 min. in an incubator (Memmert,

Universal oven UN30m, Germany). Subsequently,

detection of ammonia production by MICP process

was measured by adding 1 ml of Phenol

nitroprusside (Phenol 1% (w/v) and Sodium

nitroprusside 0.05% (w/v)) and 1 ml of Alkaline

hypochlorite (Sodium hydroxide 0.5% (w/v) and

Sodium hypochlorite 0.84% (v/v)) to catalyze

ammonia production. The solution was further

incubated at 37°C for 30 min. Then, the absorbance

of the solution using the visible spectrophotometer

at a wavelength of 626 nm. The data were further

analyzed to determine the urease activity produced

by each bacteria strain.

In the preparation of the standard ammonium

chloride solution, ammonium chloride (Carlo

Erba™, Germany) was dissolved in distilled water

to achieve concentrations of 200, 400, 600, 800, and

1000 µmol. Then, each concentration of ammonium

chloride solution was prepared the same as the

enzyme solution. The absorbance of these solutions

was measured at a wavelength of 626 nm against a

blank solution. The obtained absorbance values

were plotted on a graph representing the calibration

curve of ammonium chloride solution, as shown in

Figure 1.

Fig. 1: Calibration curve of ammonium chloride

The absorbance to analyze urease activity for

each bacteria strain was plotted on the calibration

curve. Ammonium chloride concentration (X) in

each bacteria strain was calculated using Equations

4 and 5 derived from the calibration curve of

ammonium chloride. Subsequently, the urease

activity was determined using Equation 6, utilizing

the calculated concentration (X).

(y × dilution) = ax + b (4)

x = (y × dilution) - b

a (5)

Where, y is the reading O.D.626

X is an NH4Cl (µmol)

Urease activity = X × Vt

Vsub. × ti × Vsmp.

(6)

Where, Vt is the total volume (ml)

Vsub. Is the volume of the substrate (ml)

ti is the incubation time (min.)

Vsmp. is the volume of enzyme (ml)

2.3.5 Calcium Carbonate Precipitation

Add the calcium chloride solution with a

concentration of 100-300 mM, [15], to the bacterial-

suspended solution to induce the precipitation of

calcium carbonate. After adding the calcium

chloride solution, incubate the resulting solution in

an incubator at 37°C for 1-7 days. [8], filter the

precipitate obtained using a vacuum filtration

system through a Büchner funnel with Whatman

No.4 filter paper. Then, dry the precipitate by

subjecting it to hot air and drying it at 60°C for 24

hr.

2.3.6 Criteria for Microorganism Selection

Select the concentration of calcium chloride solution

that produces the highest level of calcium carbonate

precipitation among the five bacterial strains by

weighing the dried precipitates obtained from each

strain and comparing them.

2.3.7 Chemical Composition by SEM

This experiment was conducted to observe the

microstructure and evaluate the chemical

composition of the calcium carbonate precipitated

using a Scanning Electron Microscope (SEM) with

energy-dispersive X-ray Spectroscopy (EDS) (FEI

QUANTA 450). The experiment captures the

microstructure of the calcium carbonate precipitate

at 1000x magnification.

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

298

Volume 20, 2024

2.4 Sand Sample Preparation

The sand samples obtained from Section 2.1 were

molded into cube shapes with 5 cm in width, length,

and height dimensions, as shown in Figure 2. The

sample was achieved by taking 200 g of sand per

sample, and three samples were prepared for each

bacterial strain. The sand was then mixed with the

bacterial-suspended solution obtained from Section

2.2.3, with a bacterial volume of 35 ml. The sand

was compacted into molds, providing a height of 5

cm. Subsequently, an optimum concentration of

calcium chloride solution, selected based on the

criteria outlined in Section 2.3.6, was added to the

sand. The volume of the calcium chloride solution

was also 35 ml, marking the completion of one

cycle. Following this, a mixture containing NBU

and a calcium chloride solution, each with a volume

of 35 ml, was added to the molds every 24 hr. for 7

days.

Fig. 2: Casting mold

3 Results

3.1 Growth Curve

Based on the study, growth rates were determined

by measuring the light absorbance at a wavelength

of 600 nm, and cell concentration was calculated as

shown in Table 1.

Table 1. Maximum growth rate

Microorganisms

Maximum growth rate (hr.)

Cell concentration (cells/ml)

P. mirabilis TISTR 100

1.60 x 108

B. thuringiensis TISTR 126

1.98 x 108

S. aureus TISTR 118

1.61 x 108

Bacillus sp. TISTR 658

1.75 x 108

B. megaterium TISTR 067

1.91 x 108

(a)

(b)

(c)

(d)

(e)

Fig. 3: Gram strained bacteria: (a) P. mirabilis TISTR 100, (b) B. thuringiensis TISTR 126, (c) S. aureus TISTR

118, (d) Bacillus sp. TISTR 658, and (e) B. megaterium TISTR 067

Table 2. Gram stain

Microorganisms

Gram

P. mirabilis TISTR 100

B. thuringiensis TISTR 126

S. aureus TISTR 118

Bacillus sp. TISTR 658

B. megaterium TISTR 067

Fig. 4: 96-well plate of biofilm detection. Where 1 and 12 are control (blank) 2-3: biofilm formation from P.

mirabilis TISTR 100, 4-5: B. thuringiensis TISTR 126, 6-7: B. megaterium TISTR 067, 8-9: Bacillus sp.

TISTR 658, and 10-11: S. aureus TISTR 118

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

299

Volume 20, 2024

3.2 Gram Strain

Based on the study, Gram staining following the

method of Hans Christian Gram was performed,

[24] and observations were made using a

microscope with a magnification of 100x. The

results are shown in Figure 3, indicating that among

the five bacterial strains examined, P. mirabilis

TISTR 100 was the only strain that exhibited Gram-

negative (-) characteristics, as shown in Table 2.

3.3 Biofilm Detection and Screening

From the results of the experiment on biofilm

formation, as shown in Figure 4, it was observed

that each bacterial strain could produce biofilms.

This phenomenon can be attributed to the binding of

crystal violet, which affects the adhesion of bacteria

and the resulting production of calcium carbonate.

Intense staining indicates higher biological adhesion

compared to faded staining, [25].

The measurement of O.D.570, as shown in Table

3, confirms these findings.

3.4 Urease Screening and Activity

It was observed that all bacterial strains could

produce the enzyme urease, which catalyzes the

hydrolysis of urea. This reaction occurs with the

presence of urea as the substrate in the medium,

increasing the pH of UA and UB. Consequently, the

color of the medium changes from the control (-) to

a positive (+) result, exhibiting a pinkish-red color,

[26], as depicted in Figure 5.

Table 4 shows the urease activity using

Berthelot's method. The result was found that P.

mirabilis TISTR 100 had the maximum urease

activity, approximately 2389 U/ml.

Table 3. The reading O.D.570 (nm) of biofilm

0.073

0.273

0.239

0.218

0.188

0.245

0.302

0.638

0.582

0.316

0.170

0.102

0.103

0.220

0.172

0.201

0.213

0.238

0.332

0.468

0.543

0.387

0.193

0.107

0.101

0.175

0.237

0.199

0.215

0.218

0.267

0.357

0.512

0.177

0.225

0.085

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 5: Urease screening on UA: (a) P. mirabilis TISTR 100, (b) B. thuringiensis TISTR 126, (c) S. aureus

TISTR 118, (d) Bacillus sp. TISTR 658, (e) B. megaterium TISTR 067, and (f) urease screening on UB of all

bacteria strains

Table 4. Urease activity of each microorganism

Microorganisms

Urease activity (U/ml)

P. mirabilis TISTR 100

2389

B. thuringiensis TISTR 126

1989

S. aureus TISTR 118

1589

Bacillus sp. TISTR 658

789

B. megaterium TISTR 067

589

Control (a) (b) (c) (d) (e)

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

300

Volume 20, 2024

3.5 Calcium Carbonate Precipitation

The induction of calcium carbonate precipitation

through microbial calcification was investigated in

the experiment outlined in Section 2.3.5.

Interestingly, distinct variations in the

characteristics of the calcium carbonate precipitates

were observed among the different bacterial strains,

as illustrated in Figure 6.

Furthermore, it was found that the maximum

yielding of calcium carbonate precipitation,

represented by the formation of precipitates,

differed among the various strains of bacteria in the

presence of calcium chloride as the inducing agent.

The calcium carbonate precipitation rate is directly

increased with urease activity. Therefore, the

maximum calcium carbonate precipitation rate was

found in P. mirabilis TISTR 100, followed by B.

thuringiensis TISTR 126, S. aureus TISTR 118,

Bacillus sp. TISTR 658, and B. megaterium TISTR

067. This phenomenon occurred at a pH range of

7.3 to 8.8, as depicted in Figure 7.

3.6 Chemical Composition by SEM

From the result, the calcium carbonate precipitate

from each bacteria has a different shape. The

physical shape was classified into three groups:

regular, round, and irregular compared to the

analytical grade calcium carbonate (KEMAUS). The

regular group comprises analytical grade calcium

carbonate (a small rhombohedral), B. thuringiensis

TISTR 126, and B. megaterium TISTR 067 were

found in a regular shape with a bigger particle than

the control, as shown in Figure 8a), Figure 8c) and

Figure 8f).

The round group comprises P. mirabilis TISTR

100 and Bacillus sp. TISTR 658. However, the

calcium carbonate precipitated showed a different

physical shape in the group, for P. mirabilis TISTR

100 precipitated with a small round particle, and

Bacillus sp. TISTR 658 precipitated with a round,

flat appearance, as shown in Figure 8b) and Figure

8e). The irregular group comprises S. aureus TISTR

118, which has several shapes, as shown in Figure

8d).

The chemical composition of the calcium

carbonate precipitated was determined using the

EDS method, as shown in Table 5. The elements

found in the sample were Calcium (Ca), Carbon (C),

Oxygen (O), Phosphorus (P), and Chlorine (Cl). The

analytical grade calcium carbonate contains only

Ca, C, and O. For the calcium carbonate precipitated

calcium carbonate from each bacteria found an

additional P and Cl.

(a)

(b)

(c)

(d)

(e)

Fig. 6: Precipitated calcium carbonate: (a) P. mirabilis TISTR 100, (b) B. thuringiensis TISTR 126, (c) S.

aureus TISTR 118, (d) Bacillus sp. TISTR 658, and (e) B. megaterium TISTR 067

Fig. 7: Calcium carbonate yield of each bacteria strain

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

301

Volume 20, 2024

Fig. 8: Microstructure of the precipitated calcium carbonate: (a) the analytical grade calcium carbonate, (b)

P. mirabilis TISTR 100, (c) B. thuringiensis TISTR 126, (d) S. aureus TISTR 118, (e) Bacillus sp. TISTR 658,

and (f) B. megaterium TISTR 067, [27], [28]

Table 5. The chemical composition of each bacteria

Sample

Chemical composition (% by weight)

Ca C O content (%)

Analytical grade CaCO3

41.2

7.8

51.0

0.0

100.0

P. mirabilis TISTR 100

32.2

13.4

44.5

7.7

2.2

90.1

B. thuringiensis TISTR 126

35.0

13.8

41.6

8.9

0.7

90.4

S. aureus TISTR 118

37.0

11.0

44.1

5.5

2.4

92.1

Bacillus sp. TISTR 658

41.7

11.3

42.0

3.3

1.7

95.0

B. megaterium TISTR 067

30.0

27.3

34.7

5.2

2.8

92.0

3.7 Improvement of Sand Bonding

The improvement of sand quality by selecting

calcium chloride concentrations that induce optimal

calcium carbonate precipitation was investigated.

This process resulted in the formation of the

highest level of calcium carbonate precipitates for

each bacterial strain, as observed in the

experimental results depicted in Figure 7, as shown

in Figure 9.

It was observed that the sand samples that

experienced quality improvement, which allowed

them to maintain their structure and stability most

effectively, had undergone quality improvement

through applying P. mirabilis TISTR 100, as

represented in Figure 9a. This finding aligns with

the results obtained from the investigation on the

highest capability to produce calcium carbonate

precipitates.

Using the natural microorganisms present

within the sandy soil can precipitate calcium

carbonate to form bonds in the sandy soil particles,

[29]. It was found that naturally existing bacteria

still depend on nutrients and calcium sources used

to precipitate calcium carbonate appropriately.

Using Sporosarcina pasteurii to precipitate with the

optimum conditions were able to cause the highest

calcium carbonate precipitation in sand, [30]. When

compared with the bacteria strains in this research

(according to the optimum conditions of each

strain), they can form calcium carbonate bonding,

leading to the sand maintaining its shape.

(a)

(b)

(c)

(d)

(e)

Fig. 9: Improvement of sand: (a) P. mirabilis TISTR 100, (b) B. thuringiensis TISTR 126, (c) S. aureus TISTR

118, (d) Bacillus sp. TISTR 658, and (e) B. megaterium TISTR 067

100 µm

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

302

Volume 20, 2024

4 Conclusion

In this study, we proposed and demonstrated the

MICP for sand improvement as a new eco-friendly

bio-mineralization process. The procedure involved

the selection of bacteria capable of producing

urease enzymes to facilitate the precipitation of

calcium carbonate through urea hydrolysis, which

occurred when the pH was shifted from neutral to

mildly alkaline conditions. The selected bacteria

were found to form biofilms with varying

intensities of coloration, which directly correlated

with the amount of biofilm formation and inversely

correlated with the sand's structural stability.

This investigation used the various strains of

bacteria to improve bonds in sand using the MICP

process. All bacteria strains showed the forming of

calcium carbonate bonds between the sand

particles, resulting in the sand maintaining its shape

even when de-molded. From the experimental

result, P. mirabilis TISTR 100 produces the highest

urease activity and calcium carbonate precipitation.

Therefore, the MICP process may be a method that

can reduce the usage of cement in sand

improvement.

This study has shown that the bacteria isolated

in Thailand have a similar ability to form calcium

carbonate bonding as bacteria typically used

abroad. Future research and development are

warranted to optimize the MICP process

parameters, including the selection of specific

microorganisms and their cultivation conditions,

and to evaluate the long-term performance and

durability of the MICP-treated sand in a stable

application. Nevertheless, the results of this study

highlight the potential of MICP as a viable and

environmentally friendly technique for sand

improvement, contributing to the advancement of

real-world sustainable construction practices.

Acknowledgement:

The authors are grateful to the Faculty of Applied

Science and Faculty of Engineering, King

Mongkut’s University of Technology North

Bangkok for financial support. Moreover, this

research begins with the cooperation of two

disciplines, which would involve its application in

conjunction with the use of concrete.

Declaration of Generative AI and AI-assisted

technologies in the writing process

During the preparation of this work the authors

used Grammarly in order to check the grammar of

this context. After using this tool/service, the

authors reviewed and edited the content as needed

and take full responsibility for the content of the

publication.

References:

[1] Ghalandarzadeh, S., Maghoul, P.,

Ghalandarzadeh, A., & Courcelles, B.

(2024). Effect of nanoparticle-enhanced

biocementation in kaolinite clay by

microbially induced calcium carbonate

precipitation. Construction and Building

Materials, 414, 1-16,

https://doi.org/10.1016/j.conbuildmat.2024.1

34939.

[2] Gowthaman, S., Koizumi, H., Nakashima,

K., & Kawasaki, S. (2023). Field

experimentation of bio-cementation using

low-cost cementation media for preservation

of slope surface. Case Studies in

Construction Materials, 18, 1-17,

https://doi.org/10.1016/j.cscm.2023.e02086.

[3] Umar, M., Kassim, K. A., & Ping Chiet, K.

T. (2016). Biological process of soil

improvement in civil engineering: A review.

Journal of Rock Mechanics and Geotechnical

Engineering, 8(5), 767-774,

https://doi.org/10.1016/j.jrmge.2016.02.004.

[4] Lai, H.-J., Cui, M.-J., & Chu, J. (2023).

Effect of pH on soil improvement using one-

phase-low-pH MICP or EICP biocementation

method. Acta Geotechnica, 18(6), 3259-

3272, https://doi.org/10.1007/s11440-022-

01759-3.

[5] Chen, Y., Zhao, L., Zi, J., Han, J., & Zhang,

C. (2024). Investigation on the Shear

Behavior and Mechanism of MICP-Treated

Loess Soil. Geofluids, 2024, 1-10,

https://doi.org/10.1155/2024/8001743.

[6] Lv, C., Tang, C.-S., Zhang, J.-Z., Pan, X.-H.,

& Liu, H. (2023). Effects of calcium sources

and magnesium ions on the mechanical

behavior of MICP-treated calcareous sand:

experimental evidence and precipitated

crystal insights. Acta Geotechnica, 18(5),

2703-2717, https://doi.org/10.1007/s11440-

022-01748-6.

[7] Spencer, C. A., Sass, H., & Van Paassen, L.

(2023). Increased Microbially Induced

Calcium Carbonate Precipitation (MICP)

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

303

Volume 20, 2024

Efficiency in Multiple Treatment Sand

Biocementation Processes by Augmentation

of Cementation Medium with Ammonium

Chloride. Geotechnics, 3(4), 1047-1068,

https://doi.org/10.3390/geotechnics3040057.

[8] Achal, V., & Pan, X. (2014). Influence of

Calcium Sources on Microbially Induced

Calcium Carbonate Precipitation by Bacillus

sp. CR2. Applied Biochemistry and

Biotechnology, 173(1), 307–317,

https://doi.org/10.1007/s12010-014-0842-1.

[9] Omoregie, A.I., Palombo, E.A., & Nissom,

P.M. (2020). Bioprecipitation of calcium

carbonate mediated by ureolysis: a review,

Environ. Eng. Res. 26(6), 200379,

https://doi.org/10.4491/eer.2020.379.

[10] Almajed, A., Abbas, H., Arab, M., Alsabhan,

A., Hamid, W., & Al-Salloum, Y., (2020).

Enzyme-induced carbonate precipitation

(EICP)-based methods for ecofriendly

stabilization of different types of natural

sands, J. Clean. Prod., 274, 122627,

https://doi.org/10.1016/j.jclepro.2020.122627

[11] Ivanov, V., & Stabnikov, V., (2017).

Construction Biotechnology. Biogeo-

chemistry, Microbiology and Biotechnology

of Construction Materials and Processes

Springer Singapore, Singapore.

[12] Naveed, M., Duan, J., Uddin, S., Suleman,

M., Hui, Y., & Li, H., (2020). Application of

microbially induced calcium carbonate

precipitation with urea hydrolysis to improve

the mechanical properties of soil, Ecol. Eng.,

153, 1-14,

https://doi.org/10.1016/j.ecoleng.2020.10588

[13] Chen, Y., Han, Y., Zhang, X., Sarajpoor, S.,

Zhang, S., & Yao, X. (2023). Experimental

study on permeability and strength

characteristics of MICP-treated calcareous

sand. Biogeotechnics, 1(3), 1-10,

https://doi.org/10.1016/j.bgtech.2023.100034

[14] Zeitouny, J., Lieske, W., Alimardani

Lavasan, A., Heinz, E., Wichern, M., &

Wichtmann, T. (2023). Impact of New

Combined Treatment Method on the

Mechanical Properties and Microstructure of

MICP-Improved Sand. Geotechnics, 3(3),

661-685,

https://doi.org/10.3390/geotechnics3030036.

[15] Lee, C., Lee, H., & Kim, O. (2018).

Biocement Fabrication and Design

Application for a Sustainable Urban Area,

Sustainability, 10(11), 4079,

https://doi.org/10.3390/su10114079.

[16] Choi, S. G., Chu, J., Brown, R. C., Wang, K.,

& Wen, Z. (2017). Sustainable Biocement

Production via Microbially Induced Calcium

Carbonate Precipitation: Use of Limestone

and Acetic Acid Derived from Pyrolysis of

Lignocellulosic Biomass. ACS Sustainable

Chemistry & Engineering, 5(6), 5183-5190,

https://doi.org/10.1021/acssuschemeng.7b00

521.

[17] Coffey, B.M., & Anderson, G.G. (2014).

Biofilm Formation in the 96-Well Microtiter

Plate. In: Filloux, A., Ramos, JL. (eds)

Pseudomonas Methods and Protocols, 631-

641. Humana, New York, NY.

https://doi.org/10.1007/978-1-4939-0473-

0_48.

[18] Wang, H., Xin, J., Zheng, X., Fang, Y., Zhao,

M., & Zheng, T., (2023). Effect of biofilms

on the clogging mechanisms of suspended

particles in porous media during artificial

recharge, Journal of Hydrology, 619, 1-11,

https://doi.org/10.1016/j.jhydrol.2023.12934

[19] Dhami, N.K., Alsubhi, W.R., Watkin, E., &

Mukherjee, A., (2017). Bacterial Community

Dynamics and Biocement Formation during

Stimulation and Augmentation: Implications

for Soil Consolidation, Front. Microbiol., 8,

1-17,

https://doi.org/10.3389/fmicb.2017.01267.

[20] Terzis, D., & Laloui, L. (2017). On the

application of microbially induced calcite

precipitation for soils: A multiscale study. In

A. Ferrari & L. Laloui (Eds.), Advances in

laboratory testing and modelling of soils and

shales (ATMSS) (pp. 388-394). Springer,

https://doi.org/10.1007/978-3-319-52773-

4_46.

[21] ASTM E11-22 Standard Speciﬁcation for

Woven Wire Test Sieve Cloth and Test

Sieves, [Online]. www.astm.org (Accessed

Date: June 3, 2024).

[22] ASTM D698-12 Standard Test Methods for

Laboratory Compaction Characteristics of

Soil Using Standard Effort (12,400 ft-lbf/ft3

(600 kN-m/m3)), [Online]. www.astm.org

(Accessed Date: July 5, 2021).

[23] Okwadha, G. D. O., & Li, J. (2010).

Optimum Conditions for Microbial

Carbonate Precipitation. Chemosphere,

81(9), 1143-1148,

https://doi.org/10.1016/j.chemosphere.2010.0

9.066.

[24] Paray, A.A., Singh, M., Mir, M.A., and Kaur,

A., (2023). Gram Staining: A Brief Review,

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

304

Volume 20, 2024

International Journal of Research and

Review, 10(9), 336-341,

https://doi.org/10.52403/ijrr.20230934.

[25] Daod, N., Lertworapreecha, M., and

Niyasom, C., (2015). Biofilm Formation and

Adhesin Genes in Staphylococcus aureus

Strains Isolated from Community and

Hospital Settings in Phatthalung Province,

Thailand, Thaksin. J., 18(3), 25-33.

[26] Kohta, M., Koyanagi, H., Inagaki, Y.,

Nishikawa, K., Kobayashi, N., Tamura, S.,

Ishikawa, M., Banno, Y., Takekoshi, K.,

Mano, K., and Sugama, J. (2023). Selective

detection of urease-producing bacteria on the

genital skin surface in patients with

incontinence-associated dermatitis.

International Wound Journal, 20(8), 3289-

3297, https://doi.org/10.1111/iwj.14209.

[27] Pianfuengfoo, S., Kongtunjanphuk, S.,

Zhang, H., & Sukontasukkul, P. (2024). Use

of buffer treatment to utilize local non-alkali

tolerant bacteria in microbial induced

calcium carbonate sedimentation in concrete

crack repair. Heliyon, 10(4), 1-11,

https://doi.org/10.1016/j.heliyon.2024.e2677

[28] Pianfuengfoo, S. (2022). Application of

Biocement as Cementitious Material

[Unpublished master’s thesis]. King

Mongkut’s University of Technology North

Bangkok.

[29] Cardoso, R., Pedreira, R., Duarte, S. O. D.,

and Monteiro, G. A. (2020). About calcium

carbonate precipitation on sand

biocementation. Engineering Geology, 271,

1-8,

https://doi.org/10.1016/j.enggeo.2020.10561

[30] Mirazizi, S., Khayat, N., and Nazarpour, A.

(2023). Effect of Fines Content in Silt-Sand

Mixtures with Different Saturations on

Microbial-Induced Calcium Carbonate

Precipitation. Geomicrobiology Journal,

40(3), 227-237,

https://doi.org/10.1080/01490451.2022.2141

378.

Contribution of Individual Authors to the

Creation of a Scientific Article (Ghostwriting

Policy)

The authors equally contributed in the present

research, at all stages from the formulation of the

problem to the final findings and solution.

Sources of Funding for Research Presented in a

Scientific Article or Scientific Article Itself

The authors are grateful to the Faculty of Applied

Science and Faculty of Engineering, King

Mongkut’s University of Technology North

Bangkok for financial support.

Conflict of Interest

The authors have no conflicts of interest to declare.

Creative Commons Attribution License 4.0

(Attribution 4.0 International, CC BY 4.0)

This article is published under the terms of the

Creative Commons Attribution License 4.0

https://creativecommons.org/licenses/by/4.0/deed.e

n_US

WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT

DOI: 10.37394/232015.2024.20.29

Kongtunjanphuk S.,

Pianfuengfoo S., Sukontasukkul P.

E-ISSN: 2224-3496

305

Volume 20, 2024