energies
Article
Factors Affecting Soybean Crude Urease Extraction and
Biocementation via Enzyme-Induced Carbonate Precipitation
(EICP) for Soil Improvement
Shuang Shu 1 , Boyang Yan 1 , Bin Ge 1 , Shiling Li 2 and Hao Meng 1, *
Citation: Shu, S.; Yan, B.; Ge, B.; Li, S.;
Meng, H. Factors Affecting Soybean
Crude Urease Extraction and
Biocementation via Enzyme-Induced
Carbonate Precipitation (EICP) for
Soil Improvement. Energies 2022, 15,
5566. https://doi.org/10.3390/
en15155566
Academic Editors: Qiang Tang,
Wei Liu and Yucheng Huang
Received: 22 June 2022
Accepted: 28 July 2022
Published: 31 July 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Energies 2022, 15, 5566. https://doi.org/10.3390/en15155566
1 Key Laboratory of Ministry of Education for Geomechanics and Embankment Engineering, Hohai University,
No.1, Xikang Road, Nanjing 210098, China; shuang.shu@icloud.com (S.S.); boyang.yan@icloud.com (B.Y.);
bin.ge@hhu.edu.cn (B.G.)
2 Yancheng Surveying and Design Institute Water Resources Co., Ltd., Yancheng 224000, China;
lishiling1214@163.com
* Correspondence: menghao@hhu.edu.cn
Abstract: Enzyme-induced carbonate precipitation (EICP) is a new biogeotechnical ground improve-
ment technique that uses calcium carbonate (CaCO3 ) formed by biochemical processes to increase soil
strength and stiffness. In this paper, crude urease extracted from soybeans was employed to catalyze
the precipitation of CaCO3 in sand. To optimize the urease extraction efficiency, factors affecting
the soybean crude urease extraction, including the powdered soybean particle size, concentration,
soaking time, and soaking temperature, were addressed. This paper also provided further insight
regarding the impact of the urease activity of soybean crude extract on the chemical conversion
efficiency and the biocementation performance in EICP. The findings revealed that the powdered
soybean concentration and the particle size were the two most important factors affecting the urease
activity of the soybean crude extract. The enzyme activity utilized in the EICP process might further
lead to different reactant efficiencies of urea-CaCl2 solution, and consequently, the improvement in
the physical and mechanical properties of biocemented sand. Considering the chemical conversion
efficiency and the biocementation performance, 60 g/L of powdered soybean was concluded as the
preferred quantity for extracting the crude urease, with an enzyme activity of 6.62 mM urea min−1 .
Under this condition, a chemical conversion efficiency of approximately 95% for 0.5 M urea-0.5 M
CaCl2 could be obtained in merely 12 h, and the unconfined compressive strength (UCS) of the
EICP-treated sand exceeded 4 MPa with a CaCO3 content of ~8%. As a high-efficient cost-effective
alternative to the purified enzyme for carbonate precipitation, the soybean crude urease showed
great potential for ground improvement.
Keywords: enzyme-induced carbonate precipitation (EICP); soybean crude urease; extraction
efficiency; biocementation; carbonate precipitation; ground improvement
1. Introduction
Biocementation is an innovative soil-improvement method for geotechnical and en-
vironmental engineering applications [1–7]. The most effective forms of biocementation,
including enzyme-induced carbonate precipitation (EICP) and microbially induced car-
bonate precipitation (MICP), are achieved via urease-catalyzed urea hydrolysis, whereby
soluble calcium (Ca2+ ) is converted into solid calcium carbonate (CaCO3 ) crystals that ce-
ment individual soil particles together, leading to increased soil strength and stiffness [8–10].
The biochemical process can be described by the following equations:
Urease
CO(NH2 )2 + 2H2 O → 2NH4 + + CO3 2− (1)
Ca2+ + CO3 2− → CaCO3 (s) (2)
https://www.mdpi.com/journal/energies
Energies 2022, 15, 5566 2 of 18

Urease catalyzes the hydrolysis of urea into carbonate ions and ammonium (Equa-
tion (1)). The production of ammonium results in an increase in the pH and creates an
alkaline environment favorable for carbonate formation. The precipitation of CaCO3 crys-
tals occurs when soluble calcium sources are provided (Equation (2)). The end-product
CaCO3 crystal, a natural and non-toxic mineral, can be an excellent alternative to the
traditional Portland cement, which is energy-intensive and environmentally unfriendly
during production [11].
EICP is distinguished from MICP due to its use of free urease to catalyze the hydrolysis
of urea (Equation (1)) rather than the intercellular urease in live bacteria. The free urease
enzyme has a size of 10 nm, 2–3 orders-of-magnitude smaller than the bacterial cells,
facilitating its transport in soil pores [12,13]. In addition, using free urease (EICP) instead
of live bacteria (MICP) eliminates the challenges associated with controlling the microbial
growth (activity) in soils [14].
Though the advantages of EICP are visible, some problems are yet to be fully addressed
before EICP’s further application can be implemented. The cost of purified urease enzyme,
comprising more than half of the total cost of the EICP solution, is one of the major
challenges in large-scale applications [13]. The EICP process catalyzed by the purified
urease enzyme lacks the nucleation sites for CaCO3 formation, which may cause disorder in
the precipitation morphology [15,16]. Additional stabilizers, such as powdered non-fat milk
or foreign CaCO3 crystals, are needed for calcium carbonate precipitation in the biochemical
process as nucleation points. These further increase the costs of EICP [13,17]. Furthermore,
the purified urease is relatively unstable, especially under a high temperature and extreme
pH conditions, leading to the catalyst deactivation and lower reaction efficiency of EICP
compared with MICP [9,15,18]. Soybean crude extract, containing an appreciable amount
of urease and protein, may be an appropriate alternative to the expensive and unstable
purified urease enzyme to catalyze the precipitation of CaCO3 .
This study exploited the key factors affecting the urease activity of soybean crude
extract and the performance of soybean crude urease in EICP. An optimized extraction pro-
cess of the soybean crude urease was proposed, including soybean smashing and soaking,
the salting-out of the excess protein, and liquid-solid separation. The impact of several
possible factors, including the powdered soybean particle size and concentration, soaking
time, and temperature, on the enzyme activity of soybean crude urease was investigated
with the aim of achieving the efficient use of soybeans for crude urease extraction. The
influence of the dosage of soybean crude urease on the efficiency of carbonate precipi-
tation and the physical-mechanical properties of Ottawa standard sand were evaluated
for the optimization process. Analytical methods, including X-ray diffraction (XRD) and
scanning electron microscopy (SEM), were employed to confirm CaCO3 precipitation and
characterize the microstructure development in the biocemented sand.

2. Materials and Methods

2.1. Soybean Crude Urease Preparation and Urease Activity Measurement
Soybeans were purchased from a nearby market and smashed with a grinder. The
crude urease was extracted by mixing powdered soybean and tap water (pH = 7.5). The
extraction process was as follows: (a) the powdered soybean was soaked in tap water and
fully dispersed by stirring; (b) the mixture was preserved at different storage times and
temperatures; (c) food-degraded CaSO4 ·2H2 O (CaSO4 ·2H2 O content ≥ 98%), as an additive
to salt out excess protein, was added into the powdered soybean suspension with a dosage
of 0.1 g CaSO4 ·2H2 O/g powdered soybean, and the mixture was stirred for five minutes;
(d) the mixed solution was centrifuged (4000 rpm, 15 min, and 4 ◦ C) and the supernatant
was collected; and (e) the insoluble substances in the supernatant were removed by filtration
and the soybean crude urease was obtained without further purification. Before other
experiments, the soybean crude urease was stored in a refrigerator at 4 ◦ C.
Urease activity reflecting the urea hydrolysis rate was measured based on the change of
electric conductivity of 1 M of urea at 25 ◦ C. The increased rate of conductivity (mS/min/cm)
 tion and the soybean crude urease was obtained without further purification. B
experiments, the soybean crude urease was stored in a refrigerator at 4 °C.
Urease activity reflecting the urea hydrolysis rate was measured based on
Energies 2022, 15, 5566
of electric conductivity of 1 M of urea at 25 °C. The increased 3rate of 18
of co
(mS/min/cm) was related to the urease activity (mM urea min ) with a ratio −1

according to Whiffin et al. [19]. Note that the change in electric conductivity te
was after
relatedmixing
to the urease −1 ) with a ratio of 1: 11.1, according to
stable the activity
soybean (mM urea min
crude urease and urea solution for 30 min, as
Whiffin et al. [19]. Note that the change in electric conductivity tended to be stable after
Figure 1, and the electric conductivity variation value after mixing for 30 min
mixing the soybean crude urease and urea solution for 30 min, as shown in Figure 1, and
for the
calculating the urease
electric conductivity activity.
variation Themixing
value after batchfortests were
30 min conducted
was used in triplicat
for calculating
average value (±standard deviation) was calculated and will be presented here
the urease activity. The batch tests were conducted in triplicates, and the average value
(±standard deviation) was calculated and will be presented herein.

Figure 1. Change of electrical conductivity with time after mixing the soybeans crude urease and
Figure 1. Change
urea solution (1 M).of electrical conductivity with time after mixing the soybeans crude
urea solution (1 M).
2.2. Parameters Affecting Soybean Crude Urease Extraction
To optimally use the soybeans to extract crude urease for biocalcification, a study of
2.2.several
Parameters
factors Affecting Soybean
that affect the Crude
enzymatic Urease
activity Extraction
of prepared soybean crude urease was
carried out, including
To optimally usethethe
degree to whichto
soybeans theextract
soybeanscrude
are crushed
urease(related to the pow-
for biocalcification,
dered soybean size), the powdered soybean concentration, the soaking time of powdered
several factors
soybean, and thethat affect
soaking the enzymatic
temperature. Five sets ofactivity
powderedof prepared
soybean samplessoybean
with parti-crude u
carried out,
cle size including
ranges of 0–0.075 the
mm,degree
0.075–0.25tomm,
which themm,
0.25–0.5 soybeans
0.5–1 mm,areandcrushed
1–2 mm were(related t
obtained by filtration through different sieves. The sieved powdered
dered soybean size), the powdered soybean concentration, the soaking time of soybean was soaked
in tap water, which diluted it to the desired powdered soybean concentrations (i.e., 20, 40,
soybean, and
60, 80, and 100the soaking
g/L). temperature.
The soaking Five sets
time was controlled fromof0.5
powdered
to 24 h, andsoybean
the soakingsample
ticletemperature
size ranges of from
varied 0–0.075 ◦
mm,
5 to 50 C. 0.075–0.25 mm, 0.25–0.5 mm, 0.5–1 mm, and 1–
obtained by filtration through different sieves. The sieved powdered soybean w
2.3. Sand Samples’ Preparation and Testing Scheme
in tap water, which
To investigate thediluted it to thecrude
effect of soybean desired
ureasepowdered soybean
activity on the concentrations
performance of EICP
60, in80, and 100 g/L). The soaking time was controlled from 0.5
Ottawa sand, four kinds of powdered soybean concentrations were employed to extractto 24 h, and t
the soybean crude urease, i.e.,520tog/L − 1 (4.86 mM urea min−1 ),
temperature varied from −1
50(2.15
°C. mM urea min ), 40 g/L−1
60 g/L (6.62 mM urea min ), and 80 g/L (8.17 mM urea min ), as shown in Figure 2.
During the extraction, the powdered soybean size was 0–0.25 mm, the soaking time of pow-
2.3.dered
Sandsoybean
Samples’
was Preparation and Testing
0.5 h, and the soaking Scheme
temperature was around 25 ◦ C. The cementation
solution (CS) contained 1 mol/L urea and 1 mol/L calcium chloride.
To investigate the effect of soybean crude urease activity on the performan
in Ottawa sand, four kinds of powdered soybean concentrations were employed
the soybean crude urease, i.e., 20 g/L (2.15 mM urea min −1), 40 g/L (4.86 mM u
60 g/L (6.62 mM urea min−1), and 80 g/L (8.17 mM urea min−1), as shown in Fig
ing the extraction, the powdered soybean size was 0–0.25 mm, the soaking tim
dered soybean was 0.5 h, and the soaking temperature was around 25 °C. The ce
Energies 2022, 15, x FOR PEER REVIEW
Energies 2022, 15, 5566 4 of 18

Soybean
Figure2. 2.
Figure crude
Soybean ureaseurease
crude used inused
this study.
in this study.
Poorly-graded Ottawa sand [20] with a mean size of 0.36 mm was used throughout
Poorly-graded
this study. Ottawa
Table 1 summarizes sand [20]
the physical with aofmean
properties sizesand.
the tested of 0.36 mm chloride
Polyvinyl was used thro
this study.
(PVC) Table 1molds
cylindrical summarizes were the physicaltoproperties
employed make the of the
sandtested sand. Polyvin
specimens
(diameter
ride (PVC) = 50cylindrical
mm, lengthmolds= 100 mm).
wereDry sand wastoplaced
employed makeintotheeach
sand mold at a rela- (diame
specimens
tive
mm, density
length of =
~45%
100 by pluviation.
mm). Dry sandEachwas
column was positioned
placed into each vertically
mold at with top anddensity
a relative
bottom open. A low-temperature premixed percolation method was used in this study to
by pluviation. Each column was positioned vertically with top and bottom open
bio-stabilize the sand. Biological reagents (soybean crude urease and cementation solution)
temperature
with premixed
a low temperature (4 ◦ C)percolation methodand
were firstly premixed was thenused in this introduced
immediately study to to bio-stab
sand.
the sandBiological reagents (soybean
by surface percolation crude
(from the top). Theurease andofcementation
infiltration liquid was duesolution)
to the wit
gravity and capillary forces. The low-temperature premixed method’s
temperature (4 °C) were firstly premixed and then immediately introduced to the key feature was that
the urease activity could be temporarily suppressed (see Figure 3) during the infiltration
surface percolation (from the top). The infiltration of liquid was due to the grav
phase, thus inhibiting the fast accumulation of CaCO3 precipitation in the surficial soil.
capillary
Under forces.
surface The low-temperature
percolation premixed
(fully drained) conditions, method’s
the water retentionkey feature
capacity was that th
of Ottawa
activity
sand in thecould
columnsbe was
temporarily
~72 mL. The suppressed (see
retained water wasFigure 3) during
characterized the infiltration
as capillary water pha
and adsorbed water [21]. The low-temperature premixed percolation process
inhibiting the fast accumulation of CaCO3 precipitation in the surficial soil. Under consisted of
the following four
percolation steps:
(fully drained) conditions, the water retention capacity of Ottawa san
1. A totalwas
of 72 ~72
mL tap water ◦ C) was introduced into each column to provide moist
columns mL. The(~25
retained water was characterized as capillary water
conditions for the follow-up EICP treatment;
sorbed
2.
water [21]. The low-temperature premixed percolation process consiste
A total of 36 mL soybean crude urease (4 ◦ C) and 36 mL cementation solution (4 ◦ C)
following fouradequately
were mixed steps: and then poured immediately onto the sand surface;
1. The
3. A total
sand of 72 mLwere
samples tapincubated
water (~25 °C) was
at room (~25 ◦into
introduced
temperature C) foreach column to provid
the biochemical
process to occur for 12 h;
conditions for the follow-up EICP treatment;
4. Steps (2) and (3) were performed several times (2–8) to obtain different cementation
2. levels
A total of soil
of the 36 mL soybean crude urease (4 °C) and 36 mL cementation solutio
samples.
were mixed adequately and then poured immediately onto the sand surface
Table Properties
3. 1.The sand ofsamples
sand usedwere
in this incubated
study. at room temperature (~25 °C) for the bioc
process
Name toDoccur
10 (mm)for 12
D50h;
(mm) Gs (g/cm3 ) emax emin Dr%
4.Ottawa
Steps
sand(2) and (3) were performed
0.26 0.36 several times
2.65 0.735 (2–8) to obtain different
0.467 45 cem
levels of the soil samples.
After the last EICP treatment, 500 mL of tap water was flushed through ea
column from the top to remove the residual unreacted CaCl2, urea and the by-
NH4Cl in the sand samples. During the experiments (including the washing sta
effluents collected from the outlet were used for calcium content measurement.
Energies 2022,2022,
Energies 15, x15,
FOR PEER REVIEW
5566 5 of 518of 18

Figure 3. Effect
Figure of temperature
3. Effect andand
of temperature pH pH
on the enzyme
on the activity
enzyme of soybean
activity crude
of soybean urease.
crude urease.

After the of
Table 1. Properties last EICP
sand usedtreatment, 500 mL of tap water was flushed through each sand
in this study.
column from the top to remove the residual unreacted CaCl2 , urea and the by-product
Name D10 (mm) D50 (mm) Gs (g/cm3) emax emin Dr%
NH4 Cl in the sand samples. During the experiments (including the washing stage), the
Ottawa
effluents collected from the outlet were used for calcium content measurement.45
sand 0.26 0.36 2.65 0.735 0.467

2.4.2.4.
Chemical
ChemicalConversion
ConversionEfficiency, Total
Efficiency, Calcium
Total CalciumCarbonate Content,
Carbonate Content, andand
Calcium
CalciumCarbonate
Carbonate
Distribution
Distribution in Sand
in Sand
TheThe chemical
chemical conversion
conversion efficiency
efficiency of cementation
of cementation solution
solution duringduring
the the
EICPEICP process
process
was calculated as the ratio of the chemical reactant’s
was calculated as the ratio of the chemical reactant’s (CaCl 2) mass (CaCl 2 ) mass that took part
that took part in the in the
biochemical
biochemical reaction
reaction (Equation
(Equation (2))(2)) to the
to the total
total amount
amount of CaCl
of CaCl 2 inputs
2 inputs
at the
at the endpoint
endpoint of of
each test. The quantity of calcium involved was calculated as the difference between the the
each test. The quantity of calcium involved was calculated as the difference between
amount
amount of calcium
of calcium introduced
introduced intointo
sand sand samples
samples andand
the the calcium
calcium content
content in the
in the effluent
effluent
container. Excessive HCl solution (2 M) was added to each effluent
container. Excessive HCl solution (2 M) was added to each effluent container to dissolve container to dissolve
the the possible
possible precipitated
precipitated carbonates.
carbonates. Then,
Then, the the calcium
calcium content
content waswas determined
determined usingusing
the the
EDTA titrimetric method
EDTA titrimetric method [22]. [22].
The The
totaltotal content
content of CaCO
of CaCO in the sand samples was determined by the quantity of
3 in3the sand samples was determined by the quantity of
2+
Ca2+Ca participating
participating in thein the biochemical
biochemical reaction
reaction (as mentioned
(as mentioned previously).
previously). After
After the the
sand sand
specimens were broken during the unconfined compressive test, sand fragments (~10 g
specimens were broken during the unconfined compressive test, sand fragments (~10 g
each) from the top, middle, and bottom of each sample were collected for calcium carbonate
each) from the top, middle, and bottom of each sample were collected for calcium car-
content measurement using the acid-washing method [11].
bonate content measurement using the acid-washing method [11].
2.5. Water Permeability Tests and Unconfined Compressive Tests
2.5. Water Permeability Tests and Unconfined Compressive Tests
Water permeability tests were carried out using the constant head permeability method
followingpermeability
Water tests The
Cheng et al. [23]. were carried outtests
permeability using
werethe constant inside
conducted head the
permeability
PVC column,
method following Cheng et al. [23]. The permeability tests were conducted
where the sand specimens were kept undisturbed. After the permeability tests, inside thethe
PVC
bioce-
column, where the sand specimens were kept undisturbed.
◦ After the permeability
mented samples were oven-dried for 48 h at 50 C. Then, the unconfined compressive tests tests,
the were
biocemented
conducted.samples were oven-dried
The elastic modulus, E50, for was
48 hdetermined
at 50 °C. Then, theslope
as the unconfined
of secantcom-
drawn
pressive
from the origin of the stress–strain curve through the point at 50% of peak stress [24].of
tests were conducted. The elastic modulus, E50, was determined as the slope
secant drawn from the origin of the stress–strain curve through the point at 50% of peak
2.6.[24].
stress Microscale Identification Analysis and Data Analysis
X-ray diffraction (XRD) analysis was employed to identify the crystalline phases of
2.6.the
Microscale
carbonateIdentification Analysis
precipitates. and Data Analysis
The oven-dried sand samples (after the unconfined compressive
tests)
X-raywere crushed(XRD)
diffraction for XRD analysis
analysis using
was a high-resolution
employed to identify X-ray diffractometer
the crystalline phases(PANa-
of
the lytical XPert PRO,
carbonate The Netherlands).
precipitates. Scanning
The oven-dried Electron
sand Microscopy
samples (SEM)
(after the analysis was
unconfined
 compressive tests) were crushed for XRD analysis using a high-resolution X-ray diffrac-
tometer (PANalytical XPert PRO, The Netherlands). Scanning Electron Microscopy (SEM)
Energies 2022, 15, 5566 analysis was performed to examine microstructure development and the morphology of 6 of 18
precipitates in the biocemented sand. Several small pieces of sand samples collected after
the unconfined compressive tests were oven-dried, sputter-coated with gold, and exam-
ined using a Hitachi
performed S4800microstructure
to examine Field Emissiondevelopment
Scanning Electron
and the Microscope (FE-SEM),
morphology Hita- in
of precipitates
chi the
Ltd., Tokyo, Japan.
biocemented sand. Several small pieces of sand samples collected after the unconfined
The one-way
compressive analysis
tests of variationsputter-coated
were oven-dried, (ANOVA) (P with
= 0.05) method
gold, was used using
and examined to compare
a Hitachi
the S4800
mean Field
values of experiment result. P is the probability of the outcome in a
Emission Scanning Electron Microscope (FE-SEM), Hitachi Ltd., Tokyo,statistical
Japan.
experiment.
The one-way analysis of variation (ANOVA) (P = 0.05) method was used to compare the
mean values of experiment result. P is the probability of the outcome in a statistical experiment.
3. Results and Discussion
3.1.3.Factors
Results and Discussion
Affecting Soybean Crude Urease Extraction
3.1. Factors Affecting
3.1.1. Powdered Soybean Soybean
Size and Crude Urease Extraction
Concentration
3.1.1. Powdered Soybean Size and Concentration
To investigate the effect of the powdered soybean size and concentration on soybean
To investigate
crude urease the effect
enzyme activity, theofsoaking
the powdered
time and soybean size and concentration
the temperature were set as 0.5 onhsoybean
and
25 °C, ◦respectively. These measures were taken to make efficient use of the soybeans 0.5
crude urease enzyme activity, the soaking time and the temperature were set as whileh and
25 C, respectively. These measures were taken to make efficient use of
extracting crude urease. It is evident from Figure 4 that both the size and the concentrationthe soybeans while
extracting crude urease. It is evident from Figure 4 that both the size
of the powdered soybean had a significant impact on the soybean crude urease activity. and the concentration
Forofallthe
thepowdered
powderedsoybean
soybeanhad a significant
sizes consideredimpact
in thison the soybean
study, crudecrude
the soybean urease activity.
urease
For all the powdered soybean sizes considered in this study, the soybean crude urease
activity increased with the increase in the powdered soybean concentration. Therefore,
activity increased with the increase in the powdered soybean concentration. Therefore,
the amount of soybean crude urease used in the EICP could be quantified according to
the amount of soybean crude urease used in the EICP could be quantified according to
the enzyme activity and the powdered soybean. In addition, the urease activity in the
the enzyme activity and the powdered soybean. In addition, the urease activity in the
soybean crude extract increased with increasing the degree of crushings of the soybean
soybean crude extract increased with increasing the degree of crushings of the soybean
for a given powdered soybean concentration. For instance, when the powdered soybean
for a given powdered soybean concentration. For instance, when the powdered soybean
concentration was 40 g/L, the urease activity in the soybean crude extract prepared using
concentration was 40 g/L, the urease activity in the soybean crude extract prepared using
powdered soybean with a particle size range of 0.075–0.25 mm was 4.93 mM urea min−1, −1
powdered soybean with a particle size range of 0.075–0.25 mm was 4.93 mM urea min ,
about 1.67 times higher than that of 1–2 mm. A decreased powdered soybean size resulted
about 1.67 times higher than that of 1–2 mm. A decreased powdered soybean size resulted
in a higher increasing rate of urease activity. However, the two ranges of the powdered
in a higher increasing rate of urease activity. However, the two ranges of the powdered
soybean size (i.e., 0.075–0.25 mm and 0–0.075 mm) yielded statistically similar results (p >
soybean size (i.e., 0.075–0.25 mm and 0–0.075 mm) yielded statistically similar results
0.05)
(p in termsinofterms
> 0.05) soybean crude urease
of soybean crude activity. This similarity
urease activity. might be
This similarity attributed
might to the to
be attributed
rough extraction method adopted in this study. The soybean urease
the rough extraction method adopted in this study. The soybean urease was adequatelywas adequately ex-
tracted with the powdered soybean particle size of 0.075–0.25 mm; therefore,
extracted with the powdered soybean particle size of 0.075–0.25 mm; therefore, crushing crushing the
particle size to size
the particle the range
to theof 0–0.075
range mm could
of 0–0.075 mmnot further
could improve
not further the urease
improve activity.
the urease activity.

Figure
Figure 4. Effect
4. Effect of powdered
of powdered soybeansoybean
size andsize and concentration
concentration on thecrude
on the soybean soybean crude
urease urease
enzyme
enzyme activity.
activity.

It is commonly accepted that the higher the urease activity, the faster the rate of the
biochemical reactions become and the more the CaCO3 precipitates [13,25]. During the
EICP process, the formerly-precipitated CaCO3 might act as nucleation seeds for calcite pre-
cipitation, facilitating the subsequently-precipitated CaCO3 to agglomerate and form large
 Energies 2022, 15, 5566 7 of 18

calcite clusters at the contact areas between the sand particles [26]. The large calcite clusters
(as it is known for effective CaCO3 ) that accumulated within and progressively filled the
interstitial gaps of the inter-particles bind separate sand particles together, thereby improv-
ing the already strong bonding force to demonstrate an even higher strength [1,23,25,27].
Consequently, more CaCO3 precipitation in porous materials significantly contributed to
improving the mechanical properties such as shear strength and erosion resistance. Consid-
ering the urease activity and the cost among all the degrees of crushing of the soybeans, the
optimal particle size of the powdered soybean was 0–0.25 mm. Unless particularly stated,
the particle size of the powdered soybean used hereafter was 0–0.25 mm. In addition to the
degree of crushing, the urease activity in the soybean crude extract could be improved by
substantially increasing the dosage of the powdered soybean, which would dramatically
enhance the total cost of biocalcification.

3.1.2. Soaking Time and Temperature

Energies 2022, 15, x FOR PEER REVIEW
For the application of EICP technology in large-scale fields, achieving the economical
production of urease in natural environments is of great significance. To achieve this goal, a
simple and efficient extraction strategy (i.e., under an such as appropriate soaking time and
temperature) is needed. Figure 5 plots the effect of the soaking time and temperature on the
urease activity of soybean crude extract. The powdered soybean size was within the range
of 0–0.25 and the powdered soybean concentration was set to be 40 g/L. It is indicated from
Figure 5 that the enzyme activity of the soybean crude urease extracted at the temperatures
of 5, 15, and 25 ◦ C appeared broadly similar (p > 0.05). As the soaking temperature increased
to 40 and 50 ◦ C, the urease activity in the soybean crude extract kept nearly constant in the
first 5 h and then sharply declined. The reduction in the urease activity should be due to
the deterioration of protein and the inactivation of enzyme at relatively high temperatures.
The findings revealed a strong correlation between urease activity and temperature, and
high temperatures might impede urease activity. Dilrukshi et al. [28] measured the urease
activity of crude extracts from watermelon seeds at various temperatures (from 25 to
70 ◦ C). They claimed that the activity of the crude extract decreased with time and that the
decay rate was more pronounced at high temperatures. Similar observations regarding8 of 18 the
effect of environmental temperature on the biocementation process can also be found in
Cheng et al. [23] and Sun et al. [29].

Figure
Figure 5. Effect
5. Effect of soaking
of soaking timetemperature
time and and temperature
on theonenzyme
the enzyme activity
activity of soybean
of soybean crudecrude urease.
urease.

3.2. Effect of Powdered Soybean Concentration (Urease Activity) on Chemical Conversion

Efficiency and Calcium Carbonate Content of EICP-Treated Sand
The bio-catalyzed precipitate of CaCO3 accumulates as more EICP solutions (soybean
crude urease and cementation solution) are introduced to the soil. To efficiently use the
cementation solutions for CaCO3 precipitation, a high chemical conversion efficiency is
Energies 2022, 15, 5566 8 of 18

The soaking time had no impact on the urease activity in the soybean crude extract at
room temperature, implying the feasibility of the soybean crude urease obtained in this
work for potential engineering applications. Compared with the urease-producing bacteria
(UPB) cultivation, the production of urease with soybean crude urease was more time-
saving (less than 1 h in this study) and less sensitive to seasonal (temperature) variations for
the biomineralization of CaCO3 . Based on the results in Figure 5, the soaking temperature
and the soaking time of 25 ◦ C and 0.5 h were suggested and adopted in the remainder of
this study.

3.2. Effect of Powdered Soybean Concentration (Urease Activity) on Chemical Conversion

Efficiency and Calcium Carbonate Content of EICP-Treated Sand
The bio-catalyzed precipitate of CaCO3 accumulates as more EICP solutions (soybean
crude urease and cementation solution) are introduced to the soil. To efficiently use the
cementation solutions for CaCO3 precipitation, a high chemical conversion efficiency is
required in the soybean crude urease-induced biochemical process. Figure 6a displays that
an increase in the powdered soybean concentration (urease activity) increased the efficiency
of the biological reaction. Since a larger amount of urea was hydrolyzed, a greater mass
of calcium was bio-transformed into CaCO3 . In fact, during the first two treatments (12 h
for one treatment), a powdered soybean concentration of 60 g/L (6.62 mM urea min−1
before premixing) enabled the production of almost 95% of the theoretical mass of calcium
carbonate for 0.5 M urea-0.5 M CaCl2 (after premixing) within 12 h. However, for the
same cementation solution, the use of 40 g/L (4.86 mM urea min−1 before premixing)
and 20 g/L (2.15 mM urea min−1 before premixing) powdered soybean concentration
reduced the average chemical conversion efficiency to 41.2% and 70.9% (in the first two
treatments), respectively. The chemical conversion efficiency declined with the increase
in the number of EICP treatment cycles when the powdered soybean concentrations were
60 and 80 g/L. The EICP solution retention capacity (initially 72 mL) of Ottawa sand in
the columns might decrease due to the accumulation of CaCO3 crystals in the pores and
the formation of preferential flow paths, which in turn, resulted in the leakage of excess
biological reagents and thereby the decrease in the total chemical conversion efficiency. The
increased chemical conversion efficiency with the augmentation of the EICP treatment
Energies 2022, 15, x FOR PEER REVIEW 10 offor
18
lower powdered soybean concentrations (40 and 20 g/L) might be due to the accumulation
of urease residues in the soil from the former treatments.

(a) (b)
Figure
Figure 6.
6. Effect
Effect of
of powdered
powdered soybean
soybean concentration
concentration and
and treatment
treatment cycle
cycle on
on (a)
(a) chemical
chemical conversion
conversion
efficiency
efficiency and (b) total
and (b) total CaCO
CaCO3 content.
content.
3

3.3. Effect of Powdered

The powdered Soybeanconcentration
soybean Concentrationaffected
(Ureasethe
Activity) on the
chemical Properties
conversion of
efficiency of the
Biocemented
cementationSand solution and the content of CaCO3 in the EICP-treated soil samples (Figure 6b).
To improve
3.3.1. UCS and theElastic
chemical conversion efficiency and reduce the waste of CaCl2 and urea, a
Modulus
lower urea-CaCl2 solution concentration or a longer retention time should be adopted for
The influence of the treatment cycle and the powdered soybean concentration (urease
low powdered soybean concentrations (40 and 20 g/L). However, more EICP solution was
activity) on the UCS of the biocemented soil samples is provided in Figure 7. In general,
the value of the UCS increased with the reduplicative percolation of the EICP solution, as
illustrated in Figure 7a. In addition to the treatment times, the powdered soybean concen-
tration (urease activity) was another crucial factor impacting the mechanical properties of
the biocemented sand specimens (Figure 7a). For the same treatment cycles, the UCS of
Energies 2022, 15, 5566 9 of 18

required to obtain the target level of cementation at a lower concentration of cementation

solution, further increasing the total cost of EICP. Moreover, a longer retention time could
further increase the time cost of the EICP. The statistical analysis revealed that the overall
biochemical reactants’ efficiency and the total content of CaCO3 in the EICP-treated soil
samples were similar for the powdered soybean concentration of 60 and 80 g/L (p > 0.05).
From the aspects of the reactants’ efficiency and application cost, 60 g/L of the powdered
soybean was preferred for extracting the crude urease in this study.
It has been reported that below a specific urea-CaCl2 input rate (0.042 mol/L/h) and
at a bacterial optical density (OD600 ) between 0.8 and 1.2, the reaction efficiency of MICP
is higher than 90% (Al Qabany et al., 2012). In this study, with a urea-CaCl2 input rate of
~0.042 mol/L/h (retention time: 12 h, urea-CaCl2 concentration after premixing: ~0.5 M)
and a urease activity of ~3.31 mM urea min−1 (after premixing), the reactant efficiency of
the soybean crude urease-induced CaCO3 precipitation was comparable to those obtained
from MICP treatment [30]. A review of the literature shows that the chemical conversion
efficiency of EICP varied among different research groups. Yasuhara et al. [31] premixed
urease powder with sand before injecting cementation solution and found that the chemical
conversion efficiency ranged from 30 to 60%. Almajed et al. [13] reported that when a
treatment solution consisting of 1 M urea, 0.67 M calcium chloride, and 3 g/L enzyme
(3500 U/g) was adopted to biocement sand, the chemical conversion efficiency of EICP
(after curing for 7 days) in the soil fell in the range of 70–95%. Carmona et al. [32] conducted
a series of test-tube (aqueous environments) experiments. They found that with a curing
time of 24 h and urease activity of 4000 U/L, the reactant efficiency was less than 50% for
0.5 M urea and 0.5 M CaCl2 solution. The chemical conversion efficiency of soybean crude
urease-induced CaCO3 precipitation in this study (60 g/L powdered soybean with urease
activity of ~3.31 mM urea min−1 after premixing) was higher than those obtained from
purified enzyme-induced CaCO3 precipitation within a short retention time (12 h). Two
factors accounted for this phenomenon: firstly, bacteria act as the nucleation sites for the
formation of CaCO3 crystals through their adsorption of Ca2+ and by creating localized
supersaturation, whereas using purified enzymes may lead to a lack of nucleation points
for the CaCO3 precipitation; secondly, the purified urease is unstable and can easily be
affected by high temperatures, extreme pH, and other environmental factors, limiting their
applications in field conditions [15,18].
Soybean crude urease (without further purification) contains an appreciable amount
of urease and soybean protein. The soybean protein had an isoelectric point of around
pH 4.5, as measured by an isoelectric focusing analysis. The isoelectric point is the pH
value at which the zeta potential value is zero, implying that there is no electric charge on
the surface of the soybean protein. The net charge on the protein molecule is affected by the
pH of its surrounding environment. It becomes negatively charged when the surrounding
environment’s pH exceeds the isoelectric point [33]. During the soybean crude urease-based
biochemical process, the pH of the EICP solution was much higher than the isoelectric
point of soybean protein (~pH 4.5). The negatively charged soybean proteins (similar to
the urease-producing bacteria) may also act as nucleation sites through the adsorption of
Ca2+ to their surfaces, creating a localized supersaturation that favored the precipitation of
CaCO3 . Furthermore, the macromolecular soybean proteins acted as an organic stabilizer
to protect the urease against environmental change, which was similar to the commonly
used dried non-fat milk [17].

3.3. Effect of Powdered Soybean Concentration (Urease Activity) on the Properties of

Biocemented Sand
3.3.1. UCS and Elastic Modulus
The influence of the treatment cycle and the powdered soybean concentration (urease
activity) on the UCS of the biocemented soil samples is provided in Figure 7. In general,
the value of the UCS increased with the reduplicative percolation of the EICP solution, as
illustrated in Figure 7a. In addition to the treatment times, the powdered soybean concen-
 3.3. Effect of Powdered Soybean Concentration (Urease Activity) on the Properties of
Biocemented Sand
3.3.1. UCS and Elastic Modulus
The influence of the treatment cycle and the powdered soybean concentration (urease
Energies 2022, 15, 5566 activity) on the UCS of the biocemented soil samples is provided in Figure 7. In general,
10 of 18
the value of the UCS increased with the reduplicative percolation of the EICP solution, as
illustrated in Figure 7a. In addition to the treatment times, the powdered soybean concen-
tration (urease activity) was another crucial factor impacting the mechanical properties of
tration (urease activity) was another crucial factor impacting the mechanical properties of
the biocemented sand specimens (Figure 7a). For the same treatment cycles, the UCS of
the biocemented sand specimens (Figure 7a). For the same treatment cycles, the UCS of
sand increased significantly with the increase in the powdered soybean concentration
sand increased significantly with the increase in the powdered soybean concentration from
from 20 to 60 g/L and then remained roughly constant even when a higher powdered
20 to 60 g/L and then remained roughly constant even when a higher powdered soybean
soybean concentration (80 g/L) was employed. A statistical analysis of the results indi-
concentration (80 g/L) was employed. A statistical analysis of the results indicated that the
cated that the UCS was similar (p > 0.05) when the powdered soybean concentration was
UCS was similar (p > 0.05) when the powdered soybean concentration was 60 and 80 g/L.
60 and 80 g/L. The phenomena mentioned above could be related to the precipitated
The phenomena mentioned above could be related to the precipitated CaCO3 (Figure 7b).
CaCO3 (Figure 7b). For all the powdered soybean concentrations, the UCS increased with
For all the powdered soybean concentrations, the UCS increased with the increase in the
the
CaCO increase in the CaCO3 amount, i.e., a more considerable amount of bio-mediated
3 amount, i.e., a more considerable amount of bio-mediated CaCO3 increased the
CaCO 3 increased the bonding strength between soil particles, leading to an improvement
bonding strength between soil particles, leading to an improvement in the mechanical
in the mechanical
properties (UCS) ofproperties (UCS) ofsand
the biocemented the biocemented
samples. sand samples.

(a) (b)
Figure 7. Effect
Figure 7. Effect of
of powdered
powdered soybean
soybean concentration
concentration on
on the
the unconfined
unconfined compressive
compressive strength
strength as
as aa
function of (a) cycle of treatment and (b) CaCO3 content.
function of (a) cycle of treatment and (b) CaCO3 content.

Figure 813compares
compares thethe
present andand
present previously reported
previously relationship
reported between
relationship the UCS
between the
and
UCStheandbiocatalyzed CaCOCaCO
the biocatalyzed 3 content,
3 including
content, the
includingpresent
the EICP
present results
EICP obtained
results using
obtained
soybean crude urease
using soybean crude and
ureasethe and
previously reported reported
the previously EICP (using purified
EICP (usingurease) andurease)
purified MICP
and MICP (using urease producing bacteria) results. The CaCO3 content refers to the
mass of generated CaCO3 to the mass of sand. The trend of the UCS in relation to the
CaCO3 content recorded in this work was comparable to that obtained from MICP treat-
ment [23,34] and EICP treatment [13,35]. However, the UCS value obtained in the present
work was at a noticeably higher range than that of EICP- and MICP-treated data in terms
of comparable CaCO3 precipitation levels. It is worth noting that the overall amount of
CaCO3 precipitation could not be considered as the sole factor for the biocementation
effects [36–38]. Wang et al. [27] pointed out that the precipitated CaCO3 could be catego-
rized into effective CaCO3 and non-effective CaCO3 in terms of the different nucleation
sites. The effective CaCO3 may act as solid bridges to bind separate sand particles to-
gether, thereby improving strength and stiffness [23]. In addition, the influence of other
factors (e.g., crystal polymorphs) may either influence the biocementation effect even at an
identical CaCO3 content. Dyer and Viganotti [39] suggested that the crystal morphology
dramatically affected the failure mechanism of MICP-treated samples due to the different
geometrical interferences. Consequently, in consideration of the abovementioned factors, a
microscale and microstructure analysis should be conducted for further investigation.
To investigate the possible explanation for the abovementioned difference, the crys-
talline phase and microfeatures of the precipitate within the soil matrix were examined
using an X-ray diffractometer (XRD) and scanning electron microscopy (SEM), respectively.
The XRD analysis (Figure 8) shows that quartz and calcite existed in the soybean crude
urease-based biocemented sand, while no calcite formation was observed in the original
sand. Calcite may be the most desirable crystal of biocatalyzed CaCO3 for geotechnical
applications due to its thermodynamic stability [15]. A similar CaCO3 crystal type was
observed in other enzyme-induced calcium carbonate precipitation studies [13,32], sug-
 Energies 2022, 15, 5566 11 of 18

Energies 2022, 15, x FOR PEER REVIEW
Energies 2022, 15, x FOR PEER REVIEW
gesting that soybean crude urease can be a good alternative to the purified urease for
catalyzing the precipitation of calcite for ground improvement. The small precipitated
crystals (typical crystal size of 1–3 µm) were agglomerated to form large clusters with
an approximate size range of 20–100 µm in the soybean crude urease (60 g/L powdered
soybean) based biocemented sand. One notable feature of this deposition pattern was
that although some of the large crystal clusters precipitated on the grain surface, the gaps
between the sand grains were entirely filled with clustered crystals (Figure 9), 12
which
of 18could
bridge the adjacent sand grains and provide a strong enough bonding force to bear the
high shear strength [23,40,41].
12 of 18

FigureFigure XRD analysis

XRD8.analysis
9. Figure of untreated
of untreated and EICP-treated
and EICP-treated (60 g/L(60 g/L powdered
powdered soybean)
soybean) sand. sand.
9. XRD analysis of untreated and EICP-treated (60 g/L powdered soybean) sand.

Figure 10. SEM images of EICP-treated (60 g/L powdered soybean) sand samples at eight treatments
Figure 9. SEM images of EICP-treated (60 g/L powdered soybean) sand samples at eight treatments
level with a magnification of: (a) 300 time; (b,c) 1200 time; (d) 5000 times.
level with a magnification of: (a) 300 time; (b,c) 1200 time; (d) 5000 times.
Figure 10. SEMAfter
images of EICP-treated
eight (60 g/L powdered
cycles of treatment, the typicalsoybean) sand curves
stress–strain samplesofatthe
eight treatments
biocemented
level withsoil
a magnification
samples treatedof: (a)
with300different
time; (b,c) 1200 time;
powdered (d) 5000
soybean times.
concentrations were obtained and
are presented in Figure 11a. All the EICP-treated sand samples exhibited a brittle nature
After eight cycles
comparable of treatment,
to those the
observed in thetypical stress–strain
MICP [11,34] curves
and EICP oftreatments.
[35,42] the biocemented
During
soil samples treated with
the unconfined different tests,
compressive powdered
failuressoybean
tended toconcentrations
occur within thewere
wholeobtained and
core (Figure
Energies 2022, 15, 5566 12 of 18

After eight cycles of treatment, the typical stress–strain curves of the biocemented
soil samples treated with different powdered soybean concentrations were obtained and
are presented in Figure 10a. All the EICP-treated sand samples exhibited a brittle nature
comparable to those observed in the MICP [11,34] and EICP [35,42] treatments. During the
Energies 2022, 15, x FOR PEER REVIEW 13 of 18
unconfined compressive tests, failures tended to occur within the whole core (Figure
Energies 2022, 15, x FOR PEER REVIEW 13 10b).
of 18
This result was in line with the relatively uniform distribution of CaCO3 precipitation in the
sand columns (Figure 11). A low-temperature premixed percolation strategy was employed
was
in employed
this work. Duein this work.
to the Due toactivity
low urease the lowof urease activitycrude
the soybean of theextract
soybean ◦ C, a extract
at 4crude at
period of
was employed in this work. Due to the low urease activity of the soybean crude extract at
4 °C,
lag a period
phase of in
existed lagthe
phase existedphase
percolation in theofpercolation phase of
the EICP solution, the EICP
which ensuredsolution, which
the relatively
4 °C, a period of lag phase existed in the percolation phase of the EICP solution, which
ensured distribution
uniform the relatively ofuniform distribution
the premixed of the
biological premixed
agents biological
within the agents(10
sand column within the
cm) and
ensured the relatively uniform distribution of the premixed biological agents within the
consequently
sand column (10the cm)
uniformity of the CaCO
and consequently the
3 precipitation.
uniformity of the CaCO 3 precipitation.
sand column (10 cm) and consequently the uniformity of the CaCO 3 precipitation.

(a) (b)
(a) (b)
Figure 10.
11. Results of
of EICP-treated sand
sand samples: (a) stress–strain relationship
relationship after eight
eight treatment
Figure 11. Results
Figure Results of EICP-treated
EICP-treated sand samples: (a) stress–strain
samples: (a) stress–strain relationship after
after eight treatment
treatment
cycles and (b) typical failure of sand sample.
cycles and (b) typical failure of
of sand
sand sample.
sample.

Figure 12. Calcium carbonate content distribution in EICP-treated sand samples after eight treat-
12. Calcium carbonate content distribution in EICP-treated sand samples after eight treat-
ments. 11. Calcium carbonate content distribution in EICP-treated sand samples after eight treatments.
Figure
ments.
The elastic modulus (E50), defined as the ratio of 0.5 UCS and the corresponding
The elastic modulus (E50), defined as the ratio of 0.5 UCS and the corresponding axial
axialThe elastic
strain, was modulus
used to(E50), defined
quantify the as the ratio
stiffness ofofthe
0.5biocemented
UCS and the corresponding
soil [24,40,43]. axial
The
strain, was used to quantify the stiffness of the biocemented soil [24,40,43]. The E50 of the
strain,
E50 of was used to quantify
the EICP-treated sandthe stiffness
samples of thefrom
ranged biocemented soil [24,40,43].
18.47 to 200.96 The E50 to
MPa, according of the
EICP-treated sand samples ranged from 18.47 to 200.96 MPa, according to the powdered
EICP-treated
powdered sand samples
soybean rangedand
concentration from 18.47 to cycle.
treatment 200.96 For
MPa, according
example, to eight
after the powdered
cycles of
soybean concentration and treatment cycle. For example, after eight cycles of biocemen-
soybean concentration and treatment cycle. For example, after eight cycles of biocemen-
tation treatment, the E50 increased from 87.27 to 195.12 MPa (i.e., 2.24 times) while the
tation treatment, the E50 increased from 87.27 to 195.12 MPa (i.e., 2.24 times) while the
powdered soybean concentration rose from 20 to 60 g/L (Figure 13a). As evident in Figure
powdered soybean concentration rose from 20 to 60 g/L (Figure 13a). As evident in Figure
13b, the E50 increased with the total CaCO3 content in the EICP-treated samples, which
13b, the E50 increased with the total CaCO3 content in the EICP-treated samples, which
was comparable to that observed in the previous research [24,34]. As mentioned earlier,
was comparable to that observed in the previous research [24,34]. As mentioned earlier,
Energies 2022, 15, 5566 13 of 18

biocementation treatment, the E50 increased from 87.27 to 195.12 MPa (i.e., 2.24 times)
while the powdered soybean concentration rose from 20 to 60 g/L (Figure 12a). As evident
in Figure 12b, the E50 increased with the total CaCO3 content in the EICP-treated samples,
which
Energies 2022, 15, x FOR PEER REVIEW
was comparable to that observed in the previous research [24,34]. As mentioned ear-
14 of 18
lier, the results indicated that the strength and stiffness of the EICP-treated sand exhibited
a similar trend regarding the treatment cycle and powdered soybean concentration.

(a) (b)
Figure 12.
Figure 13. Effect
Effect of
of powdered
powdered soybean
soybean concentration
concentration on
on E50
E50 as
as aa function
function of
of (a)
(a) cycle
cycle of
of treatment
treatment
and (b) CaCO 3 content.
and (b) CaCO3 content.

Biomineralization is
Biomineralization isaahighly
highlysophisticated
sophisticated process
process because
because of the involvement
of the involvement of nu-
of
cleation sites
nucleation and
sites andthethe
precipitation
precipitationofofcrystals.
crystals.ForForexample,
example,biomineral-associated
biomineral-associated macro- macro-
molecules, such as proteins and proteoglycans, may initiate and stabilize non-equilibrium
crystal polymorphs and morphologies through interactions between anionic moieties and
cations in solution
solution or or at
at the
theminerals’
minerals’surfaces
surfaces[44].[44].Therefore,
Therefore, a further
a further investigation
investigation of
this complex biocatalyzed process to find the possible reasons
of this complex biocatalyzed process to find the possible reasons for forming the pecu- for forming the peculiar
deposition
liar pattern
deposition of calcite
pattern is worth
of calcite carrying
is worth out inout
carrying theinfuture based based
the future on the on current study.
the current
It should
study. be emphasized
It should that the UCS
be emphasized andUCS
that the E50 values
and E50 from the ureolysis-based
values biocemen-
from the ureolysis-based
tation-treated soil can besoil
biocementation-treated highlycan variable,
be highlydepending on diverse experimental
variable, depending factors. The
on diverse experimental
factors.
results ofThe results of experiments
experiments in this area can in this area can
be highly be highly
variable due variable due toproperties
to the various the various of
properties of biocementation-treated
biocementation-treated soil such
soil such as relative as relative
density [45], density
particle [45],
shapeparticle
[46], and shape [46],
particle
and particle
size [34]. size [34].
In Zhao et al. In
[47],Zhao et al. value
the UCS [47], the UCS value ofsand
of MICP-treated MICP-treated
with a greater sandd50 with
was a
greater d50 was
much higher much higher
compared compared
with those with with thosed50
a smaller with a smaller
under d50 under
an identical an identical
treatment strat-
treatment strategy.
egy. Regarding RegardingMahawish
the gradation, the gradation,et al.Mahawish et al. [48]that
[48] demonstrated demonstrated that the
the biocementation-
biocementation-treated
treated column samplescolumn with thesamples with the lower
lower percentage percentage
of fine materialsof fine materials
(25%) showed a (25%)more
showed a more uniform distribution
uniform distribution of CaCO3 precipitation along of CaCO 3 precipitation along its length
its length compared to the samples, compared
to the samples,
which containedwhich
more contained
fines and amore fines and
gap-graded a gap-graded
distribution led todistribution
higher UCSled to higher
values of the
UCS values of the biocementation-treated soil. In addition, biological
biocementation-treated soil. In addition, biological agent compositions such as sources of agent compositions
such
urease as[49],
sources of urease
sources [49], sources
of calcium [50], and of calcium
urea-CaCl [50], and urea-CaCl
2 concentration 2 concentration
[45] had a significant [45]
had
impacta significant impact on the biocementation
on the biocementation process. Mujah process.
et al. [51]Mujah et al.
pointed out[51] pointed
that out that
MICP-treated
MICP-treated sand samples with lower cementitious solution
sand samples with lower cementitious solution concentrations possessed higher UCS val-concentrations possessed
higher
ues at aUCS
givenvalues
CaCO3atcontent.
a givenNevertheless,
CaCO3 content. ZhaoNevertheless,
et al. [47] statedZhao et al. [47]
an opposite stated an
observation
opposite observation between the UCS value and the cementitious
between the UCS value and the cementitious solution concentration: the higher the ce- solution concentration:
the higher concentration,
mentation the cementation theconcentration,
higher the UCS thevalue.
higher Thetheinfluence
UCS value. The influencefac-
of environmental of
environmental factors (e.g., pH and temperature) on the performance
tors (e.g., pH and temperature) on the performance of urelysis-based biocemented soil of urelysis-based
biocemented
cannot be ignoredsoil cannot
eitherbe ignored
[29,52]. either [29,52].
In conclusion, muchIn conclusion,
more effortmuch should more effort should
be made to pro-
be
vide made to provide
an insight an optimization
into the insight into the optimization
of EICP based onof EICP based
soybeans’ crude onurease
soybeans’ crude
for various
urease for various possible applications.
possible applications.

3.3.2. Permeability
The measured permeability values of the sand samples after EICP treatment were
firstly normalized by the permeability value of the original sand. As expected, permeabil-
ity reduction appeared with the advancement of the EICP treatments (Figure 14a). The
permeability was reduced from 2.4 × 10−4 m/s for untreated sand to 1.3 × 10−5 m/s for bio-
cemented sand (80 g/L) after eight cycles of treatment. Similar outcomes were reported by

Energies 2022, 15, 5566
CaCO3 may act as solid bridges to bind separate sand particles together, thereby improv-
ing strength and stiffness [23]. In addition, the influence of other factors (e.g., crystal pol-
ymorphs) may either influence the biocementation effect even at an identical CaCO3 con-
tent. Dyer and Viganotti [39] suggested that the crystal morphology dramatically affected
the failure mechanism of MICP-treated samples due to the different geometrical interfer-
ences. Consequently, in consideration of the abovementioned factors, a microscale 14 of
and18

microstructure analysis should be conducted for further investigation.

Figure 13.
Figure 8. UCS
UCSofofthe
thepresent
presentEICP
EICP results
results versus
versus previous EICP and
previous EICP and MICP
MICPdata
data.(Yasuhara et al. [31];
Neupane et al. [35]; Almajed et al. [13]; Al Qabany and Soga, [45]; Cheng et al. [23]; Hoang et al. [34]).
To investigate the possible explanation for the abovementioned difference, the crys-
3.3.2.
tallinePermeability
phase and microfeatures of the precipitate within the soil matrix were examined
usingThe an measured permeability
X-ray diffractometer values
(XRD) andof scanning
the sand electron
samples microscopy
after EICP treatment
(SEM), respec-were
firstly
tively.normalized
The XRD analysisby the permeability
(Figure 9) shows valuethat
of the original
quartz and sand.
calciteAs expected,
existed permeabil-
in the soybean
ity reduction
crude appeared
urease-based with the advancement
biocemented sand, while no of the EICP
calcite treatments
formation was(Figure
observed14a).inThethe
permeability was reduced from 2.4 × 10 −4 m/s for untreated sand to 1.3 × 10−5 m/s for
original sand. Calcite may be the most desirable crystal of biocatalyzed CaCO 3 for ge-
biocemented
Energies 2022, 15, x FOR PEER REVIEW sand (80 g/L)
otechnical applications dueafter eight
to its cycles of treatment.
thermodynamic stabilitySimilar
[15]. Aoutcomes were reported
similar CaCO crystal
3 15 of 18
by
type was observed in other enzyme-induced calcium carbonate precipitationwith
[23], wherein the permeability of the biocementation treated sand decreased the
studies
increase in the content
[13,32], suggesting that of CaCO3crude
soybean precipitation.
urease can Figure
be a 14b
goodindicated
alternative thattoalthough
the purified the
reduction
urease for in permeability
catalyzing the of the EICP-treated
precipitation of sand
calcite for samples
ground was mainly controlled
improvement. The smallbypre-
the
CaCO3 precipitation content, the concentration of the powdered soybean (urease activity)
CaCO
cipitated precipitation
crystals content, the concentration of the powdered soybean (urease activity)
EICP (typical crystal
had size of 1–3 μm) were agglomerated to form largeofclusters
3
used in the treatment a non-negligible impact. For the same amount CaCO3
used
with in the EICP treatment had aofnon-negligible impact. For the same amount of CaCO
precipitate, the permeability reduction of biostabilized sand treated with (60
an approximate size range 20–100 μm in the soybean crude urease g/L pow- 3
higher pow-
precipitate,
dered the permeability
soybean)concentrations reduction
based biocemented of biostabilized
sand. One notable sand treated
feature with
of thisthan higher
deposition powdered
pattern
dered soybean
soybean concentrations (for (for example,
example, 60 and 80 60g/L)
and 80
wasg/L)
morewasthan
more that thatsand
ofgrain
the of the sand
treated
was that
treated although
with lower some of
powdered the large
soybeancrystal clusters
concentrations precipitated
(for on
example, the
20 and surface,
40 g/L). the
The
with
gaps lower
between powdered
the sandsoybean
grains concentrations
were entirely (for example,
filled with 20 and crystals
clustered 40 g/L).(Figure
The higher10),
higher activity
urease urease activity
(60 and (60g/L)
80 and may
80 g/L) may
cause thecause
rapid the rapid precipitation
precipitation CaCO3ofin
of enough CaCO 3 in the
the top area
which
top areacould
of bridge
the sand the adjacent
samples, sand
which grains
would and
block provide
the soil a strong
pores and lead to bonding
clogging force
dur-
of
to the
bear sand
the samples,
high which
shear strength would block
[23,40,41]. the soil pores and lead to clogging during the
ing the EICP
EICP treatment [53].treatment [53].

(a) (b)
Figure 14.
Figure 14. Effect
Effectof
ofpowdered
powderedsoybean
soybeanconcentration
concentrationononthe
the normalized
normalized permeability
permeability as as a function
a function of
of (a) cycle of treatment and (b) CaCO3 content.
(a) cycle of treatment and (b) CaCO3 content.

3.4.
3.4. Implications
Implications of
of Soybeans
Soybeans Crude
Crude Urease
Urease for
for Ground-Improvement
Ground-ImprovementApplications
Applications
The
The application of
of biocementation
biocementationfor
forground
groundstabilization
stabilizationrequires
requiresa great
a great degree
degree of
of cementing
cementing of of
thethe calcium
calcium carbonate
carbonate between
between thethe
soilsoil grains
grains to produce
to produce an adequate
an adequate im-
provement in strength. The cost of urease-producing bacteria or urease enzymes is one of
the main obstacles in adopting the bio-mediated CaCO3 technique in large-scale practice.
For instance, nutrients in the culture medium (urease-producing bacteria) are expensive
and can account for as high as 60% of the total operation cost of MICP [54]. In contrast,
Energies 2022, 15, 5566 15 of 18

improvement in strength. The cost of urease-producing bacteria or urease enzymes is one

of the main obstacles in adopting the bio-mediated CaCO3 technique in large-scale practice.
For instance, nutrients in the culture medium (urease-producing bacteria) are expensive
and can account for as high as 60% of the total operation cost of MICP [54]. In contrast,
the cost of commercially available urease enzyme comprises 57–98% of the total cost of
the EICP treatment [13]. Thus, low-cost urease sources are highly desirable for the large-
scale application of biocementation. Soybeans can be a cost-effective alternative source of
urease to catalyze the precipitation of CaCO3 crystals. It is also crucial to produce greater
amounts of crude urease from soybeans and to make efficient use of the enzyme for cost
minimization. The findings obtained from the present work show that powdered soybean
concentration and particle size are the two crucial factors affecting the urease activity of
soybean crude extract. The enzyme activity used in EICP process can further result in
different reactant efficiencies of urea-CaCl2 solution, and consequently, the improvement
in the physical and mechanical property of biocemented sand. For ground improvement
purposes, 60 g/L of the powdered soybean was preferred for extracting the crude urease
with an enzyme activity of 6.62 mM urea min−1 , leading to a chemical conversation
efficiency of approximately 95% for 0.5 M urea-0.5 M CaCl2 over 12 h. In this study,
considering the current price of soybeans, the material cost of the crude urease enzyme
(powdered soybean concentration: 60 g/L) was about USD 0.036/L−1 , which was much
lower than that of the commonly used Sporosarcina pasteurii and purified urease enzyme.
Furthermore, the cost can be further reduced if the remaining soybean dregs after extracting
the crude urease are used to produce soya meal, which is rich in protein and can be
used to feed animals or culture microorganisms. This work uses the low temperature
premixed percolation method, which can be used wherever the surficial soil needs to be
treated, such as strengthening the surface of slopes, preventing shallow ground liquefaction,
and controlling wind or water induced erosion. Although the study demonstrates the
feasibility of using soybean crude urease for sand consolidation in short columns, its meter-
scale performance is still unknown. In addition, the soil conditions, such as the grain
size and mineral composition, vary considerably in different regions, which may have
a non-negligible impact on the ground reinforcement of EICP. Furthermore, the effects
of the residual unreacted CaCl2 and urea as well as the by-product of NH4 Cl on the soil
microenvironment remain to be seen. Many more efforts must be made to address the
practical problems mentioned above, which are critical for the large-scale application
of EICP.

4. Conclusions
This study placed emphasis on the key factors affecting the urease activity of soybean
crude extract and the performance of soybean crude urease in EICP. Soybean crude ure-
ase was extracted simply by mixing powdered soybean and tap water, and a modified
method was proposed to optimize the urease extraction efficiency. The powdered soybean
concentration and particle size were the two critical factors affecting the urease activity of
soybean crude extract. To make the most of soybeans in terms of crude urease extraction,
the powdered soybean particle size should be less than 0.25 mm. Under this condition,
soybean crude urease can be fully extracted at room temperature in less than 1 h.
The soybean power concentration can be used as an indicator of the extracted soybean
crude urease activity due to the positive correlation. A series of sand column tests were
performed to investigate the effects of soybean power concentration on the chemical
conversion efficiency and mechanical properties of the biocemented ASTM-graded Ottawa
sand. When the soybean power concentration increased to 60 g/L (6.62 mM urea min−1 ),
a chemical conversion efficiency of approximately 95% for 0.5 M urea-0.5 CaCl2 could be
obtained in merely 12 h. Furthermore, with a CaCO3 content of ~8% (after eight cycles of
treatment), the UCS and E50 of EICP-treated sand exceeded 4 and 190 MPa, respectively.
The formation of large, agglomerated calcite crystal clusters filled the gaps between
the sand grains, showing the high efficiency of soybean crude urease in improving the
Energies 2022, 15, 5566 16 of 18

strength and stiffness of EICP-treated sands. This study indicates that soybean crude
urease, as a cost-effective and more efficient alternative to the purified enzyme and
urease-producing bacteria, can be used to catalyze the precipitation of calcite in sands for
ground improvement.

Author Contributions: Conceptualization, S.S., B.G. and H.M.; Data curation, B.Y. and S.L.; Inves-
tigation, S.S. and B.Y.; Methodology, S.S., B.G. and H.M.; Supervision, H.M.; Visualization, B.G.;
Writing—original draft, S.S. and B.Y.; Writing—review & editing, S.L. and H.M. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China under
grant numbers 51978244 and 51979088.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data models, or code that support the findings of this study are
available from the corresponding author upon reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. DeJong, J.; Mortensen, B.; Martinez, B.; Nelson, D. Bio-mediated soil improvement. Ecol. Eng. 2010, 36, 197–210. [CrossRef]
2. Tang, Q.; Tang, X.; Li, Z.; Chen, Y.; Kou, N.; Sun, Z. Adsorption and desorption behaviour of Pb (II) on a natural kaolin:
Equilibrium, kinetic and thermodynamic studies. J. Chem. Technol. Biot. 2009, 84, 1371–1380. [CrossRef]
3. Van Paassen, L.A.; Daza, C.M.; Staal, M.; Sorokin, D.Y.; Der Zon, W.V.; Van Loosdrecht, M.C. Potential soil reinforcement by
biological denitrification. Ecol. Eng. 2010, 36, 168–175. [CrossRef]
4. Soon, N.W.; Lee, L.M.; Khun, T.C.; Ling, H.S. Improvements in engineering properties of soils through microbial-induced calcite
precipitation. KSCE J. Civ. Eng. 2013, 17, 718–728. [CrossRef]
5. Tang, Q.; Gu, F.; Zhang, Y.; Zhang, Y.Q.; Mo, J.L. Impact of biological clogging on the barrier performance of landfill liners.
J. Environ. Manag. 2018, 222, 44–53. [CrossRef]
6. Lee, S.; Kim, J. An experimental study on enzymatic-induced carbonate precipitation using yellow soybeans for soil stabilization.
KSCE J. Civ. Eng. 2020, 24, 2026–2037. [CrossRef]
7. Meng, H.; Gao, Y.; He, J.; Qi, Y.; Hang, L. Microbially induced carbonate precipitation for wind erosion control of desert soil:
Field-scale tests. Geoderma 2021, 383, 114723. [CrossRef]
8. Chu, J.; Stabnikov, V.; Ivanov, V. Microbially induced calcium carbonate precipitation on surface or in the bulk of soil. Geomicrobiol.
J. 2012, 29, 544–549. [CrossRef]
9. Gao, Y.; He, J.; Tang, X.; Chu, J. Calcium carbonate precipitation catalyzed by soybean urease as an improvement method for
fine-grained soil. Soils Found. 2019, 59, 1631–1637. [CrossRef]
10. Stocks-Fischer, S.; Galinat, J.K.; Bang, S.S. Microbiological precipitation of CaCO3 . Soil Biol. Biochem. 1999, 31, 1563–1571.
[CrossRef]
11. Choi, S.G.; Chu, J.; Brown, R.C.; Wang, K.; Wen, Z. Sustainable biocement production via microbially induced calcium carbonate
precipitation: Use of limestone and acetic acid derived from pyrolysis of lignocellulosic biomass. ACS Sustain. Chem. Eng. 2017, 5,
5183–5190. [CrossRef]
12. Krajewska, B.; Ureases, I. Functional, catalytic and kinetic properties: A review. J. Mol. Catal. B-Enzym. 2009, 59, 9–21. [CrossRef]
13. Almajed, A.; Tirkolaei, H.K.; Kavazanjian, E. Baseline investigation on enzyme-induced calcium carbonate precipitation. J. Geotech.
Geoenviron. Eng. 2018, 144, 04018081. [CrossRef]
14. Khodadadi, T.H.; Kavazanjian, E.; van Paassen, L.; DeJong, J. Bio-grout materials: A review. In Proceedings of the Grouting 2017,
Honolulu, Hawaii, 9–12 July 2017; ASCE: Reston, VA, USA, 2017. [CrossRef]
15. Almajed, A. Enzyme Induced Carbonate Precipitation (EICP) for Soil Improvement. Ph.D. Thesis, Arizona State University,
Phoenix, AZ, USA, 2017.
16. Zhang, Y.; Tang, Q.; Shi, P.; Katsumi, T. Influence of bio-clogging on permeability characteristics of soil. Geotext. Geomembr. 2021,
49, 707–721. [CrossRef]
17. Hamdan, N.; Kavazanjian, E.J. Enzyme-induced carbonate mineral precipitation for fugitive dust control. Géotechnique 2016, 66,
546–555. [CrossRef]
18. Poźniak, G.; Krajewska, B.; Trochimczuk, W. Urease immobilized on modified polysulphone membrane: Preparation and
properties. Biomaterials 1995, 16, 129–134. [CrossRef]
19. Whiffin, V.S.; Van Paassen, L.A.; Harkes, M.P. Microbial carbonate precipitation as a soil improvement technique. Geomicrobiol. J.
2007, 24, 417–423. [CrossRef]
Energies 2022, 15, 5566 17 of 18

20. ASTM D2487; Standard Practice for Classification of Soils for Engineering Purposes (Unified Soil Classification System). ASTM
International: West Conshohocken, PA, USA, 2018. [CrossRef]
21. Cheng, L.; Cord-Ruwisch, R. In situ soil cementation with ureolytic bacteria by surface percolation. Ecol. Eng. 2012, 42, 64–72.
[CrossRef]
22. He, J.; Gao, Y.; Gu, Z.; Chu, J.; Wang, L. Characterization of crude bacterial urease for CaCO3 precipitation and cementation of
silty sand. J. Mater. Civ. Eng. 2020, 32, 04020071. [CrossRef]
23. Cheng, L.; Shahin, M.A.; Mujah, D. Influence of key environmental conditions on microbially induced cementation for soil
stabilization. J. Geotech. Geoenviron. Eng. 2017, 143, 04016083. [CrossRef]
24. Xiao, Y.; Stuedlein, A.W.; Ran, J.; Evans, T.M.; Cheng, L.; Liu, H.; Van Paassen, L.A.; Chu, J. Effect of particle shape on strength
and stiffness of biocemented glass beads. J. Geotech. Geoenviron. Eng. 2019, 145, 6019016. [CrossRef]
25. Tang, Q.; Shi, P.; Yuan, Z.; Shi, S.; Xu, X.; Katsumi, T. Potential of zero-valent iron in remediation of Cd (II) contaminated soil:
From laboratory experiment, mechanism study to field application. Soils Found. 2019, 59, 2099–2109. [CrossRef]
26. Meng, H.; Shu, S.; Gao, Y.; Yan, B.; He, J. Multiple-phase enzyme-induced carbonate precipitation (EICP) method for Soil
Improvement. Eng. Geol. 2021, 294, 106374. [CrossRef]
27. Wang, Y.; Soga, K.; Dejong, J.T.; Kabla, A.J. A microfluidic chip and its use in characterising the particle-scale behaviour of
microbial-induced calcium carbonate precipitation (MICP). Géotechnique 2019, 69, 1086–1094. [CrossRef]
28. Dilrukshi, R.; Nakashima, K.; Kawasaki, S. Soil improvement using plant-derived urease-induced calcium carbonate precipitation.
Soils Found. 2018, 58, 894–910. [CrossRef]
29. Sun, X.; Miao, L.; Tong, T.; Wang, C. Study of the effect of temperature on microbially induced carbonate precipitation. Acta
Geotech. 2018, 14, 627–638. [CrossRef]
30. Al Qabany, A.; Soga, K.; Santamarina, C. Factors affecting efficiency of microbially induced calcite precipitation. J. Geotech.
Geoenviron. Eng. 2012, 138, 992–1001. [CrossRef]
31. Yasuhara, H.; Neupane, D.; Hayashi, K.; Okamura, M. Experiments and predictions of physical properties of sand cemented by
enzymatically-induced carbonate precipitation. Soils Found. 2012, 52, 539–549. [CrossRef]
32. Carmona, J.P.S.F.; Oliveira, P.J.V.; Lemos, L.; Pedro, A.M.G. Improvement of a sandy soil by enzymatic calcium carbonate
precipitation. Geotech. Eng. 2017, 171, 1–13. [CrossRef]
33. Tan, E.S.; Ying-Yuan, N.; Gan, C.Y. A comparative study of physicochemical characteristics and functionalities of pinto bean
protein isolate (PBPI) against the soybean protein isolate (SPI) after the extraction optimisation. Food Chem. 2014, 152, 447–455.
[CrossRef]
34. Hoang, T.; Alleman, J.; Cetin, B.; Choi, S.G. Engineering properties of biocementation coarse- and fine-grained sand catalyzed by
bacterial cells and bacterial enzyme. J. Mater. Civ. Eng. 2020, 32, 4020030. [CrossRef]
35. Neupane, D.; Yasuhara, H.; Kinoshita, N.; Ando, Y. Distribution of mineralized carbonate and its quantification method in
enzyme mediated calcite precipitation technique. Soils Found. 2015, 55, 447–457. [CrossRef]
36. Tang, Q.; Katsumi, T.; Inui, T.; Li, Z. Membrane behavior of bentonite-amended compacted clay. Soils Found. 2014, 54, 329–344.
[CrossRef]
37. Lin, H.; Suleiman, M.T.; Brown, D.G.; Kavazanjian, E. Mechanical behavior of sands treated by microbially induced carbonate
precipitation. J. Geotech. Geoenviron. Eng. 2016, 142, 04015066. [CrossRef]
38. Tang, Q.; Liu, W.; Wang, H.Y.; Cheng, R.; Qian, Y.F. Membrane behavior of bentonite-amended Fukakusa clay in K, Na and Ca
solutions. J. Cent. South Univ. 2016, 23, 3122–3131. [CrossRef]
39. Dyer, M.; Viganotti, M. Oligotrophic and eutrophic MICP treatment for silica and carbonate sands. Bioinspir. Biomim. Nan. 2017, 6,
168–183. [CrossRef]
40. Bian, X.; Zeng, L.L.; Li, X.Z.; Hong, J.T. Deformation modulus of reconstituted and naturally sedimented clays. Eng. Geol. 2021,
295, 106450. [CrossRef]
41. Bian, X.; Zeng, L.L.; Li, X.Z.; Shi, X.S.; Zhou, S.M.; Li, F.Q. Fabric changes induced by super-absorbent polymer on cement and
lime stabilized excavated clayey soil. J. Rock Mech. Geotech. 2021, 13, 1124–1135. [CrossRef]
42. Kavazanjian, E.; Hamdan, N. Enzyme induced carbonate precipitation (EICP) columns for ground improvement. In Proceedings
of the IFCEE 2015, San Antonio, TX, USA, 17–21 March 2015; ASCE: Reston, VA, USA, 2015; pp. 2252–2261. [CrossRef]
43. Bian, X.; Zhang, W.; Li, X.Z.; Shi, X.S.; Deng, Y.F.; Peng, J. Changes in strength, hydraulic conductivity and microstructure of
superabsorbent polymer stabilized soil subjected to wetting-drying cycles. Acta Geotech. 2022. [CrossRef]
44. Fu, G.; Qiu, S.R.; Orme, C.A.; Morse, D.; De Yoreo, J.J. Acceleration of calcite kinetics by abalone nacre proteins. Adv. Mater. 2005,
17, 2678–2683. [CrossRef]
45. Al Qabany, A.; Soga, K. Effect of chemical treatment used in MICP on engineering properties of cemented soils. Géotechnique 2013,
63, 331–339. [CrossRef]
46. Ismail, M.; Joer, H.; Randolph, M.; Meritt, A. Cementation of porous materials using calcite. Géotechnique 2002, 52, 313–324.
[CrossRef]
47. Zhao, Q.; Li, L.; Li, C.; Li, M.; Amini, F.; Zhang, H. Factors affecting improvement of engineering properties of MICP-treated soil
catalyzed by bacteria and urease. J. Mater. Civ. Eng. 2014, 26, 04014094. [CrossRef]
48. Mahawish, A.; Bouazza, A.; Gates, W.P. Effect of particle size distribution on the bio-cementation of coarse aggregates. Acta
Geotech. 2017, 13, 1019–1025. [CrossRef]
Energies 2022, 15, 5566 18 of 18

49. Nafisi, A.; Safavizadeh, S.; Montoya, B.M. Influence of microbe and enzyme-induced treatments on cemented sand shear response.
J. Geotech. Geoenviron. 2019, 145, 06019008. [CrossRef]
50. Liu, L.; Liu, H.; Xiao, Y.; Chu, J.; Xiao, P.; Wang, Y. Biocementation of calcareous sand using soluble calcium derived from
calcareous sand. Bull. Eng. Geol. Environ. 2017, 77, 1781–1791. [CrossRef]
51. Mujah, D.; Cheng, L.; Shahin, M.A. Microstructural and geomechanical study on biocemented sand for optimization of MICP
process. J. Mater. Civil Eng. 2019, 31, 4019025. [CrossRef]
52. Tang, C.S.; Yin, L.Y.; Jiang, N.J.; Zhu, C.; Zeng, H.; Li, H.; Shi, B. Factors affecting the performance of microbial-induced carbonate
precipitation (MICP) treated soil: A review. Environ. Earth Sci. 2020, 79, 94. [CrossRef]
53. Bian, X.; Zeng, L.L.; Ji, F.; Xie, M. Plasticity role in strength behaviour of cement-phosphogypsum stabilized soils. J. Rock Mech.
Geotech. 2022. [CrossRef]
54. Yoosathaporn, S.; Tiangburanatham, P.; Bovonsombut, S.; Chaipanich, A.; Pathom-Aree, W. A cost effective cultivation medium
for biocalcification of Bacillus pasteurii KCTC 3558 and its effect on cement cubes properties. Microbiol. Res. 2016, 186, 132–138.
[CrossRef]