Mechanical behaviour and microstructure of granite residual bio-cemented soil by microbially induced calcite precipitation with different cementation-solution concentrations

Abstract

Microbially induced calcite precipitation (MICP) stands as an environmentally friendly and promising technique for enhancing the performance of soil. Bacteria catalyze the hydrolysis of urea, prompting calcium ions to react with carbonate ions, ultimately forming calcium carbonate precipitation as a cement within soil grains. However, studies of using MICP to enhance granite residual soil (GRS) that is recognized as a problematic soil because of its wide grain size distribution are relatively rare. In this present study, bio-cemented GRS samples were prepared through grouting with Sporosarcina pasteurii as the colony and a mixture of urea and calcium chloride as the cementation solution. The effect of cementation-solution concentrations on the mechanical properties of the bio-cemented samples was analyzed through unconfined compression and triaxial shear tests. Furthermore, X-ray computerized tomography, scanning electron microscopy, and X-ray diffraction experiments were performed to reveal the mechanism of MICP from a microscopic perspective. The experimental results indicate that an optimal concentration of 2 mol/L achieved the highest level of cementation, resulting in an impressive 47.15% increase in the unconfined compressive strength of the GRS samples. The triaxial shear strength and stress paths of bio-cemented samples were affected by the cementation level. The variation of porosity indicated that CaCO 3 precipitation improves soil densification by filling the macropores among the soil grains. The CaCO 3 precipitates from the MICP treatment predominantly exist in the form of calcite crystals, serving to fill, wrap, and cement within the soil structure, thereby enhancing the cohesive and frictional forces exerted by the bio-cemented grains.

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Environmental Earth Sciences (2024) 83:31
https://doi.org/10.1007/s12665-023-11352-w
ORIGINAL ARTICLE
Mechanical behaviour andmicrostructure ofgranite residual
bio‑cemented soil bymicrobially induced calcite precipitation
withdifferent cementation–solution concentrations
RanAn1,3 · HaodongGao2,3 · XianweiZhang3 · XinChen2· YixianWang1· HaoXu2
Received: 9 June 2023 / Accepted: 29 November 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023
Abstract
Microbially induced calcite precipitation (MICP) stands as an environmentally friendly and promising technique for enhanc-
ing the performance of soil. Bacteria catalyze the hydrolysis of urea, prompting calcium ions to react with carbonate ions,
ultimately forming calcium carbonate precipitation as a cement within soil grains. However, studies of using MICP to enhance
granite residual soil (GRS) that is recognized as a problematic soil because of its wide grain size distribution are relatively
rare. In this present study, bio-cemented GRS samples were prepared through grouting with Sporosarcina pasteurii as the
colony and a mixture of urea and calcium chloride as the cementation solution. The effect of cementation–solution concentra-
tions on the mechanical properties of the bio-cemented samples was analyzed through unconfined compression and triaxial
shear tests. Furthermore, X-ray computerized tomography, scanning electron microscopy, and X-ray diffraction experiments
were performed to reveal the mechanism of MICP from a microscopic perspective. The experimental results indicate that
an optimal concentration of 2mol/L achieved the highest level of cementation, resulting in an impressive 47.15% increase
in the unconfined compressive strength of the GRS samples. The triaxial shear strength and stress paths of bio-cemented
samples were affected by the cementation level. The variation of porosity indicated that CaCO3 precipitation improves soil
densification by filling the macropores among the soil grains. The CaCO3 precipitates from the MICP treatment predomi-
nantly exist in the form of calcite crystals, serving to fill, wrap, and cement within the soil structure, thereby enhancing the
cohesive and frictional forces exerted by the bio-cemented grains.
Keywords MICP· Granite residual soil· Mechanical behaviour· Microstructure· Cementation–solution concentration
Introduction
Used extensively in infrastructure construction in southern
China, granite residual soil (GRS) has a formation process
that is influenced mainly by the prevailing subtropical and
tropical climatic conditions and the properties of the parent
rock (An etal. 2023), and GRS is characterized by having
a wide particle distribution from clay to gravel and a com-
plex primary pore and crack structure (Mohamedzein and
Aboud 2006). Compacted residual soils are commonly used
as filling materials in highway, foundation, and slope con-
struction, and their geotechnical properties play an important
role in engineering stability. However, physical and chemical
weathering greatly affects the properties of residual soils,
* Ran An
anran@wust.edu.cn
* Xianwei Zhang
xwzhang@whrsm.ac.cn
Haodong Gao
ghd0301@outlook.com
Xin Chen
cx2022611@126.com
Yixian Wang
wangyixian2012@hfut.edu.cn
Hao Xu
3321419310@qq.com
1 School ofCivil andHydraulic Engineering, Hefei University
ofTechnology, Hefei, People’sRepublicofChina
2 School ofUrban Construction, Wuhan University ofScience
andTechnology, Wuhan, People’sRepublicofChina
3 State Key Laboratory ofGeomechanics andGeotechnical
Engineering, Institute ofRock andSoil Mechanics,
Chinese Academy ofSciences, Wuhan430071,
People’sRepublicofChina

Environmental Earth Sciences (2024) 83:31
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31 Page 2 of 13
resulting in low strength, high porosity, high permeability,
and easy disintegration in water (An etal. 2022a, b; Liu
etal. 2022). Traditionally, fibers, cements, or polymers are
added to GRS to improve its engineering properties (Okonta
and Nxumalo 2022; Yuan etal. 2022). However, although
using these materials to reinforce GRS is beneficial, doing
so has some negative economic and environmental implica-
tions. Therefore, it is important to seek an environmentally
friendly, energy-saving, and effective method to improve soil
properties.
In 1997, Ferris etal. (1997) investigated Sporosarcina
pasteurii which hydrolyzes urea by producing urease and
has become the most commonly used bacteria for micro-
bially induced calcite precipitation (MICP). The carbon-
ate ions produced by the hydrolysis reaction continuously
increase the alkalinity of the reaction solution and com-
bine with calcium ions to produce CaCO3, thereby enhanc-
ing the mechanical properties of the soil (Stocks-Fischer
etal. 1999). In recent years, bio-cementation based on
MICP technology has emerged as an effective and environ-
mentally friendly comprehensive soil utilization technique
(Wani and Mir 2020). The technique of MICP that involves
a complicated biochemical reaction is now used widely
in soil improvement, dust stabilization, wastewater treat-
ment, and heavy-metal remediation (DeJong etal. 2010;
Jain and Dali 2019; Rajasekar etal. 2021; Wani and Mir
2021a), and its microscopic mechanism is briefly depicted
in Fig.1. First, the Sporosarcina pasteurii hydrolyzes urea
into ammonium ions and hydroxide ions in an alkaline
environment. Then, the calcium ions provided by CaCl2
in the cementation solution combine chemically with the
carbonate ions to induce CaCO3 precipitation, which is
an insoluble product with a cementation effect (Canakci
etal. 2015). Finally, the CaCO3 precipitation deposited on
the surface of soil particles enhances the bonding force of
grains (Sasaki and Kuwano 2016). Similarly, MICP tech-
nology can also serve as an effective method for heavy
metal precipitation from wastewater (Torres-Aravena etal.
2018).
In previous cases when MICP was used for soil reinforce-
ment, it was mostly for sandy soils, which have larger grain
size and pore throat size and are more geometrically com-
patible with CaCO3, making it easier to perform MICP by
grouting (Karimian and Hassanlourad 2022). Mujah etal.
(2019) studied the combined effect of bacterial culture and
cementation solution on bio-cemented sand and concluded
that the combination of 3.2mg/mL bacterial cultural and
0.25mol/L cementation solution produced the highest
strength; this is because effective CaCO3 crystals are gener-
ated under these conditions. Chek etal. (2021) treated beach
sand with high-concentration bacterial solution and cemen-
tation solution using surface spraying and percolation, and it
was observed that higher optical density and more bacteria
resulted in lower erodibility and greater crust depth. Jain
and Das (2023) conducted further analysis on the impact of
equal MICP treatment on the biocementation and bioclog-
ging of sands with varying grain sizes and concluded that
the relative size of the soil particles and biominerals deter-
mines the soil strength. Furthermore, due to the formation of
CaCO3 precipitates, MICP can also reduce the permeability
Fig. 1 Microbially induced
calcite precipitation (MICP) by
urease-producing bacteria in the
presence of urea and calcium
chloride

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of sandy soil by decreasing pore volume and pore size (Gao
etal. 2019; Wani and Mir 2021c).
However, the transport, adsorption, and nucleation
of microorganisms are challenging in fine-grained soils
because of their comparatively tiny particle size and pore
throat size, thus making MICP treatment for fine-grained
soils more challenging. As curing technology has developed,
the microbial treatment of fine-grained soils has achieved a
certain amount of progress. Cardoso etal. (2018) used MICP
in sand containing clay; the results indicated that there was
a chemical interaction between the clay minerals and the
feeding solution, which makes applying MICP to clay more
complicated. Islam etal. (2020) observed that CaCO3 pre-
cipitation increased with increasing clay content, suggesting
that MICP is more effective in soils with higher clay content;
the explanation was the presence of more bacteria in the
clay, and thus it was confirmed that bio-stimulated CaCO3
precipitation can be applied in clay.
The concentration of the cementation solution affects the
cementing effect of MICP to a great extent, including the
production and distribution of CaCO3 precipitation (Stocks-
Fischer etal. 1999; Soon etal. 2014). Al Qabany and Soga
(2013) investigated the effects of cementation–solution con-
centration in the range of 0–1mol/L on the strength and
permeability of cemented soils, and they found that (i) the
unconfined compressive strength (Su) increased with increas-
ing concentration and (ii) low concentration resulted in a
sharp decrease in permeability, whereas high concentra-
tion resulted in a gradual decrease in permeability. Zhao
etal. (2014) discovered that increasing the cementation
concentration from 0.25 to 0.5mol/L increased the Su of
bio-cemented soils tenfold from 0.13 to 1.36MPa, and
increasing the concentration from 0.5 to 1.5mol/L nearly
doubled the Su from 1.36 to 2.13MPa. Besides the strength
properties, Al Qabany etal. (2011) discovered that the
sedimentation pattern is also influenced by concentration.
They observed that lower concentrations tend to distribute
the precipitate better for the same amount of precipitation,
particularly at lower cementation levels. An optimal cemen-
tation–solution concentration is not only a precondition for
large production of CaCO3 precipitation but also provides
the best survival environment for microorganisms during the
MICP treatment process.
Given the numerous adverse engineering properties of
residual soils and their lack of research to date, MICP tech-
nology may be a promising direction in soil enhancement.
To date, numerous studies have focused on improving the
mechanical properties and engineering characteristics of
residual soils using MICP. Soon etal. (2013) compared the
strength enhancement of residual soil and sand by MICP,
and they found that the shear strength of residual soil was
improved significantly more than that of sand under the same
MICP treatment, but the hydraulic conductivity of residual
soil was lower than that of sand. Lee etal. (2013) reported
that MICP treatment led to a noticeable improvement in the
strength and stiffness of residual soil, and they showed that
there was a good correlation between CaCO3 content and
the compression index, peak stress, and total settlement
of the soil, but a poor relationship with the compression
index. However, although some progress has been made in
studying the strength properties and microstructure of soils
after MICP treatment, the factors affecting the MICP effec-
tiveness of GRS are yet to be studied fully, especially the
concentration of the cementation solution. Therefore, more
attention should be paid to applying this method to residual
soils, especially GRS, which is recognized as a problematic
and special soil because of its grain size distribution.
In the present study, the MICP technique was used to
investigate the cementation of GRS by Sporosarcina pas-
teurii with six different concentrations of cementation solu-
tion. The rules of cementation–solution concentrations
affected the mechanical behavior and pore distribution were
analyzed by conducting unconfined compression and triaxial
shear tests, and X-ray computerized tomography. Further-
more, scanning electron microscopy (SEM) experiments
were conducted to reveal the mechanism of soil enhance-
ment by CaCO3 crystals induced by microorganisms, and the
CaCO3 type was identified via X-ray diffraction (XRD) tests.
Materials
Granite residual soil
The samples of GRS used for MICP treatment were col-
lected from a slope in Guilin in Guangxi Province in
China. A set of experiments was conducted to measure
the physical properties of the soil, including its moisture
content (w), dry density (ρd), specific gravity (Gs), void
ratio (e), and Atterberg limits (wp, wL, and Ip). In addition,
the mineral composition was obtained by an XRD test per-
formed according to ASTM D4926-2015 (2015). Table1
lists the basic physical properties and mineral composi-
tion of the studied GRS, which consisted primarily of clay
Table 1 Physical parameters
and mineral composition of
studied GRS
w
(%)
ρd
(g.cm−3)
Gse wL
(%)
wp
(%)
IpMineral composition (%)
Quartz Kaolinite Illite Hematite
35.2 1.49 2.68 1.05 50.5 25.6 24.9 53.8 37.7 5.6 2.9

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minerals (37.7% kaolinite) and non-clay minerals (53.8%
quartz), indicating that it had undergone intense weather-
ing. The grain size distribution was measured according to
ASTM D422-63 (2002), and as shown in Fig.2, the GRS
contained high contents of clay, silt, and sand particles and
so was characterized as a mixed-grain soil.
Bacterial solution
The bacteria used in this experiment was Sporosarcina pas-
teurii (ATCC 11859) purchased from the China National
General Microbial Species Collection Management Center
(CGMCC). The bacterial solution contained 15-g/L tryp-
tone, 5-g/L peptone, 5-g/L NaCl, and 20-g/L urea, adjusted
to pH7.3 with a 1mol/L NaOH solution. The tryptone,
peptone, and NaCl were autoclaved, and the urea was steri-
lized by ultraviolet light. The bacteria were inoculated into
the medium and then shaken in a constant-temperature
oscillating incubator at 30°C and 130rpm for 36h. The
bacterial medium was centrifuged twice at 4000rpm for
30min, and the suspension was discarded. Fresh medium
was retained for final optical density measurements by a
spectrophotometer at 600nm (OD600 = 0.91). The urease
activity was determined indirectly by measuring the rate
of ammonia production via the Nessler method (Cheng
etal. 2016). The urease activity prepared for this study
was 14.6 U/mL, representing urease present in 1mL of
culture solution could hydrolyze 14.6μmol of urea per
minute. To maintain the viability of the bacterial solution,
it was stored under refrigeration at 4°C and utilized within
1 week (Konstantinou etal. 2021).
Cementation solution
The cementation solution was a mixture of CaCl2 and urea
solution, which provided a calcium source for microbial
cementation and a nitrogen source for microbial growth dur-
ing the MICP process. Urea is hydrolyzed by urease pro-
duced by bacteria to produce carbonate ions, which react
chemically with calcium ions provided by calcium chloride
to produce CaCO3 precipitate. In this study, six different
cementation concentrations with an equal molar ratio of urea
to CaCl2 (0, 0.5, 1.0, 1.5, 2.0, and 2.5mol/L) were used
for the preparation of bio-cemented samples at an ambient
temperature of 20°C and 50% relative humidity.
Methods
MICP treatment
As shown in Fig.3, the soil samples of 50mm diameter and
100mm height for the tests were prepared in a full-contact
PVC pipe with 5-mm-thick filter layers at both ends to pre-
vent seepage of soil. The soil column was compacted by
being vibrated until it reached a dry density of 1.49g/cm3.
Bacterial cultures were introduced into the GRS samples as
an aqueous solution in this study (Fu etal. 2023). To prevent
the clogging of CaCO3 near the injection site, a two-phase
grouting with a peristaltic pump was employed. The effec-
tiveness of this technique has been extensively confirmed by
Whiffin etal. (2007). Initially, the dried sample underwent
an injection of a bacterial solution at a flow rate of 10mL/
min, allowing for the transportation of bacteria through soil
pores via advection and diffusion (Cui etal. 2020). Sub-
sequently, the bacteria were adsorbed onto soil particles.
To enable the bacteria to be in full contact with the soil,
the sample was left for 6h and then injected with one pore
volume of cementation solution at the same rate. After 6h
of curing, the sample was turned upside down carefully and
the above injection procedures were repeated to improve the
uniformity of CaCO3 distribution. A round of grouting was
considered complete when both ends of the sample were
injected, and each sample was subjected to six rounds of
grouting. After the last round of grouting, the chemicals
remaining on the surface of the sample were flushed out with
five pore volumes of deionized water. Finally, the weak parts
of the soil column at both ends were removed and polished
to make a standard sample for mechanical tests.
Unconfined compression strength tests
Unconfined compression strength (UCS) tests performed
according to ASTM D2166/D2166M-16 (2016) were used
to investigate the compressive strength and failure modes of
Fig. 2 Grain-size distribution and soil classification of studied GRS

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untreated and bio-cemented samples. The UCS instrument
used for these tests was a WDW-100E microcomputer-con-
trolled universal testing machine. During a test, a cylindri-
cal sample was loaded at a rate of 1mm/min until it was
damaged; the stress–strain curve was recorded by the data
acquisition system, and the failure pattern of the sample was
captured by a digital camera.
Consolidated undrained triaxial compression tests
To investigate the mechanical characteristics of the bio-
cemented samples, a GDS triaxial shear device was used to
conduct consolidated undrained compression tests accord-
ing to ASTM D4767-11 (2020). Before testing, the standard
cylindrical samples were washed with distilled water, then
vacuum saturated for 24h, then placed in a pressure chamber
for 24h to consolidate at confining pressures of 50, 100,
200, and 400kPa. Finally, the specimens were subjected to
axial loading at an axial strain rate of 0.01%/min until the
axial strain reached 15%.
X‑ray computerized tomography tests
To investigate the geometrical morphology and distribution
of pores and cracks of untreated and bio-cemented samples,
a series of X-CT tests were performed using a high-resolu-
tion PhoenixV X-CT scanner (General Electric; Wunstorf,
Germany). Grayscale images with a spatial resolution of
20LP/mm were obtained from each scan, and the spatial
porosity was observed after binarisation and three-dimen-
sional (3D) reconstruction of the grayscale images based on
the watershed algorithm.
Scanning electron microscopy tests
The microstructural characteristics and the distribution
of precipitated CaCO3 in the bio-cemented samples were
investigated using SEM. Before testing, all specimens under-
went deionization flushing and were subsequently dried
for 48h. Following this, subsamples with fresh surfaces
were excised from the dehydrated SEM samples and sput-
ter coated with gold alloy to improve their conductivity for
obtaining high-quality SEM images. Finally, the prepared
subsamples were observed via SEM to generate a series of
microscopic images with varying magnifications.
X‑ray diffraction tests
The mineral compositions of both untreated and bio-
cemented samples were analyzed using an X-ray diffractom-
eter (Shimadzu XRD-6000) with Cu Ka radiation at 40kV
and 30mA. Scans were performed from 15° to 65° in steps
of 0.02° at a rate of 0.5s per step.
Results anddiscussion
Effect ofcementation–solution concentration
onunconfined compressive strength
To investigate the strength characteristics of GRS treated
by MICP, untreated and bio-cemented samples with differ-
ent cementation–solution concentrations were subjected
to UCS tests, the results of which are shown in Fig.4. As
can be seen, the stress–strain curves exhibit typical strain-
softening characteristics: initially, the stress of each sample
increases with strain until the peak, after which it decreases
with continually increasing strain. The peak stresses of the
bio-cemented samples are significantly higher than that
of the untreated sample, and the peak stress of the bio-
cemented sample with 2mol/L cementation solution is the
highest among all the samples. This indicates that there
is an optimum cementation solution of 2mol/L for a bio-
cemented sample to obtain a maximum peak stress as the
Fig. 3 a Schematic of MICP treatment; b photograph of grouting devices

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cementation–solution concentration is increased from zero
to 2.5mol/L.
Figure5 shows the effects of the cementation–solution
concentration on the Su and elastic modulus (E) of the bio-
cemented samples. As can be seen, applying MICP with
the optimum cementation–solution concentration greatly
improves the strength characteristics of GRS. As shown in
Fig.5, Su and E of the bio-cemented sample with 2mol/L
cementation solution increased by 47.15% and 82.08%,
respectively. The increases in Su and E indicate the forma-
tion of CaCO3 crystals between soil particles, which can
develop structural continuity in the appropriate alkaline
environment. However, the Su and E of the bio-cemented
sample with 2.5mol/L cementation solution decreased. This
may be due to the inhibition of the generation of enzymes
necessary for bacterial functional metabolism. In addition
to inhibiting microbial activity, a higher concentration
cementation solution also impairs the efficiency of CaCO3
precipitation (Kunst and Rapoport 1995; Rivadeneyra etal.
2020). Previous investigations have demonstrated that bacte-
rial growth and metabolism can be inhibited in urea–Ca2+
cementation solutions when concentrations exceed a specific
threshold (Nekolny and Chaloupka 2000; Tang etal. 2020).
For instance, in the case of dredged river silt, the greatest
enhancement in strength and elastic modulus was observed
when the concentration of the cementing solution reached
1.5mol/L, and for soft clay and sandy soil, optimal cemen-
tation–solution concentrations were found to be 0.5mol/L
and 1mol/L, respectively (Van Paassen 2009; Xiao etal.
2020; Wang etal. 2022). However, in this experiment, the
Su and E of the bio-cemented sample decreased when the
cementation–solution concentration exceeded 2mol/L, and
the soil type, microbe species, optical densities, and ambient
temperature are considered to be factors influencing these
differences (Bosak etal. 2004; Ferris etal. 2004; Dhami
etal. 2013; Kim etal. 2013; Wani and Mir 2021b; Mir and
Wani 2021; Jain 2023).
Moreover, as a result of microbial cementation, MICP
transformed the failure mode of the GRS from plastic to
brittle, aligning with Tang etal. (2020) who observed that
MICP-treated soils generally exhibit more brittle charac-
teristics. Figure6 shows damage photographs and failure
patterns of the bio-cemented samples with different cemen-
tation levels. The untreated and weakly cemented samples
exhibited failure patterns of local cracks due to insufficient
cohesion (Fig.6a, b). When the concentration was increased
slightly, the plastic failure of the bio-cemented samples was
markedly weakened (Fig.6c, d). Comparing the failure pat-
terns of the two bio-cemented samples shown in Fig.6e, f
shows that the one with 2mol/L cementation solution exhib-
ited a more significant brittle failure (although both samples
showed similar integral shear damage), which confirms that
the optimum cementation–solution concentration promotes
the microbial cementing capacity, contributing to the full
performance of MICP in enhancing GRS. Taken together,
these results indicate that bio-cemented samples treated with
cementation–solution concentrations increasing from zero
to 2.0mol/L develop higher compressive parameters and,
therefore, form a stronger structure.
Effect ofcementation–solution concentration
onshear strength
With shear strength being a governing design parameter
for geotechnical engineering structures, there have been
numerous studies of microbial cementation for improving
the shear strength properties of soils (Arpajirakul etal.
0
50
100
150
200
0123456
Axial stress (kPa)
Axial strain (%)
0 mol/L
0.5 mol/L
1.0 mol/L
1.5 mol/L
2.0 mol/L
2.5 mol/L
Fig. 4 Stress–strain curves of untreated and bio-cemented samples
Unconfined compressive strength
Elastic modulus
0 0.5 1 1.5 2 2.5
100
120
140
160
180
200
220
Concentration (mol/L)
S
u
(kPa)
0
2
4
6
8
10
E (MPa)
Fig. 5 Effects of cementation–solution concentration on parameters
from UCS tests

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Page 7 of 13 31
2021; Sharma etal. 2022). Figure7 indicates that the
stress–strain curves for untreated and bio-cemented sam-
ples all exhibit strain-hardening (Cui etal. 2017). In gen-
eral, with increasing axial strain, the axial stress increases
sharply initially and then stabilizes. The stress–strain
curves of all samples are featured by strain-hardening
and are influenced markedly by the confining pressure;
specifically, as the confining pressure is increased from
50 to 400kPa, the stress–strain curves move consistently
upward.
Fig. 6 Failure modes of bio-cemented samples with different cementation concentrations: a zero; b 0.5 mol/L; c 1.0 mol/L; d 1.5 mol/L; e
2.0mol/L; f 2.5mol/L
Fig. 7 Stress–strain curves of bio-cemented samples with various cementation–solution concentrations: a zero; b 0.5 mol/L; c 1.0 mol/L; d
1.5mol/L; e 2.0mol/L; f 2.5mol/L

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The theory of Mohr–Coulomb provides the basis for cal-
culating the shear strength parameters of soils. In the case of
testing data for the untreated and bio-cemented with 2mol/L
cementation solution samples, the Mohr circles and shear
strength envelopes are presented in Fig.8. The Mohr circles
exhibited that as the mean stress increases, the shear strength
rises linearly. In addition, the shear envelope moves upward
with increasing cementation level. The slope of the stress
paths increases, because the soil densification was enhanced
under the impact of microbial cementation (DeJong etal.
2010). The six sets of effective stress parameters (c' and φ')
calculated based on the Mohr–Coulomb theory are listed
in Table2. The effective cohesion of the bio-cemented
samples with different cementation–solution concentra-
tions (0.5, 1.0, 1.5, 2.0, 2.5mol/L) increases by 29.15%,
66.36%, 94.85%, 127.62%, and 122.27%, respectively. In
the range of 0.5–2.5mol/L for the cementation solution, the
internal friction angle is higher than that of the untreated
sample, increasing by 9.97%, 19.01%, 26.53%, 36.50%, and
33.19%, respectively. Note that the bio-cemented sample
with 2mol/L concentration solution has the greatest increase
in shear strength, which is consistent with the analysis of its
Su and E. The results show that MICP treatment enhances
the shear behavior of GRS by increasing both its effective
cohesion and internal friction angle. This is because CaCO3
precipitates can be considered as particle-wrapping materials
that improve the surface roughness of soil particles and thus
increase the effective internal friction angle. Meanwhile,
CaCO3 crystals can also be considered as particle-binding
materials that increase the effective cohesion because of the
enhanced binding strength between soil particles.
Effect ofcementation–solution concentration
onpore size distribution
Through X-ray computed tomography scans of the bio-
cemented samples, changes in their densification can be
estimated by visualization and quantitative analysis. 3D
models of the pore structures in Fig.9 were reconstructed
from X-CT images. It is clear that the pore sizes of the bio-
cemented samples are reduced greatly and the total pore vol-
umes are smaller when the concentration exceeds 1.5mol/L.
Elliot etal. (2007) used the pore size classifications of
5–30μm, 30–100μm, 100–1000μm, and 1000–2000μm,
an approach that has been used widely. Combined with the
present X-CT test results, the 3D pore sizes were divided
into four intervals: micropores (≤ 100 μm), mesopores
(100–500μm), macropores (500–1000μm), and intercon-
nected cracks (≥ 1000μm). The distribution of pore sizes
was affected by the cementation level for the bio-cemented
samples, as shown in Fig.10. In general, compared to the
untreated sample, the bio-cemented samples had fewer
macropores and interconnected cracks, but more mesopores
and micropores. In addition, the cracks in bio-cemented
samples gradually transformed into mesopores and micropo-
res with the increase of concentration–solution concentra-
tion. In addition, this change was most obvious when the
concentration reached 2mol/L. The reason for the change in
pore size distribution is that the CaCO3 precipitates mainly
occupy macropores and cracks to reduce pore sizes. As a
result, this eventually appears as an enhancement in the soil
densification. The analysis of pore size distribution shows
the feasibility of filling the voids in GRS bio-cemented by
MICP treatment.
Effects ofcementation–solution concentration
onmicrostructure andmineral composition
To reveal the mechanism for the solidification of MICP
treatment, the microscopic characteristics of an untreated
sample and one bio-cemented with 2mol/L cementation
solution were analyzed using SEM images obtained with dif-
ferent magnifications. As shown in Fig.11a for the untreated
sample, the soil particles were arranged loosely and the fis-
sures were larger. By contrast, as shown in Figs.11b and
11c for the bio-cemented sample, CaCO3 crystals were inte-
grated into the entire soil structure, cementing adjacent soil
0 300 600 900 1200 1500
0
200
400
600
800
1000
τ´(kPa)
σ´ (kPa)
C0-50 kPa
C0-100 kPa
C0-200 kPa
C0-400 kPa
C2.0-50 kPa
C2.0-100 kPa
C2.0-200 kPa
C2.0-400 kPa
τ´=44.66+σ´·tan32.16°
τ´=19.62+σ´·tan24.56°
Fig. 8 Shear strength envelope and Mohr circles of soils from triaxial
shear tests
Table 2 Parameters of strength
characteristics of untreated and
bio-cemented samples
Concentra-
tion (mol/L)
c´ (kPa) φ´ (°)
0 19.62 23.56
0.5 25.34 25.91
1.0 32.64 28.04
1.5 38.23 29.81
2.0 44.66 32.16
2.5 43.61 31.38

Environmental Earth Sciences (2024) 83:31
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Page 9 of 13 31
particles tightly together and there were fewer fissures in
the bio-cemented sample. This is also consistent with the
microscopic observations made by Wani and Mir (2020),
which indicate that CaCO3 binds to stabilize soil parti-
cles and increases flocculation. As shown in Fig.11d, the
CaCO3 crystals—which are mostly irregular spheres—are
distributed mainly in the internal pores, on the surfaces,
and contact points of the soil particles. Formed from MICP,
the CaCO3 crystals play the roles of filling, wrapping, and
cementing (Tian etal. 2022): filling means that CaCO3 crys-
tals are precipitated in the internal pores between coarse
particles, which makes the soil more compact and thus
increases its shear strength; wrapping refers to CaCO3 crys-
tals on the surfaces of soil particles improving the roughen-
ing of the soil, thereby increasing its internal friction angle;
cementing means that the CaCO3 precipitation enlarges the
contact regions of the soil particles, thereby increasing the
cohesive strength of the soil microstructure.
The changes in mineral composition were confirmed by
XRD tests. Previous work showed that three main types of
CaCO3 are produced during MICP, i.e., calcite, vaterite, and
amorphous (Zhang etal. 2020). The physical and mechani-
cal properties of calcite are the most stable, but bacterial
activation and the rate of urea hydrolysis may affect the crys-
tallization of CaCO3. To analyze the mineral types of the
microbially induced crystals, XRD tests were performed on
an untreated sample and one bio-cemented with 2.0mol/L
cementation solution. As shown in Fig.12, the mineral com-
position of the untreated sample was mainly quartz, kaolin-
ite, illite, and hematite; after MICP treatment, calcite with
Fig. 9 3D pore models of various bio-cemented samples: a zero; b 0.5mol/L; c 1.5mol/L; d 2.0mol/L

Environmental Earth Sciences (2024) 83:31
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31 Page 10 of 13
a content of 4.9% appeared in the bio-cemented sample, but
its XRD spectrum shows no vaterite or amorphous CaCO3,
which confirms that the CaCO3 precipitates in MICP-treated
GRS are calcite crystals.
Conclusions
To examine the potential of using MICP to enhance GRS,
laboratory tests were performed on samples of GRS bio-
cemented with six groups of cementation–solution concen-
trations. The optimum cementation–solution concentration
was determined for the highest improvement in the mechani-
cal properties of the GRS. The pore distribution and distri-
bution and types of CaCO3 crystals produced during MICP
were studied to analyze the mechanism of MICP action.
Based on the experimental results, the following conclu-
sions are drawn.
(1) From uniaxial compression tests, the largest
increases in Su and E were 47.15% and 82.08%, respec-
tively, for the bio-cemented sample with 2 mol/L
Fig. 10 Pore size distributions of untreated and bio-cemented samples
Fig. 11 SEM micrographs and schematic: a untreated sample (2000 × magnification); b bio-cemented sample (2000 × magnification); c bio-
cemented sample (5000 × magnification); d distribution of CaCO3 in soil

Environmental Earth Sciences (2024) 83:31
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Page 11 of 13 31
cementation solution, this being the optimum concen-
tration for MICP treatment. The compressive strength
decreased as the cementation–solution concentration was
increased up to 2.5mol/L, this being because higher con-
centration not only inhibited the efficiency of CaCO3 for-
mation but also affected the activity of the microorganisms
and the production of enzymes required for their metabo-
lism. The failure pattern of untreated and bio-cemented
samples under uniaxial compression changed from plastic
to brittle, indicating that CaCO3 precipitation enhanced
the integrity of the samples.
(2) The triaxial test results showed that the cementa-
tion level had a significant effect on stress paths of the bio-
cemented samples. Specifically, the bio-cemented sample
with the optimum concentration had the largest increase in
ultimate deviator stress compared to that of the untreated
sample. Being ascribed to the enhancing bonding degree
of soil particles, the increases in its effective cohesion and
internal friction angle of the bio-cemented samples with
2mol/L cementation solution were the largest, at 127.62%
and 36.50%, respectively.
(3) The X-ray CT-based analysis of the pore size dis-
tribution of GRS before and after MICP treatment showed
that the CaCO3 precipitation mainly filled macropores and
interconnected cracks. This variation of porosity indicates
that CaCO3 precipitation improved the densification of the
GRS samples.
(4) The action mechanism for the change in mechanical
properties of GRS after MICP was analyzed microscopi-
cally. CaCO3 precipitation was distributed mainly in pores
or on the surfaces and contact points of particles, acting as
filling, wrapping, and cementing, respectively. Besides, the
results of XRD spectra confirmed that the CaCO3 precipi-
tates were calcite crystals, which have stable physical and
mechanical properties.
Acknowledgements The present study had the financial support of the
National Natural Science Foundation of China (Grant Nos. 12102312
and 42177148), Institute of Rock and Soil Mechanics, Chinese Acad-
emy of Sciences (SKLGME021018), and the Open Fund of State Key
Laboratory of Geohazard Prevention and Geoenvironment Protection
(SKLGP2021K011). The authors are grateful for the help form anony-
mous editors and reviewers.
Authors' contributions All authors contributed to the study concep-
tion and design. Writing—original draft preparation: RA, HG, XZ;
writing—review and editing: RA, HG, XC; data collection and analy-
sis: RA, HG, YW; methodology: RA, HG, HX. All authors read and
approved the final manuscript.
Data Availability Data will be made available on request.
Declarations
Competing interests The authors declare no competing interests.
Conflict of interest The authors declare that they have no known com-
peting financial interests or personal relationships that could have ap-
peared to influence the work reported in this paper.
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