Acta Geotechnica (2017) 12:971–986
DOI 10.1007/s11440-017-0574-9
RESEARCH PAPER
Influence of cementation level on the strength behaviour
of bio-cemented sand
Ming-Juan Cui1 • Jun-Jie Zheng1 • Rong-Jun Zhang1 • Han-Jiang Lai1 • Jun Zhang2
Received: 8 March 2016 / Accepted: 19 July 2017 / Published online: 8 September 2017
Ó Springer-Verlag GmbH Germany 2017
Abstract Microbially induced calcite precipitation
(MICP) is used increasingly to improve the engineering
properties of granular soils that are unsuitable for con-
struction. This shows MICP technique significant advan-
tages such as low energy consumption and environmentally
friendly feature. The objective of the present study is to
assess the strength behaviour of bio-cemented sand with
varying cementation levels, and to provide an insight into
the mechanism of MICP treatment. A series of isotropic
consolidated undrained compression tests, calcite mass
measurement and scanning electron microscopy tests were
conducted. The experimental results show that the strength
of bio-cemented sand depends heavily on the cementation
level (or calcite content). The variations of strength
parameters, i.e. effective friction angle u0 and effective
cohesion c0 , with the increase in calcite content can be well
evaluated by a linear function and an exponential function,
respectively. Based on the precipitation mechanism of
calcite crystals, bio-clogging and bio-cementation of cal-
cite crystals are correlated to the amount of total calcite
crystals and effective calcite crystals, respectively, and
contributed to the improvement in the effective friction
angle and effective cohesion of bio-cemented sand,
separately.
& Jun-Jie Zheng
zhengjj@hust.edu.cn
1
Institute of Geotechnical and Underground Engineering,
Huazhong University of Science and Technology, Wuhan
430074, Hubei, China
2 of the complex MICP technique in practice. One of the
Key Lab of Highway Construction and Maintenance
Technology in Loess Region, Shanxi Transportation
Research Institute, Taiyuan 030000, Shanxi, China
Keywords Bio-cemented sand  Cementation level 
Isotropic consolidated undrained compression test 
Mechanism analysis  Shear strength  Scanning electron
microscopy
1 Introduction
Application of a new emerging ground treatment technique,
namely microbially induced calcite precipitation (MICP), in
geotechnical engineering is gaining increasing attention and
popularity in recent years. For the MICP technique, bacteria
with high urease activity hydrolyse urea into carbonate ions
and then produce calcite crystals in the presence of calcium
ions [19, 32], which is a spontaneous process with environ-
mentally friendly characteristics in comparison with the
conventional chemical grouting method. However, the
chemical grouting method commonly involves additives
such as resins, lignin, acrylate, urethane and asphalt [28],
some of which may be toxic and cause groundwater con-
tamination to some extent [9, 17]. Some researchers have
dedicated to investigate the effectiveness of MICP treatment
[9, 35]. It is widely accepted that MICP treatment is able to
decrease the permeability of porous media [27], increase the
strength and stiffness [18, 25] and mitigate liquefaction of
loose sand [23, 24]. MICP treatment is also a feasible method
to construct an aquaculture pond in sand [4, 30], immobilise
the sand dust and related pollutants [31] and improve the
strength of soft marine clay [16].
Some studies have focused on investigating the potential
influencing factors of engineering properties of bio-ce-
mented sands [1, 2, 14, 20, 26] to promote the application
most fundamental factors is the cementation level [22],
which is defined as chemical treatment times in the present
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study. Feng and Montoya [11] experimentally investigated
the drained shear strengths of bio-cemented sand speci-
mens with four different cementation levels (i.e. untreated,
lightly treated, moderately treated and heavily treated). It
was found that higher cementation level produced higher
stiffness and strength. Montoya and DeJong [22] also
conducted research on the stress–strain behaviour of bio-
cemented sand with five different cementation levels at a
confining pressure of 100 kPa and pointed out that the
increase in strength could be attributed to the rise of fric-
tion based on the assumption that the cohesion of bio-
cemented sand was zero. In contrast, Ivanov and Chu [15]
believed that the enhancement of strength was mainly
caused by the bio-cementation of calcite crystals. In a
word, the intrinsic strength enhancing mechanism of bio-
cemented sand is still unclear in the previous studies.
Therefore, further experimental investigations are needed
to provide more information for analysing the strength
behaviour of bio-cemented sand with varying cementation
levels in both macro- and micro-scale perspectives.
To better reveal the mechanism of MICP treatment, more
extensive experimental investigations are conducted through
isotropic consolidated undrained compression (CIUC) tests at
three different confining pressures on eight sets of bio-ce-
mented sand specimens with varying chemical treatment
times. The shear characteristics, such as stress–strain rela-
tionship, stress path, strength parameters and the failure mode
of the bio-cemented sand specimens, are analysed in detail to
evaluate the influence of cementation level from a macro-
perspective. Then, effort is made to qualitatively analyse the
microstructure of bio-cemented sand via scanning electron
microscopy (SEM) observation. Finally, the strength
enhancement is further discussed through establishing the
relation between the strength parameters and the precipitation
mechanism of calcite crystals.
2 Materials and methods
Three types of laboratory experiments were performed,
including CIUC tests, calcite mass measurement and SEM
tests. The CIUC tests were performed to acquire shear strength
of bio-cemented sand with different cementation levels. The
calcite mass measurement was conducted in an effort to obtain
the mass of calcite crystals. SEM tests were carried out to
demonstrate the microstructure of bio-cemented sand.
2.1 Materials
2.1.1 Sand
The sand used in this study is a type of CHINA ISO
standard sand, and its particle size distribution is shown in
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Fig. 1 Particle size distribution of the sand used in this study
Fig. 1. The coefficient of uniformity Cu is 5.08, the coef-
ficient of curvature Cc is 1.05 and the specific gravity is
2.65. Other characteristics are Dmax = 2.5 mm,
D10 = 0.13 mm and D50 = 0.52 mm. The maximum and
minimum dry densities are 1.924 and 1.662 g/cm3,
respectively.
2.1.2 Bacteria
The type of bacteria used in the present study is Spor-
osarcina pasteurii (ATCC 11859). Before MICP treatment,
Sporosarcina pasteurii was incubated in ammonium–yeast
extract medium (ATCC 1376) to gain the desired concen-
tration of bacteria. The incubating medium was in essence
a mixture of three different ingredients, namely 20 g L-1
yeast extract, 10 g L-1 (NH4)2SO4 and 0.13 M tris buffer
(pH = 9.0). Each ingredient stored in a flask was sterilized
in an autoclave at 121 °C for 25–30 min prior to mixing
them together. After Sporosarcina pasteurii was inoculated
into the medium, the flask was kept in a shaking bed with
rotation speed of 150 rpm and temperature of 30 °C until
the desired concentration of the bacteria was obtained. The
concentration of bacteria was monitored by an ultraviolet
spectrophotometer at wavelength of 600 nm (namely
optical density OD600). In this experimental investigation,
OD600 is finally determined as 1.8, and the urease activity
of bacteria is 46.7 mM urea/h. For brevity, the medium
containing incubated Sporosarcina pasteurii is referred to
as bacteria solution from hereon.
2.2 Testing program and specimen preparation
The experimental program is detailed in Table 1 and each
group includes 3–4 specimens. A preliminary investigation
into the influence of cementation level on the strength
behaviour of bio-cemented sand was based on Tests 1–8.
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Table 1 Experiment details

Test Treatment Initial relative Initial dry density Final dry density Average calcite content Number of specimens
group times (N) density (%) (g/cm3) (g/cm3) Cm_av (%)
CIUC Acid SEM
testa treatmenta test

1 2 30 1.73 1.76 1.59 3 3 1

2 4 30 1.73 1.78 2.89 3 3 1
3 6 30 1.73 1.80 4.11 3 3 –
4 8 30 1.73 1.83 5.69 3 3 1
5 10 30 1.73 1.86 7.15 3 3 –
6 12 30 1.73 1.88 8.76 3 3 1
7 14 30 1.73 1.90 9.91 3 3 –
8 16 30 1.73 1.94 11.87 3 3 1
9b 0 30 1.73 1.73 – 3 3 1
10b 16 30 1.73 1.73 – – 3 –
11c 0 80 1.87 1.87 – 3 – –
a
Bio-cemented sand specimens used in CIUC tests are also used for calcite content measurement
b
Tests 9–10 are loose sand samples
c
Test 11 is dense sand sample. No bacteria are introduced in Tests 9–11

Tests 9 and 10 were performed to examine whether the
MICP process indeed took place in the specimens via the
acid treatment, SEM measurement and energy-dispersive
X-ray (EDX) analysis. Meanwhile, Tests 1–9 and 11 were
utilised to reveal the strength enhancing mechanism of bio-
cemented sand.
For the specimen preparation, split mould was used to
fabricate sand columns (Fig. 2), and each sand column was
39.1 mm in diameter and 80 mm in height [33]. The
preparation of loose and dense sand was controlled by the
relative density. For a target relative density (30 or 80%),
the weight of dry sand filled into each mould can be cal-
culated beforehand by the weight–volume relationships for
soil aggregate. Specimens were prepared by dry pluviation
to a desired relative density. During preparing sand spec-
imens, three calibration lines in each mould along the
column were marked in advance to better control the uni-
formity of sand specimen. After filling each layer, the dry
Fig. 2 Schematic of MICP test setup
sand was compacted to the prescribed calibration line to
achieve the desired relative density. After filling the sand
into the mould, distilled water was flushed through the sand
column repeatedly to saturate the samples.
It is well known that the uniformity of calcite distribu-
tion is of significant importance to the strength enhance-
ment of bio-cemented soil because the non-uniform calcite
distribution would cause small shear resistance [1]. How-
ever, many previous studies found that the calcite distri-
bution was still non-uniform when only injecting pure
bacteria solution [1, 11, 20]. To improve the calcite dis-
tribution, a two-step biological injection method was used
for the injection of bacteria solution to prepare the bio-
cemented sand specimens (i.e. Tests 1–8). After several
comparison experiments (referring to Fig. 3, samples are
sheared at a rate of 1 mm/min), it has been justified that the
optimised two-step biological injection method (i.e. vol-
ume of the mixed bacteria solution: volume of the pure
bacteria solution = 0.6:0.4) can substantially improve the
strength of bio-cemented sand and its procedure is pre-
sented as follows:
1. A prescribed volume (0.4 times the initial pore
volume) of pure bacteria solution is introduced into
each sand specimen using a peristaltic pump at a flow
rate of 5 mL/min.
2. Upon completing the injection of the pure bacteria
solution, a desired volume (0.6 times the initial pore
volume) of the mixed bacteria solution is immediately
injected into each sand specimen with the same flow
rate.

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Fig. 3 Unconfined compressive strength and calcite content of bio-
cemented sands: different volume ratios of mixed and pure bacteria
solution of two-step biological injection method
The pure bacteria solution was prepared by the
ammonium–yeast extract medium (ATCC 1376), while
the mixed bacteria solution was a mixture of the CaCl2
solution (at a concentration of 0.5 mol/L) and the pure
bacteria solution (the volume ratio of CaCl 2 solution to
the pure bacteria solution is 1:10). The total volume of
the pure bacteria solution and the mixed bacteria
solution was equal to the initial pore volume of each
sand specimen. When the injection of bacteria solution
was finished, the specimens were cured for 8 h to let
the bacteria adsorb on the sand particles. After the
curing time, cementation solution (i.e. urea and cal-
cium chloride solution) with concentration of 0.5 mol/L
was then injected into the sand specimens using a
peristaltic pump at a flow rate of 10 mL/min. Different
rounds of cementation solution injection were consid-
ered in this investigation. For simplicity, the number of
rounds of cementation solution injection is termed as
treatment times (i.e. N) below. The interval between
two successive rounds of cementation solution injection
was 12 h. The volume of cementation solution injected
in each round was equal to the initial pore volume of
the specimen. Upon the completion of each treatment,
the bottom of the sand column was sealed using a
small rubber plug to avoid the leakage of solution (as
shown in Fig. 2). All the specimens were prepared
under an ambient temperature of 30 ± 2 °C, which can
provide favourable environmental condition for bacte-
ria metabolism. It is worth mentioning that Test 10 is
only treated by cementation solution with 16 times, but
without the introduction of bacteria solution.
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2.3 Test procedure
2.3.1 Undrained triaxial compression test
GDS triaxial testing system was utilised to perform the
undrained triaxial compression tests. Before the test, all the
fabricated specimens were washed by distilled water to
eliminate the substances that may exist in the pore space of
the specimens. Vacuum saturation was used to saturate all
the specimens for about 24 h, and then they were
demoulded and put in position in the test apparatus. When
the specimen was in position, distilled water was filled into
the pressure chamber and hydraulic saturation was con-
ducted first and then back pressure saturation was carried
out to saturate the specimen with the Skempton’s B value at
least 0.95. Finally, the specimen was axially loaded at the
rate of 0.5 mm/min.
2.3.2 Calcite mass measurement
To gain the amount of calcite crystals, acid treatment was
adopted. Firstly, all fractions of each bio-cemented sand
specimen after completing CIUC tests were collected. For
each test group, one of the crushed bio-cemented speci-
mens in each test group after CIUC test was cut by a
mechanical saw into three approximately equal parts (i.e.
top, middle and bottom) along the axial direction for calcite
content measurement to quantitatively analyse the distri-
bution of calcite crystals. Each part and other entire cru-
shed specimens were fully dried in the oven, respectively,
at 60 °C [29], and the corresponding dry mass was denoted
as Md. Secondly, 2 M hydrochloric acid was used to dis-
solve calcite crystals until no small block particles and air
bubbles were observed. Then, the residual sand was
washed by tap water and dried by oven until its mass was
kept constant. The dry mass of the sand was subsequently
measured and denoted as Msand. Hence, the mass of calcite
crystals, denoted as MCa, can be determined as:
MCa ¼ Md  Msand ð1Þ
The calcite content, Cm, is defined as the ratio of the dry
mass of calcite crystals and sand, which can be expressed
as:
MCa
Cm ð%Þ ¼  100% ð2Þ
Msand
In addition, for the dissected specimen, the total mass of
calcite, as well as sand, can be determined as the sum of the
dry mass of three parts. Then, the total mass content of
calcite of the dissected specimen can be calculated.
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2.3.3 SEM test
To better understand MICP treatment, SEM tests are usu-
ally used to clarify the microstructure of bio-cemented sand
[19]. Each specimen used for SEM test was first dried in
the oven at 60 °C. Small cubic blocks were then cut from
the core of bio-cemented sand. For the loose sand speci-
men, a small amount of sand particles was fixed on the
surface of a cube. All the test samples were carbon-coated
for 450 s. Then, the samples were scanned using the
Quanta 200 Electron Microscope (Netherlands). Mean-
while, EDX detection was also conducted for the elemental
identification.
3 Results
3.1 Calcite precipitation
The effect of cementation level on the calcite precipitation
is evaluated through analysing calcite content and the
chemical efficiency. The total mass content of calcite
crystals in each bio-cemented sand specimen together with
the loose sand is presented in Fig. 4a. The average amount
of soluble substances in the loose sand untreated (i.e. Test
9) and only treated by cementation solution (i.e. Test 10) is
about 0.18 and 0.24%, respectively. That is, the soluble
substances in sand can be ignored. However, it can be seen
that the calcite content Cm increases gradually with
increase in treatment times in bio-cemented sand, meaning
that the precipitated calcite crystals in the bio-cemented
sand are indeed induced by MICP treatment. The calcite
content along the axial direction of the sand column is
depicted in Fig. 4b, showing all the possibilities for the
calcite content to be higher on top, at the bottom or in the
middle part of the bio-cemented sand specimen. This is
mainly caused by the randomly adsorbed bacteria in sand
specimen. The difference in the calcite content among top,
middle and bottom parts in each bio-cemented sand spec-
imen is actually insignificant (the maximum relative dif-
ference is 10.68% for N = 2, 10.46% for N = 4, 5.53% for
N = 6, 4.13% for N = 8, 2.92% for N = 10, 2.74% for
N = 12, 1.67% for N = 14 and 3.11% for N = 16). This
relative difference is considered to be acceptable in view of
the inevitable experimental error. Hence, it can be con-
sidered that the distribution of calcite crystals produced by
the two-step biological injection method is relatively uni-
form. The chemical conversion efficiency (as shown in
Fig. 4c), which is calculated through comparing the mass
of calcite precipitation with the amount of calcium injected
[1, 2], only presents slight decrease during the entire MICP
treatment. In addition, it should be noted that the chemical
conversion efficiency during the entire MICP treatment is
975
less than 85%, which is relatively small. This may be due
to the fact that when calculating the chemical conversion
efficiency, the calcite crystals precipitated in porous stones
and the surface of the split mould are ignored.
3.2 Shear characteristics
3.2.1 Stress–strain–pore pressure responses
The stress–strain–pore pressure responses of MICP-
treated sand with different cementation levels, together
with the untreated loose sand and dense sand at a con-
fining pressure of 100 kPa, is illustrated in Fig. 5. As
shown in Fig. 5a, peak deviatoric stress of bio-cemented
sand is markedly enhanced compared to the untreated
loose sand and increases with the increase in cementation
level. When the loose sand is treated more than six times,
peak deviatoric stress of bio-cemented sand is higher than
that of dense sand. Figure 5a also shows that the shear
responses of the untreated loose sand and dense sand are
strain hardening and strain softening, respectively. Nev-
ertheless, for bio-cemented sand, there is a tendency from
strain hardening towards strain softening as the cemen-
tation level increases, which is consistent with the finding
of Montoya and DeJong [22]. For the bio-cemented sand
with two treatment times, stress increases with the
increase in axial strain continuously, showing the strain-
hardening behaviour. While, for the loose sand treated
more than six times, stress increases with the increase in
axial strain until peak deviatoric stress is reached, and
then softening tendency occurs. Obviously, there is a
transition state between strain hardening and strain soft-
ening because the stress–strain behaviour of the bio-ce-
mented sand with four treatment times presents strain
softening to a certain degree but not evident.
Pore pressures of all specimens present similar trend
with the increase in axial strain. Specifically, positive pore
pressure is formed initially and then increases to its peak
value. Afterwards, pore pressure begins to decrease, and
eventually negative pore pressure is reached at a certain
axial strain (as shown in Fig. 5b). The axial strain corre-
sponding to the occurrence of negative pore pressure tends
to decrease with the increase in cementation level, indi-
cating that bio-cemented sand will be more inclined to
dilation. This may be related to the brittle behaviour, which
will be analysed in Sect. 3.3.
3.2.2 Peak deviatoric stress
To better investigate the strength enhancement of bio-ce-
mented sand, the results from the triaxial compression tests
are re-plotted in Fig. 6 to present the variation of peak
deviatoric stress with the calcite content (i.e. cementation
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Fig. 4 Calcite precipitation of bio-cemented sand: a total calcite content in each specimen; b calcite content distribution in a specimen; and
c chemical efficiency
level) and confining pressure. As shown in Fig. 6a, the
peak deviatoric stress increases exponentially with the
increase in cementation level, which is highly influenced
by cementation level. This is consistent with the test results
of van Paassen et al. [34]. Meanwhile, the confining pres-
sure has an effect on the peak deviatoric stress, presenting
that the higher confining pressure, the higher peak devia-
toric stress. To better reveal the effect of cementation level
and confining pressure, peak deviatoric stress of bio-ce-
mented sand (q0 p) is normalised by that of the uncemented
loose sand (q0 p_s), which is regarded as strength improve-
ment (as shown in Fig. 6b). It can be found that within four
treatment times in this study, peak deviatoric stress of bio-
cemented sand is slightly improved in comparison with that
of the untreated loose sand. However, when cementation
level is higher, the enhancement of peak value will be
much larger.
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3.2.3 Stress path
To further investigate the shear characteristics of the bio-
cemented sand under axial compression, the stress path for
the bio-cemented sand with varying cementation levels is
illustrated in Fig. 7. The deviatoric stress, q0 , and mean
effective stress, p0 , can be calculated via the following
equations:
q0 ¼ r01  r03 ð3Þ
p0 ¼ ðr01 þ 2r03 Þ=3 ð4Þ
where r01 and r03 are the major and minor principal stresses,
respectively.
Corresponding to the analysis of stress–strain relation,
the stress path analysis also only focuses on the results of
specimens at a confining pressure of 100 kPa herein. It can
Acta Geotechnica (2017) 12:971–986 977

Fig. 5 Shear response of specimens at a confining pressure of 100 kPa: a stress–axial strain; b pore pressure–axial strain

Fig. 6 Effect of confining pressure on the strength: a peak deviatoric stress; b strength improvement

be seen from Fig. 7 that the stress path of loose sand pre-
sents an increase to peak deviatoric stress, but no stress
reversal occurs, indicating that the loose sand shows strain-
hardening behaviour. However, stress reversal can be
found in the dense sand when the peak deviatoric stress is
obtained. For bio-cemented sand, cementation level has
great influences on the stress path. When cementation level
is lower (e.g. less than four treatment times), the variation
of stress is similar to that of the untreated loose sand,
presenting strain-hardening behaviour. As the cementation
level increases (e.g. more than four treatment times), the
stress tends to increase relatively quickly to a certain value
and then continue to gradually increase to peak stress.
Afterwards, stress reversal occurs. Note that for the bio-
cemented sand treated more than 12 times, the stress path
linearly increases to the peak deviatoric stress and then
stress reversal quickly occurs, presenting a significant
strain-softening behaviour. The variation of stress path
indicates that the cementation level has a significant
influence on the shear behaviour of bio-cemented sand.
The relations between the peak deviatoric stress (q0 p)
and mean effective stress (p0 ) with different cementation
levels are depicted in Fig. 8, which are in essence the
failure envelopes of bio-cemented sand. It can be found
that the failure envelopes are approximately linear for all
the bio-cemented sand specimens under three different

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Fig. 7 Effective stress path for all the test samples
Fig. 8 Failure envelopes for all the test samples
confining pressures. In other words, the strength parameters
can be obtained by linear criteria, i.e. Mohr–Coulomb
failure criterion [8], which is a widely applicable method in
geotechnical engineering. Meanwhile, it provides an easy
way to calculate the strength parameters of soil, including
cement-treated soil [5], lime-treated soil [7] and bio-ce-
mented soil [22].
3.2.4 Strength parameters: effective friction angle
and effective cohesion
Based on Mohr–Coulomb failure criterion, the effective
friction angle u0 and effective cohesion c0 can be acquired
to characterise the strength of bio-cemented sand (Fig. 9).
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Fig. 9 Shear strength envelope of undrained triaxial compression test
(N = 6, Test 3)
The strength of bio-cemented sand is closely related to the
amount of calcite crystals, and the effective friction angle
and effective cohesion of sand after MICP treatment would
be markedly different in comparison with the untreated
loose sand. The variations of effective friction angle and
effective cohesion of bio-cemented sand with the average
calcite content are presented in Fig. 10a, b, respectively.
Moreover, the effective friction angles of loose sand
(u0 = 35.65°) and dense sand (u0 = 40.2°) (shown in
Fig. 10a) are used as benchmarks. It can be found that the
effective friction angle has perceivable linear correlation
with the average calcite content, Cm_av. The observation
conforms to the research of Montoya and DeJong [22]. It is
worth mentioning that the effective friction angle of bio-
cemented sand with eight treatment times (i.e. 40.02°) is
similar to that of dense sand (i.e. 40.2°). The effective
cohesion has the best exponential correlation with the
average calcite content (Fig. 10b). The difference in the
variation of effective friction angle and effective cohesion
with the increase in calcite content is mainly associated
with the mechanism of bio-clogging and bio-cementation,
which will be discussed in Sect. 4.3.
3.3 Failure mode
After the triaxial compression tests were finished, failure
modes of bio-cemented sand were observed. Only the
typical modes are shown in Fig. 11. Meanwhile, failure
mode of the untreated loose sand is also given to be used as
a benchmark. For the bio-cemented sand with two treat-
ment times (Fig. 11b), barrelling failure mode can be
observed, which is similar to that of the untreated loose
sand (Fig. 11a), indicating that the failure mode of bio-
cemented sand with lower cementation level can be scar-
cely changed. However, failure mode will be markedly
changed with the increase in cementation level, as shown
in Fig. 11b–e, and a well-defined shear band can be
Acta Geotechnica (2017) 12:971–986 979

Fig. 10 Variation of strength parameters of bio-cemented sand with average calcite content: a effective friction angle; b effective cohesion

Fig. 11 Specimens failed on undrained triaxial compression test (confining pressure 100 kPa): a loose sand, Test 9; b N = 2, Test 1; c N = 8,
Test 4; d N = 12, Test 6; and e N = 16, Test 8

gradually formed, presenting a certain slope instead of

local bulging. This indicates that there is a transition from
barrelling failure mode to the failure mode with a well-
defined shear zone, which is consistent with the research of
Montoya and DeJong [22]. It further confirms the transition
state of shear behaviour as indicated by the stress–strain
relationship.
To quantitatively evaluate the shear behaviour, i.e.
ductile or brittle failure, brittleness index (IB) is adopted
which is proposed by Consoli et al. [6]. The effect of
calcite content on brittleness index is plotted in Fig. 12.
According to the research of Consoli et al. [6], brittleness
index (IB) can be calculated as follows:
qf Fig. 12 Effect of calcite content on brittleness index (IB) of bio-
IB ¼ 1 ð5Þ cemented sand
qult

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where qf and qult are the peak and ultimate strength of
specimens, respectively. In this study, qf is the peak devi-
atoric stress, and qult is regarded as the deviatoric stress at
the axial strain of 15%. As shown in Fig. 12, it can be
clearly found that the higher cementation level, the larger
brittleness index. In other words, the specimen behaves
more brittle as the cementation level increases, which is
consistent with the failure mode of the specimens.
3.4 Microstructure of bio-cemented sand
SEM images of the untreated loose sand and bio-cemented
sand were obtained (shown in Figs. 13, 15, respectively) to
illustrate the morphology, precipitation and distribution
characteristics of the calcite crystals. EDX analysis was
conducted to determine the chemical components of the
untreated and bio-cemented sand.
For the untreated sand (as shown in Fig. 13a), it can be
observed from Fig. 13b that no cementing substances (e.g.
calcite crystals) adsorb on the sand particle surface or in the
particle–particle contacts. EDX analysis (presented in
Fig. 13c) shows that the untreated sand mainly contains
silicon. However, for the bio-cemented sand, a large
amount of crystalline substances is precipitated on the sand
particle surface (presented in Fig. 14), and EDX analysis
indicates that the crystalline substance mainly contains
calcium. Hence, it can be speculated that the substance is
calcite crystals in the bio-cemented sand. In addition,
Fig. 14 also shows that microstructure in each section of
bio-cemented sand is similar, and almost all the pores
among sand particles are filled with calcite crystals. These
results further confirm that calcite crystals occurred in the
bio-cemented sand are completely induced by the bacteria
during MICP treatment.
As shown in Fig. 15, it can be found that calcite crystals
are precipitated on the sand particle surface in the form of
clusters for the bio-cemented sand specimens. If the bio-
Fig. 13 Sand used in the test: a sand particles; b SEM image; and c EDX analysis
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Acta Geotechnica (2017) 12:971–986
cemented sand is slightly treated (see Fig. 15a), only small
amount of calcite crystal clusters with smaller size is pre-
cipitated. Almost all the precipitated calcite crystals are
sparsely adsorbed on the surface of sand particles, and those
precipitated in the particle–particle contacts are tiny. Nev-
ertheless, the size of the calcite crystal clusters would
increase gradually as the MICP process continues, which
may cause the bond of the calcite crystal clusters between the
adjacent sand particles and further improve the cementation
of sand (Fig. 15b). The higher the cementation level, the
more remarkable the calcite cementation (Fig. 15c–e).
4 Discussion
4.1 Calcite precipitation
For MICP treatment, the produced calcite crystals bond the
soil particles and fill the pores [15] and thus enhance the
soil properties [12, 23, 24, 27, 36, 37]. Hence, the amount
of calcite crystals has a significant influence on the strength
of bio-cemented sand [11]. Meanwhile, due to the inho-
mogeneity in calcite cementation, the local deformation of
bio-cemented specimens under compression would occur,
and a small shear resistance can be obtained [1]. This
suggests that the well uniform calcite distribution in bio-
cemented soil is very important to perform the analysis of
strength of the bio-cemented specimen with different
cementation levels.
In the present study, a two-step biological injection
method is proposed to improve the uniformity of bacterial
adsorption in sand specimens. Figures 14 and 4b show that
the distribution of calcite crystals in bio-cemented sand is
relatively uniform. In addition, Fig. 3 indicates that the
proposed two-step biological injection method (i.e. volume
of the mixed bacteria solution: volume of the pure bacteria
solution = 0.6:0.4) can substantially improve the strength
Acta Geotechnica (2017) 12:971–986
Fig. 14 Analysis of bio-cemented sand (N = 16, Test 8)
of bio-cemented sand. In this regard, the calcite distribution
and strength improved by two-step biological injection
method in the present study provide supports for the pos-
sible field application of MICP treatment to avoid the
potential challenges (e.g. bearing failure, local instability)
caused by the non-uniform distribution of calcite crystals.
4.2 Precipitation mechanism of calcite crystals
As the shear characteristics (macro-scale perspective) of
bio-cemented sand are closely related to the precipitation
of calcite crystals (micro-scale perspective), investigation
on the precipitation mechanism of calcite crystals would be
conducive to improving MICP treatment. In this section,
the precipitation mechanism is revealed by analysing the
role of calcite crystals based on the precipitation process of
calcite crystals.
During MICP treatment, bacterial cells are used as
deposition sites of calcite crystals [9, 10, 13, 21]. The
981
newly precipitated calcite crystals will adsorb on the ones
formed previously, termed as accumulation in this study,
and the size of calcite crystal clusters increases continu-
ously (Fig. 15a–e). The process of formation–accumula-
tion–growth for calcite crystals is exactly the precipitation
process of calcite crystals of bio-cemented sand.
According to the role of calcite crystals in the strength
enhancement of bio-cemented sand, calcite crystals can be
classified as effective calcite crystals and non-effective
calcite crystals. Here, calcite crystals used for bonding the
sand particles are termed as effective calcite crystals, while
calcite crystals precipitated on the surface of sand particles,
being unable to bond the sand particles, are termed as non-
effective calcite crystals in the current study (as shown in
Fig. 16). In essence, the spatially distributed effective
calcite crystals within bio-cemented sand, together with the
sand particles, constitute a reticular ‘‘structure’’ (referring
to ‘‘structure’’ herein). Namely, bio-cemented sand can be
regarded as a type of ‘‘structural soil’’, and its shear
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Fig. 15 SEM images of bio-cemented sand with different cementation levels: a N = 2, Test 1; b N = 4, Test 2; c N = 8, Test 4; d N = 12, Test
6; and e N = 16, Test 8
strength depends on the stability of the ‘‘structure’’, which
is mainly influenced by the amount of effective calcite
crystals.
The formation of effective calcite crystals can be clas-
sified into two modes: (a) particle-contact cementation
(caused by the calcite crystals precipitated at the particle–
particle contacts, as shown in Fig. 16c); and (b) calcite–
calcite cementation (caused by the calcite crystal clusters
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Acta Geotechnica (2017) 12:971–986
between the adjacent sand particles, as shown in Fig. 16b).
At lower cementation level, the size of calcite crystal
clusters is relatively small and most of them are precipi-
tated on the sand particle surface (Fig. 15a). The effective
calcite crystals are mainly in the form of particle-contact
cementation and its amount is relatively small, resulting in
low strength of bio-cemented sand (Fig. 6). This is due to
the fact that small amount of effective calcite crystals
Acta Geotechnica (2017) 12:971–986 983

Fig. 15 continued

Fig. 16 Schematic diagrams of non-effective and effective calcite crystals in bio-cemented sand: a non-effective calcite crystals; b calcite–
calcite cementation; and c particle-contact cementation

123
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causes weak ‘‘structure’’ of a bio-cemented sand (as shown
in Fig. 15a). As the MICP process continues, the size of
calcite crystal clusters increases with the increase in
cementation level, which may cause the bond of the calcite
crystal clusters between the adjacent sand particles forming
the calcite–calcite cementation. As a result, some of the
non-effective calcite crystals at the relatively lower
cementation level would be transformed into effective
calcite crystals (as shown in Fig. 16b), further improving
the cementation among sand particles (as shown in
Fig. 15b). The transition of non-effective calcite crystals
into effective calcite crystals would cause the decrease in
non-effective calcite crystals and increase in effective
calcite crystals. In this regard, the amount of effective
calcite crystals would present a significant increase with
the increase in cementation level. Hence, the ‘‘structure’’ of
bio-cemented sand is improved (Fig. 15) and the strength
enhancement of bio-cemented sand is increasingly signifi-
cant (as shown in Fig. 6b).
4.3 Strength enhancing mechanism of bio-cemented
sand
Focusing on the possibility of field applications of MICP
treatment, many geotechnical problems could be solved by
the bio-cemented sand with appropriate shear strength for
the infrastructure construction (e.g. highways and rail-
ways). Hence, the strength enhancing mechanism of MICP
technique must be well understood prior to field
implementation.
According to the precipitation mechanism of calcite
crystals, the function of calcite crystals can be classified as
bio-clogging and bio-cementation [15]. To better reveal the
strength enhancing mechanism of bio-cemented sand,
detailed bio-clogging and bio-cementation mechanism
analyses for the calcite crystals would be performed as
follows:
a. Bio-clogging. All the precipitated calcite crystals can
be regarded as pore-filling materials [15], and bio-
cemented sand will become denser as the calcite
Fig. 17 Degradation modes for MICP-treated sands: a calcite–calcite; b calcite–silica
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Acta Geotechnica (2017) 12:971–986
content increases. Hence, it can be considered that bio-
clogging of calcite crystals exists in the whole MICP
process. Due to bio-clogging, the porosity of sand is
decreased [15], causing the increase in friction angle
(Fig. 10a). Note that even though the calcite cemen-
tation is broken (as shown in Fig. 17), the precipitated
calcite crystals still attach on the surface of sand
particles. This indicates that the roughness of sand
particles can be improved. Due to the ‘‘accumulation’’
process, both the size and amount of calcite crystals
clusters increase (as shown in Fig. 15) with the
increase in cementation level, causing larger roughness
of sand particles. Thus, larger particle roughness
causes larger friction angle [3] because more energy
is needed to overcome the interlocking of particles
during the shear process. Hence, it can be concluded
that bio-clogging is associated with the total content of
calcite crystals, which is mainly contributed to
improving the effective friction angle.
b. Bio-cementation. As Ivanov and Chu [15] clarified, the
produced calcite crystals can also be regarded as
particle-binding materials. That is to say, bio-cemen-
tation exactly exists in the calcite crystals (i.e.
effective calcite crystals) to bond the sand particles.
As discussed in Sect. 4.2, the non-effective calcite
crystals would be transformed into effective calcite
crystals, causing the increasingly significant increase
in the effective calcite crystals. Note that the cohesion
of bio-cemented sand is the major difference in
comparison with the untreated loose/dense sand, and
the effective cohesion of bio-cemented sand presents
an exponential increase with the calcite content
(Fig. 10b). In this regard, it can be inferred that bio-
cementation is related to the content of effective
calcite crystals, focusing on enhancing the effective
cohesion of bio-cemented sand.
For bio-cemented sand with lower cementation level,
insufficient constraints would be formed around the sand
particles, resulting in an unstable ‘‘structure’’ to bear the
load. Hence, the increase in strength of bio-cemented sand
Acta Geotechnica (2017) 12:971–986
is relatively slow at the relatively lower cementation level
and the bio-cemented sand can be regarded as ‘‘weakly
structural soil’’, leading to low strength of bio-cemented
sand (as shown in Fig. 6). However, bio-cementation will
be pronounced with the increase in cementation level due
to the increase in effective calcite crystals, and then the
bond among sand particles can be improved, forming a
stable ‘‘structure’’ (Fig. 15). The strength of bio-cemented
sand increases remarkably at the relatively higher cemen-
tation level and the bio-cemented sand can be regarded as
‘‘strongly structural soil’’.
5 Conclusions
The strength of bio-cemented sand with varying cementa-
tion levels was investigated through undrained triaxial
compression tests. Subsequently, the mechanism of MICP
treatment was revealed with the aid of SEM tests. Based on
the analysis of the experimental results, the following
conclusions can be drawn:
1. The variations of the effective friction angle and
effective cohesion of bio-cemented sand are in the
linear and exponential form with the increase in calcite
content, respectively. Cementation level has significant
influences on the stress path and brittle behaviour of
bio-cemented sand.
2. Calcite crystals are precipitated in the form of clusters,
and its size increases continuously with the increase in
cementation level. During this process, some of the
non-effective calcite crystals would be transformed
into effective calcite crystals. At the relatively lower
cementation level, the effective calcite crystals are
mainly in the form of particle-contact cementation. As
the cementation level increases, the effective calcite
crystals in the form of calcite–calcite cementation
would occur and further improve the cementation
among sand particles.
3. Bio-clogging of calcite crystals exists in the whole
MICP process and is associated with the total calcite
content, contributed to improving the effective friction
angle. Bio-cementation of calcite crystals is caused by
the effective calcite crystals, focusing on enhancing
the effective cohesion of bio-cemented sand.
Acknowledgements This work is funded by the National Key
Research and Development Program of China (No.
2016YFC0800208), the National Natural Science Foundation of
China (NSFC) (Grant Nos. 51478201 and 51308241) and the China
Postdoctoral Science Foundation (Grant No. 2016M600595). The
authors are grateful for the financial supports. Special thanks go to the
Analytical and Testing Centre at Huazhong University of Science and
Technology (HUST) for providing Scanning Electron Microscope.
985
The authors would also like to acknowledge the invaluable cooper-
ation and assistance offered by Prof. Xiao-Yu Zhang’s research team
at College of Life Science and Technology, HUST, particularly Mr.
Xiao Zhang and Wen-Feng Wang.
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