Acta Geotechnica

https://doi.org/10.1007/s11440-021-01384-6 (0123456789().,-volV)(0123456789().
,- volV)

RESEARCH PAPER

Modified one-phase-low-pH method for bacteria or enzyme-induced

carbonate precipitation for soil improvement
Ming-Juan Cui1,2 • Han-Jiang Lai2 • Tung Hoang2,3 • Jian Chu4

Received: 23 June 2021 / Accepted: 10 October 2021

Ó The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
One of the latest developments in biocementation is the use of one-phase-low-pH MICP or EICP method as a more
effective and efficient alternative to the traditional two-phase method for the treatment of sandy soil. However, the one-
phase-low-pH method has its own limitation. The pH of the solution has to be adjusted before every treatment. In this
study, a modified one-phase-low-pH MICP or EICP method is proposed in order to simplify the treatment procedure to
improve the efficiency of biotreatment in real constructions. In this method, the low-pH bacteria or urease solution is only
used together with the cementation solution (i.e., CaCl2 and urea) for the first treatment and for the subsequent treatment,
only cementation solution is used. The test results show that using the modified one-phase-low-pH MICP method with a
bacterial solution of a volume ratio of 0.75 is comparable to that using the original one-phase-low-pH method, and 80% of
the calcium conversion efficiency can be maintained for up to 5 treatments. However, the modified one-phase-low-pH
EICP method may only be used for the first 2 or 3 treatments due to the relatively poor durability of urease enzyme in
inducing calcium carbonate during the subsequent injections of cementation solution.

Keywords Bacteria  Calcium conversion efficiency  Soil improvement  Unconfined compressive strength 
Urease

1 Introduction and (b) enzyme-induced carbonate precipitation (EICP)

Use of biocement for soil improvement has attracted great
attention in recent years [2, 3, 34, 44]. At present, two
approaches have been established for biocementation or
bioclogging: (a) microbially induced carbonate precipita-
tion (MICP) via urease-producing bacteria [2, 3, 25, 41]
& Jian Chu
CJCHU@ntu.edu.sg
1 be implemented on-site. Therefore, as an improvement to
College of Civil Engineering, Fuzhou University,
Fuzhou 350108, Fujian, China
2 mentation, the one-phase-low-pH method was proposed by
Formerly School of Civil and Environmental Engineering,
Nanyang Technological University, Blk N1, 50 Nanyang
Avenue, Singapore 639798, Singapore
3
Faculty of Road and Bridge Engineering, The University of
Danang-University of Science and Technology, 54 Nguyen
Luong Bang Street, Danang 550000, Vietnam
4
School of Civil and Environmental Engineering, Nanyang
Technological University, Blk N1, 50 Nanyang Avenue,
Singapore 639798, Singapore
using free urease enzyme from either bacteria or plants
[7, 17, 19, 29, 45]. For the treatment of sandy soil using
either approach, the pre-mixing method [4, 31–33, 45, 46]
and percolation method [21, 26, 30, 43] are used. For
example, two-phase injection method has been commonly
adopted in which the bacteria (or enzyme) solution is
injected into soil first before cementation solution con-
sisting of normally calcium ions and urea is injected
[3, 21, 41]. The above-mentioned methods usually involve
in multiple treatments, which are complex and difficult to
simplify the treatment procedure for large-scale imple-
Cheng et al. [10] for the MICP-based approach and by Cui
et al. [14] for the EICP-based approach. This method is to
inject bacterial (or enzyme) solution with pH adjustment,
urea and calcium chloride solution (named as MICP or
EICP solution hereafter) into sand simultaneously.
One of the main features of the one-phase-low-pH
method is to inhibit the bioflocculation to create a lag

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Table 1 Details of solution test

Test Bacteria (or urease) volume ratio Urease activity, Cementation solution concentration, Number of Stirringc
# VRa U/mL M treatmentsb

MICP
SM1 0.75 16 1.0 1 No
SM2 0.50 1 No
SM3 0.25 1 No
SM4 0.50 3 Yes
EICP
SE1 0.75 40 1.0 1 No
SE2 0.50 1 No
SE3 0.25 1 No
SE4 0.50 3 Yes
a
VR refers to the volume ratio of bacteria or urease in MICP or EICP solution
b
For Tests SM4 and SE4, the original solution was replaced by equal volume of 1.0 M cementation solution (i.e., CaCl2 and urea) at 24 h
intervals
c
For Tests SM/SE1-SM/SE3, the biotreatment solution was kept unstirred. For Tests SM4 and SE4, the solution was thoroughly stirred after each
measurement

period for the precipitation of calcium carbonate at low pH,
as reported by Cheng et al. [10]. This method can solve the
problem of the one-stage method [36] (without pH
adjustment) that when the bacterial solution and cementa-
tion solution is mixed, bioflocculation and rapid CaCO3
precipitation will occur immediately, resulting in local
clogging during the grouting process. Wu et al. [44] have
proved that the strength of fine aggregates using the one-
phase-low-pH method is higher than those using one-stage
method due to the pH adjustment.
As the strength of the treated soil is dependent on the
amount of calcium carbonate and the calcium carbonate
production is limited for per treatment, repeated treatments
are required for both MICP- and EICP-based method.
However, we may not need to inject bacterial or urease
solutions every time if the activity of bacteria or urease is
sufficiently long to last for a duration of 2 to 3 treatments.
In this study, a modified one-phase-low-pH method is
proposed with the intention to simplify the one-phase-low-
pH MICP or EICP method even further by reducing the
number of injections of bacterial or urease enzyme solu-
tions. Experimental data are presented to verify the effec-
tiveness of the modified one-phase-low-pH method. A
comparison between the original and modified one-phase-
low-pH MICP or EICP methods is also made to highlight
the advantages and disadvantages of each method for either
MICP- or EICP-based approach.
2 Materials and methods
2.1 Materials
In this study, the soil used was clean Ottawa 20–30 sand
(Gs = 2.65, D50 = 0.6 mm, Cu = 1.2, emax = 0.742 and
emin = 0.502). For MICP treatment, Sporosarcina pasteurii
(OD600 = 3.6 ± 0.3, urease activity UA = 16 ± 1 U/mL)
was used to prepare the biocemented sand columns. For
EICP treatment, urease was extracted from bacterial cells
via ultrasonication with the ‘‘run-cool’’ method [21] fol-
lowed by centrifuging. The activity of the urease solution
was 40 U/ml. Equal mole of CaCl2 and urea with a con-
centration of 1.0 M was used as cementation solution
(hereafter abbreviated as CS).
2.2 Testing programs
2.2.1 Solution tests
As a preliminary study, tests in solution were carried out.
The test program is summarized in Table 1.
The MICP solution and EICP solution used for the
solution tests were prepared by mixing cementation solu-
tion, deionized water and bacterial (or urease) solution in a
certain volume ratio, respectively. Three different MICP or
EICP solutions with various volume ratios of bacteria or
urease were prepared for the solution tests, all of which had
a calcium concentration of 1.0 M. In this study, VR refers
to the ratio of bacteria or urease in the MICP or EICP
solution in terms of volume. For Tests SM/SE1–SM/SE3,

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Table 2 Scheme of sand column treatment test

Test Injection method Bacteria volume Urease volume Urease activity UA, Cementation solution Number of
ratio, VR ratio, VR U/mL concentration, M treatments

CM1 Modified one-phase- 0.75 – 16 1.0 5

CM2 low-pH 0.50 –
CM3 MICP 0.30 –
CM4 0.15 –
CM5 0.10 –
CM6 0.05 –
CE1 Modified one-phase- – 0.75 40 1.0 5
CE2 low-pH – 0.50
CE3 EICP – 0.30
CE4 – 0.15
CE5 – 0.10
CE6 – 0.05

the solution was kept unstirred throughout the test (72 h in
total). For Tests SM/SE4, a thoroughly stirred was con-
ducted after each measurement to disturb the precipitated
calcium carbonate, and the original solution would be
replaced by equal volume of 1.0 M cementation solution at
24 h intervals. During the test, the calcium concentration of
the solution was measured using the titration method (re-
ferring to Sect. 2.3).
2.2.2 Sand column tests
The test program for sand column is given in Table 2. All
the specimens in each test were triplicates to make sure the
repeatability of the test results. The mold made by poly-
vinyl chloride (PVC) tube was used to prepare the
cemented sand column with 50 mm in diameter and
100 mm in height. In terms of the modified one-phase-low-
pH method, it includes two steps: (1) using one-phase-low-
pH method to inject MICP or EICP solution for the first
treatment and (2) repeated injections of cementation solu-
tion without pH adjustment (or pH & 7) for the subsequent
treatments. Herein, in terms of one-phase-low-pH method
[10], the MICP or EICP solution, which is a mixture of
urease or bacteria (pH = 6.5) and cementation solution
(i.e., CaCl2 and urea), is injected into each sand column.
That is, for Tests CM/CE1–CM/CE6 (as given in Table 2),
different volume ratios of the bacteria or urease are
injected only once with repeated injections of 1.0 M
cementation solution at 24 h intervals for the subsequent
treatments.
2.3 Property evaluation tests
For all the tests, the concentration of calcium ions in the
solution obtained from the supernatant of the solution tests
and the effluent of the sand columns was measured using
the EDTA (ethylenediaminetetraacetic acid) titration
method [12]. After the completion of MICP or EICP
treatment, all the specimens were rinsed by the distilled
water, demolded and saturated for 24 h. Unconfined com-
pression (UC) tests were conducted using an axial loading
rate of 1.0 mm/min [6] to determine the unconfined com-
pressive strength (quc). When the UC test was completed,
each specimen was collected and dried in the oven to
measure its calcium carbonate content (CCC). The CCC of
the treated sand columns was also measured by dissolving
into acid [15] to decompose the precipitated calcium car-
bonate. Moreover, scanning electron microscopy (SEM)
analysis was carried out on the treated sand columns and
the precipitates of the solution tests.
3 Modified one-phase-low-pH method
for MICP and EICP treatment
One of the major limitations of the MICP or EICP method
is the need for multiple treatments. When the one-phase-
low-pH method is used for multiple treatments [14], the pH
has to be adjusted before every treatment. This is not
desirable. One modification to this method is to inject
bacterial or urease solution once in the first treatment and
then introduce cementation solution only for the subse-
quent treatments. To evaluate the effectiveness of the

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Fig. 1 Calcium conversion efficiency of MICP and EICP in solution tests: a unstirred cases with one-time treatment and b stirred cases with one
treatment of bacteria or urease and repeated treatments of cementation solution
proposed method and optimize the treatment process, tests
in both solution and sand column were carried out using
bacteria or urease with different volume ratios (VRs). As
pointed out before, VR is defined as the ratio of bacteria or
urease in the MICP or EICP solution in terms of volume
and used as a parameter to measure the effect of VR on the
MICP- or EICP-treated sand. The upper limit of the volume
ratio is controlled within 0.75 in this study as it means 75%
of the solutions is already bacterial or urease solution. The
tests in solution were to evaluate the calcium conversion
efficiency, and the tests on sand columns were to evaluate
the geotechnical properties of sand treated using the pro-
posed method.
3.1 Calcium conversion efficiency
To evaluate the amount of calcium consumed during either
a MICP or EICP process, the calcium conversion effi-
ciency, CCE, defined as follows, was adopted:
 
Cmc
CCE% ¼ 1   100% ð1Þ
CCS
where Cmc is the measured calcium concentration in the
effluent solution; CCS is total calcium supplied for each
treatment. MICP or EICP tests in solution were carried out
for this purpose as only in this type of tests the calcium
content during the experiments could be measured.
Figure 1 shows the variation of CCE of the solutions
versus time during the MICP and EICP tests with different
volume ratios for both stirred and unstirred cases, respec-
tively. The CCE increases gradually over time and finally
tends to be stable, as shown in Fig. 1a. As expected, the
larger the volume ratios (VR), the greater the initial CCE.
The CCE of MICP is greater than that of EICP under the
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same volume ratio, although the urease activity of the
bacterial solution (16 U) is lower than that of urease
solution (40 U). A possible explanation is that both bacteria
and urease could be encapsulated by the precipitated cal-
cium carbonate, preventing bacteria or urease from con-
tacting urea for hydrolysis. This encapsulation effect seems
to affect the EICP process much more than the MICP
because the size of urease (approximately 12 nm [8]) is
much smaller than that of bacteria (500–3000 nm [40]).
This conjecture is supported by the results of Tests SM4
and SE4 (as shown in Fig. 1b) in which the solutions were
both stirred during the tests, but the different variations of
CCE for both MICP and EICP treatment. Figure 1b also
shows that the CCE for both MICP and EICP processes
was affected, but the decrease in the CCE for the EICP test
during the second and third treatments is much more. In
summary, the CCE in the subsequent EICP treatments is
more affected than that in the subsequent MICP treatment
when using the modified one-phase-low-pH method.
Furthermore, the urease activity of the supernatant in
Tests SM/SE1–SM/SE3 at 72 h and in Tests SM/SE4 at
24 h was measured and found that the urease activity
values were negligible. It implies that there are almost no
free bacteria or urease in the supernatant after a certain
amount of calcium carbonate is precipitated within the
reaction period. In other words, as the bacterial cells or
urease enzyme were used as nucleation sites [20, 38],
almost all the bacteria or the urease in the solution would
have been adsorbed by the precipitated calcium carbonate.
Before the bacterial cells or urease were fully covered by
the calcium carbonate crystals, they would continue to
induce newly formed calcium carbonate, which can be
proved by the continuous increase in CCE of Tests SM4
and SE4 (Fig. 1b).
Acta Geotechnica
Fig. 2 SEM images of solution tests: a SM1, VR = 0.75; and b SE1, VR = 0.75
Fig. 3 Calcium conversion efficiency CCE of sand column treated by
the modified one-phase-low-pH MICP method with bacteria of
different volume ratios (VRs)
Figure 2 illustrates the SEM images of the calcium
carbonate crystals formed in the solution tests using MICP
and EICP. Overall, both the spherical and rhombic calcium
carbonate crystals can be observed in each case. However,
the sizes of the calcium carbonate crystals formed in the
MICP tests are greater than that of EICP, which agrees well
with the finding of Wen et al. [42]. This may be caused by
the different sizes of bacterial cells or urease enzyme.
3.2 Modified one-phase-low-pH MICP method
for sand
The modified one-phase-low-pH MICP method was used to
treat sand columns to study the effectiveness of the
method. The CCE obtained from tests using different
volume ratios is plotted versus the number of treatments in
Fig. 3. The CCE decreases gradually with the increase in
the number of treatments, N. This was due to the reduction
in the bacterial cells consumed in the previous treatment as
well as possibly encapsulation of the bacterial cells by the
precipitated calcium carbonate. This is the reason why the
higher the initial volume ratios of the bacterial solution in
the test, the greater the CCE obtained. Thus, for the
modified one-phase-low-pH MICP method, a high volume
ratio such as VR = 0.75 should be used within this study.
With the use of MICP treatment using bacterial solution
with VR = 0.75, about 80% of the CCE can be maintained
for up to 5 treatments. If a VR = 0.50 is used, a CCE of
about 70% is maintained for 4 treatments. Thus, VR = 0.50
may also be used if the total number of treatments required
is no more than 4.
Figure 4a shows the calcium carbonate content CCC of
sand columns treated using the modified one-phase-low-pH
MICP method after 5 treatments with bacteria of different
volume ratios. It can be seen that CCC decreases gradually
with the decrease in volume ratios in the MICP treatment.
It is worth mentioning that the CCC of the treated sand
using the modified one-phase-low-pH method is 9.3% for
the case that volume ratio is 0.50. If the one-phase-low-pH
MICP method is used to treat sand using volume ratio of
0.30 for each treatment (MICP-1, N = 4 [14]), the CCC of
the treated sand is 8.7% at 4 treatments (total volume ratio
of 1.2). This indicates that for the MICP treatment, the
modified one-phase-low-pH method can not only reduce
the amount of bacterial solution, but also produce similar
amount of calcium carbonate compared with the treatment
using the original one-phase-low-pH method.
Figure 4b shows the UC strength (quc) of the sand
treated by MICP using the modified one-phase-low-pH
injection method. Similar to the variation of the CCC
(Fig. 4a), the quc of all the treated sand columns decreases
with the decrease in the volume ratio of bacterial solution.
To further analyze the variation of UC strength, the quc
versus CCC for all the treated sand using the modified one-
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Fig. 4 Calcium carbonate content CCC and UC strength quc of MICP-treated sand: a calcium carbonate content CCC versus VR; b UC strength
quc versus VR; and c relationship between quc and CCC
Fig. 5 Calcium conversion efficiency CCE of sand column treated by
EICP under different volume ratios of urease enzyme
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phase-low-pH MICP treatment is plotted in Fig. 4c. For
comparison, the data of the MICP-treated sand after 1 to 4
number of treatments using the original one-phase-low-pH
method are also shown in Fig. 4c. Overall, quc increases
with CCC for all the treated sand samples using both
method (i.e., the original one-phase-low-pH and modified
one-phase-low-pH method). As mentioned before, the CCC
of the treated sand for the modified one-phase-low-pH
MICP method (Fig. 4a) using the volume ratio of 0.50
(Test CM2) is similar to that of the treated sand using the
original one-phase-low-pH method (Fig. 4c), the UC
strength is also similar, and they are 829.6 kPa (Test CM2)
and 903.5 kPa (MICP-1, N = 4 [14]), respectively. This
indicates that the modified one-phase-low-pH MICP
method is comparable to that of the original one-phase-
Acta Geotechnica
Fig. 6 Calcium carbonate content CCC and UC strength quc of EICP-treated sand columns: a calcium carbonate content CCC versus VR; b UC
strength quc versus VR; and c relationship between quc and CCC
low-pH method with the advantage of using less amount of
bacterial solution, but similar number of treatments.
3.3 Modified one-phase-low-pH EICP method
for sand
The calcium conversion efficiency (CCE) of the treated
sand for modified one-phase-low-pH EICP method is
shown in Fig. 5. Similar to the CCE of MICP-treated sand
(Fig. 3), the CCE of EICP-treated sand also decreases with
the decrease in volume ratio of urease solution. It should be
noted that this decrease in CCE for EICP treatment is faster
than that of MICP treatment after the second cementation
solution injection, regardless of the amount of the injected
urease, when the modified one-phase-low-pH method is
used. For example, the CCE of Test CE1 (VR = 0.75)
reduces to half the original value at the second treatment
(Fig. 5), while the CCE of Test CM1 (VR = 0.75) only
reduces by 10% (Fig. 3). The CCE for EICP treatment is
generally smaller than that for MICP under the same vol-
ume ratio, although the urease activity of the urease solu-
tion is greater than that of bacteria (Table 2). This indicates
that the modified one-phase-low-pH method is not suit-
able for the EICP treatment, due to the poor durability of
urease enzyme.
The CCC of the sand treated by the modified one-phase-
low-pH EICP method is shown in Fig. 6a. It can be seen
that the CCC also decreases with the decrease in volume
ratio of urease solution. However, the CCC of the sand
treated by EICP is smaller than that of MICP under the
same volume ratio (Fig. 4). The smaller CCC in EICP
treatment is mainly caused by the lower CCE of EICP
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(Fig. 5) during the duration of urease. For example, the
CCC of the sand treated by the modified one-phase-low-pH
EICP method is only 5.3% after 5 injections of cementation
solution (Fig. 6a), when the VR of urease solution is 0.75.
However, if the original one-phase-low-pH method is used
for EICP treatment with smaller volume ratio of urease
solution (i.e., VR = 0.30), the CCC can be up to 4.4% with
2 injections of EICP solution (EICP-1, N = 2 [14]). This
indicates that the conversion of calcium ions into calcium
carbonate is inefficient when using the modified one-phase-
low-pH method for EICP treatment. The lower CCC in the
modified one-phase-low-pH method for EICP treatment
indicates the necessity of repeated injections of urease
solution using the original one-phase-low-pH method for
EICP-treated soil.
The UC strength of the sand treated by the modified one-
phase-low-pH EICP method is shown in Fig. 6b. It can be
seen that the strength also decreases with the decrease in
volume ratio of urease solution. A rapid decrease in the
strength occurs when the volume ratio is smaller than 0.75.
That is, if the modified one-phase-low-pH EICP method is
used, the volume ratio of urease solution should be higher.
The relationship between UC strength and CCC of
EICP-treated sand under different volume ratios of urease
solution is shown in Fig. 6c. Meanwhile, the data of the
treated sand using the one-phase-low-pH EICP method
conducted by Cui et al. [14] are also presented in Fig. 6c
for comparison. It can be found that the UC strength of all
the treated sand increases with the increase in CCC.
Compared with the one-phase-low-pH EICP treatment
(VR = 0.30, N = 2 [14]), similar strength of the treated
sand can be obtained using the modified one-phase-low-pH
method for EICP treatment (VR = 0.75), but it needs three
more number of treatments and extra urease solution with
the volume ratio of 0.15. Therefore, the modified one-
phase-low-pH EICP method may not be beneficial if high
CCC or high UC strength is required.
Although the modified one-phase-low-pH EICP method
is inefficient in inducing calcium carbonate, the bioce-
mentation effect of EICP is more efficient in enhancing the
strength of sand. The quc of the sand treated by EICP and
MICP is similar when VR is 0.75 (Figs. 4b and 6b), while
the calcium carbonate content for EICP-treated sand is
significantly lower than that of MICP (Figs. 4a and 6a).
This can be further confirmed by the results that the quc of
EICP is higher compared with MICP at the similar CCC
(e.g., Tests CE3 and CM5, as shown in Figs. 4a and 6a,
respectively). The greater quc of sand treated by EICP than
that of MICP under the same CCC has also been reported
by Almajed et al. [5] and Hoang et al. [21].
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3.4 Microstructure and mechanism analysis
of MICP- and EICP-treated sand
3.4.1 Microstructure analysis
Figure 7 illustrates the SEM images of MICP- and EICP-
treated sand with different volume ratios of bacteria or
urease using the modified one-phase-low-pH method.
Overall, the volume ratio (VR) of bacteria or urease barely
influences the distribution of calcium carbonate crystals in
MICP- and EICP-treated sand. The calcium carbonate
crystals were adsorbed on the surface of sand particles.
Two morphological patterns of the precipitated calcium
carbonate crystals were observed, which also have been
reported in previous researches: (a) spherical
[16, 22, 27, 28]; (b) rhombic [1, 2, 9, 11, 15, 35]. The
spherical crystals are more likely to appear in the sand with
low CCC (e.g., Fig. 7e, f), but rhombic crystals at higher
CCC. The sizes of crystals are different between MICP and
EICP. For the EICP treatment, it generally produces
smaller size of calcium carbonate crystals in the treated
sand compared with the MICP treatment at the same VR
(Fig. 7a, b) or similar CCC (Fig. 7d, e). In this regard, more
contact points for cementation will be formed by the
smaller calcium carbonate crystals in the EICP-treated sand
compared with those in MICP under the similar CCC. As
biocementation is mainly through the calcium carbonate
crystals between the adjacent soil particles, more contact
points will lead to higher strength and stiffness of the
treated soil [23].
3.4.2 Mechanism analysis
The experimental results show that the modified one-
phase-low-pH method is effective for MICP, but not for
EICP. This is mainly caused by the difference in the
duration of urea hydrolysis. One of the factors affecting the
duration of urea hydrolysis is the sizes of bacterial cells or
urease enzyme. As bacterial cells are used as the nucleation
sites of calcium carbonate in a MICP [13, 18] process,
living bacterial cells can continue to hydrolyze urea until
the nutrient is diminished due to mineralization on the
surface of bacterial cells [39]. Similarly, the urease enzyme
can also be regarded as nucleation sites and incorporated in
the precipitates as confirmed by the finding of Sondi and
Matijević [37]. Thus, both bacterial cells and urease
enzyme can continue to induce calcium carbonate until
encapsulated. This is consistent with the viewpoint of Lai
et al. [24]. However, the sizes of bacterial cells (within the
range of 500–3000 nm [40]) are much larger than that of
urease enzyme (about 12 nm [8]). The relatively large size
and high surface-to-volume ratios of bacterial cells can
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Fig. 7 SEM images of sand treated by the modified one-phase-low-pH MICP and EICP method: a CM1, VR = 0.75, CCC = 10.2%; b CE1,
VR = 0.75, CCC = 5.3%; c CM4, VR = 0.15, CCC = 6.0%; d CE4, VR = 0.15, CCC = 2.6%; e CM6, VR = 0.05, CCC = 2.4%; and f CE6,
VR = 0.05, CCC = 1.0%

provide an enormous surficial biomineralization potential
[39] and require much more calcium carbonate crystals to
encapsulate the bacterial cells. In this regard, bacterial cells
would be more difficult to be encapsulated by the precip-
itated calcium carbonate compared with the urease
enzyme. In other words, the duration of urea hydrolysis for
bacterial cells should be much longer than that of urease
enzyme. This conjecture could be further confirmed by the
size of calcium carbonate crystals (Figs. 2 and 7) and
variation of calcium conversion efficiency, CCE (Figs. 3
and 5).
The SEM shown in Figs. 2 and 7 indicates that the sizes of
calcium carbonate crystals induced by the MICP process are
usually larger compared with those by the EICP process.

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However, the nanoscale calcium carbonate crystals can be
formed on the surface of bacterial cells, as reported by Ghosh
et al. [18]. That is, the microscale crystals observed in the SEM
images should be formed by the continuous accumulation of
newly precipitated nanoscale calcium carbonate crystals using
the bacterial cells or urease enzyme as the nucleation sites.
This is consistent with the viewpoint of Cui et al. [15] that the
precipitation of calcium carbonate crystals is a process of
formation–accumulation–growth. In other words, the sizes of
the calcium carbonate crystal clusters would increase gradu-
ally with the continuous accumulation of the newly formed
calcium carbonate crystal until the bacterial cells or urease
enzyme is encapsulated by the precipitated calcium carbonate.
Thus, the size of the calcium carbonate crystal clusters pre-
cipitated with the MICP or EICP process depends highly on
the sizes of bacterial cells or urease enzyme, which affects the
duration of urea hydrolysis process.
For the calcium conversion efficiency (CCE), the CCE
of the EICP process should be greater than that of MICP
theoretically. This is because the urease can hydrolyze urea
directly and induce calcium carbonate when urea binds to
the urease metallocenter. By contrast, the urea hydrolysis
in the MICP process is more complex. Urea needs to dif-
fuse into bacterial cells first and then binds to the intra-
cellular urease to be hydrolyzed. The produced ammonia
and carbonic acid are released into the solution, forming
calcium carbonate crystals on the surface of bacterial cells.
However, the test results shown in Fig. 1 suggest that the
measured CCE of the EICP process is significantly lower
than that of the MICP process one hour after the commence
of the process. One of the possible explanations is that most
of the urease enzyme is encapsulated by calcium carbonate
rapidly, resulting in a shorter duration of urea hydrolysis
for urease enzyme compared with that of the bacterial cells.
Another possible reason for the longer duration for the
modified one-phase-low-pH MICP method is because
bacterial cells can proliferate, but not urease enzyme.
4 Conclusions
A modified one-phase-low-pH MICP or EICP method is
proposed in this paper to simplify the injection process
used for construction. In this method, the bacterial or
urease solution is injected into soil only once using the one-
phase-low-pH method and the subsequent rounds of treat-
ments will be done by injecting cementation solution.
Based on the test results and discussion, the following
conclusions can be drawn:
1. The volume ratio of bacterial or urease in the MICP or
EICP solution has a significant effect on the calcium
conversion efficiency. The larger the volume ratio, the
123
Acta Geotechnica
greater the calcium conversion efficiency. When a
bacterial solution with a high volume ratio of 0.75 is
used, the modified one-phase-low-pH MICP method
can be as effective as the original one-phase-low-pH
MICP method for up to 5 number of treatments.
2. Compared with the original one-phase-low-pH EICP
method, the modified one-phase-low-pH EICP method
is not as efficient in the production of calcium
carbonate even when urease solution with a volume
ratio of 0.75 is used. The calcium conversion efficiency
decreases rapidly during the subsequent injections of
cementation solution, leading to less production of
calcium carbonate. Thus, more number of treatments is
required to obtain the same amount of calcium
carbonate or the same UC strength as compared with
the original method.
3. The durability of calcium carbonate production during
MICP is longer than that during EICP. This is mainly
caused by the difference in the sizes of bacteria and
urease. The large sizes and high surface-to-volume
ratios of bacterial cells require more calcium carbonate
to encapsulate the surface of bacterial cells fully.
However, the sizes of urease are much smaller and thus
can be encapsulated by calcium carbonate much
quicker, causing less active urease available for
production of calcium carbonate through EICP.
Acknowledgements The authors would like to thank the financial
supports provided through Grant No. MOE2015-T2-2-142 by the
Ministry of Education, Singapore, and the Grant No. SMI-2018-MA-
02 by the Singapore Maritime Institute which are gratefully
acknowledged.
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