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One-phase-low-pH enzyme induced carbonate precipitation (EICP) method

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Article  in  Acta Geotechnica · July 2020

DOI: 10.1007/s11440-020-01043-2

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RESEARCH PAPER

One-phase-low-pH enzyme induced carbonate precipitation (EICP)

method for soil improvement
Ming-Juan Cui1 • Han-Jiang Lai1 • Tung Hoang1 • Jian Chu1

Received: 25 March 2020 / Accepted: 18 July 2020

Ó Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Enzyme induced carbonate precipitation (EICP) is an emerging soil improvement method using free urease enzyme for
urea hydrolysis. This method has advantages over the commonly used microbially induced carbonate precipitation (MICP)
process as it does not involve issues related to bio-safety. However, in terms of efficiency of calcium carbonate production,
EICP is considered lower than that of MICP. In this paper, a high efficiency EICP method is proposed. The key of this new
method is to adopt a one-phase injection of low pH solution strategy. In this so-called one-phase-low-pH method, EICP
solution consisting of a mixture of urease solution of pH = 6.5, urea and calcium chloride is injected into soil. The test
results have shown that the one-phase-low-pH method can improve significantly the calcium conversion efficiency and the
uniformity of calcium carbonate distribution in the sand samples as compared with the conventional two-phase EICP
method. Furthermore, the unconfined compressive strength of sand treated using the one-phase-low-pH method is much
higher than that using the two-phase method and the one-phase-low-pH method is also simpler and more efficient as it
involves less number of injections.

Keywords Bacteria
Calcium conversion efficiency
Soil improvement
Strength
Urease

1 Introduction In the previous studies, there are mainly two methods

Biocementation is emerging to be a new soil improvement
method. This method relies mainly on the production of
calcium carbonate in soil as the cementing material to
cement the soil particles together to increase the shear
resistance [42] and fill in the pores to reduce the perme-
ability of soil. There are mainly two approaches for the
calcium carbonate production via urea hydrolysis: a)
microbially induced carbonate precipitation (MICP) with
urease-producing bacteria [1, 2, 22, 28, 36, 40, 46, 47]; and
b) enzyme induced carbonate precipitation (EICP) using
free urease enzyme [4, 15, 18, 19, 21, 31, 33, 37, 41, 45].
Compared with the MICP method, the EICP method is free
from issues related to bio-safety and oxygen availability
and can be used for soil with finer particles [21, 24, 45].
& Jian Chu
CJCHU@ntu.edu.sg
1
School of Civil and Environmental Engineering, Nanyang
Technological University, 10 Blk N1, 50 Nanyang Ave.,
Singapore 639798, Singapore
used for EICP treatment: (a) pre-mixing method
[3, 4, 35, 39, 49]; and (b) percolation method
[17, 20, 21, 32–34]. The pre-mixing method was conducted
by mixing urease powder or EICP solution (mixture of
urease, calcium and urea) with soil and then putting the
mixed soil into a sampling mould. This method is difficult
to be used for treating soil in situ [49]. The percolation
method was conducted by injecting urease solution and
cementation solution either one solution after another [21]
or both solutions simultaneously [3]. The former is called a
two-phase method and the later a one-phase method.
Hoang et al. [21] reported that the amount of precipitated
calcium carbonate of EICP-treated sand was only about
half that of MICP for the same number of treatments. The
calcium conversion efficiency of EICP treatment reported
in the literature was generally low. Almajed et al. [3]
reported that the calcium conversion efficiency of EICP
was about 70–95% after 7 days of curing using cementa-
tion solution containing 0.67 M calcium and 1.0 M urea.
Thus, more treatments are required for EICP than for MICP
to precipitate the same amount of calcium carbonate. The

123

EICP based soil improvement methods can be much
improved if the efficiency of the calcium carbonate pro-
duction in EICP can be increased.
In this paper, a one-phase-low-pH method was adopted
to improve the efficiency of calcium carbonate production
in the EICP process. To verify the effectiveness of the one-
phase-low-pH method hereafter, a comparative study
between sand columns treated using one-phase-low-pH and
two-phase methods was conducted. The data obtained from
this study were also compared with published data using
EICP-based method. The scanning electron microscopy
(SEM) images were examined to reveal the microscopic
difference between the EICP- and MICP-treated sand.
2 Materials and methods
2.1 Materials
In this study, Sporosarcina pasteurii (OD600 = 3.6 ± 0.3,
urease activity UA = 20 ± 1 U/mL) was used for MICP
treatment and urease (UA = 40 ± 1 U/mL) extracted from
bacteria via ultrasonication was used for EICP treatment.
750 Watt ultrasonic processor (VCX 750) with 20 kHz in
frequency was used in this study to extract urease from
bacteria through the ‘‘run-cool’’ cycle. During the extrac-
tion, the temperature of bacterial solution was kept lower
than 35 °C. The soil used was clean Ottawa 20–30 sand
(Gs = 2.65, D50 = 0.72 mm, Cu = 1.2, emax = 0.742 and
emin = 0.502). Equal mole of CaCl2 and urea were used as
cementation solution which will be termed as CS hereafter.
2.2 Sand specimen treatment
The test program is summarised in Table 1. Poly vinyl
chloride (PVC) tube was used as the mould to prepare sand
specimens of 50 mm in diameter and 100 mm in height. All
tests were triplicated for repeatability. The one-phase-low-
pH injection method [7] was adopted in this study for both
MICP and EICP treatment. Hydrochloric acid (HCl) with a
concentration of 2.0 M was used to adjust the pH of bacterial
or urease solution. The procedure for the application of the
Table 1 Sand column treatment test details
Test Injection method Bacteria volume ratio VRa Urease volume ratio VR
MICP-2b Two-phase 1.0
EICP-2 1.0
MICP-1 One-phase-low-pH 0.3
EICP-1 0.3
a
The bacteria or urease volume ratio VR is the ratio of the injected bacteria or urease to the pore volume of sand column
b
MICP-2 or EICP-2 refers two-phase MICP or EICP method and MICP-1 or EICP-1 one-phase-low-pH MICP or EICP method
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Acta Geotechnica
one-phase-low-pH MICP or EICP method is as follows: (1)
preparing MICP (or EICP) solution by mixing the bacterial
solution (pH = 6.5) [or urease solution (pH = 6.5)] with CS
(2.0 M) and distilled water using a volume ratio of bacterial
(or urease) solution: CS: distilled water = 0.3: 0.5: 0.2 to
achieve a CS final concentration of 1.0 M; (2) injecting
MICP (or EICP) solution into a sand column immediately
and incubating the sand at room temperature (25 ± 1 °C) for
24 h; and (3) repeating the above steps until achieving the
required number of treatments. For comparison, the two-
phase method was also used to treat sand columns using
either EICP or MICP by injecting one pore volume of bac-
terial (or urease) solution, followed by injecting the same
volume of CS of 1.0 M after 6 h of fixation time for bacteria
(or urease).
2.3 Properties tests
Titration method was adopted to measure the calcium
concentration of the effluent after each treatment using a
standard solution of EDTA (ethylenediaminetetraacetic
acid) [9]. After completing the EICP or MICP treatment,
all samples were flushed by distilled water at least 5 cycles
to remove the residual substances. The samples were then
saturated and used for unconfined compression (UC) tests
to measure the unconfined compressive strength under a
loading rate of 1.0 mm/min [5]. After the UC test, all
fractions of the tested sample were collected and dried in
an oven with a temperature of 105 °C. The acid dissolving
method [12, 44] was adopted to determine the Calcium
Carbonate Content (CCC) of each sample. Scanning elec-
tron microscopy (SEM) tests were also conducted.
3 Results and discussion
3.1 Comparison of one-phase-low-pH and two-
phase method
Figure 1 shows the results of EICP and MICP treatment for
sand using either one-phase-low-pH or two-phase methods.
It can be seen that the Calcium Conversion Efficiency
Calcium concentration, M Number of treatments, N
1.0 1, 2, 3, 4
1.0 1, 2, 3, 4
Acta Geotechnica

(CCE) (Fig. 1a), which is the ratio of calcium consumed to
total calcium supplied for each treatment, and Calcium
Carbonate Content (CCC) (Fig. 1b) of sand treated by
EICP and MICP using the one-phase-low-pH method were
higher than that using the two-phase method. However, the
amount of bacteria or urease for each injection using two-
phase method is greater than that using one-phase-low-pH
method. The low CCE and CCC for the two-phase method
are possibly due to the fact that most of the injected bac-
teria or urease would be flushed out during the CS
injection.
In general, there are three mechanisms for the bacteria
or urease to be retained in soil: (1) the adsorption capacity
of bacteria or urease; (2) the filtration capacity of soil; and
(3) the retention ability of other media such as the pre-
cipitated calcium carbonate. The adsorption capacity of
bacterial cells or urease enzymes depends on its own
characteristics. The bacterial cells are considered as nega-
tively charged [50] and thus will be attracted to the soil
particle surface. The filter capacity of soil relies on its
physical properties of the soil (e.g., grain sizes and particle
gradation) and the sizes of bacterial cells. Soil with smaller
particle sizes and good gradation has a better filter capac-
ity. The larger bacterial cells are more likely to be trapped
in soil. For the one-phase-low-pH method, all three
mechanisms will contribute to the retention of bacterial
cells or urease enzymes. For the two-phase method, the
bacterial cells or urease enzymes are retained through the
first two mechanisms during the injection of bacterial or
urease solution. However, during the injection for CS, the
seepage force causes the flushing out of some bacterial
cells or urease enzymes.
Moreover, for the tests using the two-phase method, the
CCE for the EICP treatment (Test EICP-2) is much lower
than that for the MICP treatment (Test MICP-2). However,
when the one-phase-low-pH method is used, the CCE are
high for both the EICP treatment (Test EICP-1) and MICP
treatment (Test MICP-1). The possible reason for the low

Fig. 1 Comparison of EICP and MICP treatments of sand using either the two-phase or the one-phase-low-pH method: a calcium conversion
efficiency (CCE) versus number of treatments; b calcium carbonate content (CCC) versus number of treatments; c calcium carbonate content
(CCC) in each test after 4 treatments; and d unconfined compressive strength versus number of treatments

123

Fig. 2 Comparison of Calcium carbonate content (CCC) in sand treated by EICP and MICP using the one-phase-low-pH method: a CCC versus
number of treatments; and b CCC distribution at different positions within a treated sand column
Fig. 3 Comparison of the relationship between unconfined compres-
sive strength and calcium carbonate content for EICP- and MICP-
treated sand columns using one-phase-low-pH method
CCE for the two-phase method using the EICP treatment is
that more (or higher proportion of) urease would be flushed
out of the sand column during the CS injection. This can be
explained by the following two reasons: (1) the attachment
of urease to sand grains is weaker than bacterial cells and
(2) the sizes of urease are smaller than the sizes of bacterial
cells and thus the retention for urease is lower.
The CCC distribution of EICP- and MICP-treated sand
using either one-phase-low-pH or two-phase methods is
shown in Fig. 1c. It can be seen that the CCC distribution of
EICP and MICP treatment using one-phase-low-pH method
is uniform, but non-uniform for the two-phase method. The
CCC at the top (i.e., the region close to injection point) of the
sand column is the lowest for both EICP and MICP treatment
using two-phase method. The different distribution patterns
of calcium carbonate for both methods indicate that the one-
phase-low-pH method contributes to improve the uniformity
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Acta Geotechnica
of calcium carbonate distribution. Thus, the unconfined
compressive strength of sand column treated by EICP and
MICP using the one-phase-low-pH method is higher than
that using the two-phase method (Fig. 1d).
In conclusion, the higher CCE and more uniform dis-
tribution of calcium carbonate obtained from tests using the
one-phase-low-pH method produce higher unconfined
compressive strength compared with the two-phase method
for both the MICP and EICP treatments. However, it has to
be pointed out that not all the one-phase methods will
work. This is because MICP (or EICP) will take place as
soon as the bacterial (or urease) solution is mixed with the
CS solution. This will cause clogging at the injection side
and prevent the transmission of the CS solution in the soil,
resulting in non-uniform of calcium carbonate distribution
[19, 26, 27, 48]. However, this difficulty can be overcome
by using low pH (6.5) bacterial or urease solutions as
suggested by Cheng et al. [7] or using low activity bacterial
solution as demonstrated by Chu and Wen [11].
3.2 Comparison of EICP and MICP treatment
using the one-phase-low-pH method
Figure 2a shows a comparison of the CCC obtained after
each of EICP and MICP treatment of sand columns
using the one-phase-low-pH method in different number
of treatments. The CCC obtained in both treatments are
almost identical. It can be seen that the CCC increases
with the increase in the number of treatments for both
the EICP- and MICP-treated sand. The calcium in the
effluent of each EICP and MICP treatment were ignor-
able, meaning that the injected calcium solution for each
treatment could be converted to calcium carbonate
almost completely after 24 h incubation via the one-
Acta Geotechnica

Fig. 4 Comparison of SEM images of sand treated by EICP and MICP using the one-phase-low-pH method: a EICP-1 at CCC= 2.4% after one
treatment; b MICP-1 at CCC= 2.3% after one treatment; c EICP-1 at CCC= 4.4% after two treatments; d MICP-1 at CCC= 4.3% after two
treatments; e EICP-1 at = 8.4% after four treatments; f MICP-1 = 8.7% after four treatments

phase-low-pH method. Moreover, the distribution of
CCC in EICP- and MICP-treated sand columns is similar
and uniform (Fig. 2b).
The unconfined compressive strength (quc) of EICP- and
MICP-treated sand columns is plotted versus calcium car-
bonate content (CCC) in Fig. 3. As expected, quc increases
with increasing in CCC for both. However, quc obtained
from the EICP treatment is greater than that from the MICP
treatment for the same CCC. Similar observations have
also been reported by Almajed et al. [4] and Hoang et al.
[21].
One explanation for the above difference in the quc
versus CCC relationships was given by Hoang et al. [21] as
the difference in the deposition location of the precipitated
calcium carbonate. However, this deposition difference is
not significant in the current study. SEM images of both the
EICP- and MICP-treated sand using the one-phase-low-pH
method at different CCC are compared in Fig. 4. It can be
seen that the calcium carbonate crystals were formed on
sand particles in both cases. Two major differences have
been observed from the SEM images. Firstly, the mor-
phology of the calcium carbonate crystals formed in the

123

Fig. 5 Comparison of one-phase-low-pH EICP with published EICP treatments for sand: a relationship between calcium carbonate content
(CCC) and number of treatments N; and b relationship between unconfined compressive strength quc and CCC; c relationship between
unconfined compressive strength quc and number of treatments N
two different treatments is different. For EICP treatment,
mainly rhombic calcium carbonate crystals are observed
(Fig. 4a, c, e), whereas for MICP treatment, both rhombic
and spherical calcium carbonate crystals dominate
(Fig. 4b, d, or f). Both types of calcium carbonate crystals
have been observed by other researchers
[1, 8, 14, 23, 29, 38]. By examining the crystals in Fig. 4a
more closely, it can be seen that each rhombic crystal
consists of a number of smaller cubic or prismatic shaped
calcium carbonate crystals, suggesting the rhombic crystals
were more closely related to calcite as also pointed by
Declet et al. [13]. Gebauer et al. [16] have reported that
calcite is a more stable polymorph of calcium carbonate
and likely possesses a higher binding strength in the clus-
ters than other types. Khodadadi et al. [25] also reported
that the less stable calcium carbonate mineral phases may
not be as effective at improving the mechanical properties
of the soil. Furthermore, the surface of the spherical crys-
tals is smooth and thus its contribution to interlocking
might not be as high as the rhombic type of crystals.
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Acta Geotechnica
Secondly, the sizes of calcium carbonate crystals are also
different. The sizes of the calcium carbonate crystals
induced by EICP are smaller than that by MICP at the
similar CCC, as shown by a comparison of Fig. 4c with
Fig. 4d, which is consistent with the finding of Nafisi et al.
[30]. The difference in the sizes of the crystals is related to
the sizes of bacterial cells or urease enzymes and the
crystallisation rate of calcium carbonate. As the sizes of
bacterial cell (within the range of 500–3000 nm [43]) are
much bigger than those of urease enzyme (about 12 nm
[6]), the calcium carbonate crystals formed with bacterial
cells as nuclei [10] in MICP are much bigger than those
with urease enzyme as nuclei in EICP. In terms of crys-
tallisation rate, as the urea hydrolysis rate in EICP is
quicker, but shorter than that in MICP, the crystallisation
rate of calcium carbonate by EICP treatment would be
faster and the duration would be shorter than that of MICP.
Consequently, the calcium carbonate crystals formed dur-
ing each EICP treatment (Fig. 4c) would be smaller. If the
same amount of calcium carbonate content (CCC) is
Acta Geotechnica

Table 2 Summary of the published EICP methods

Urease Treatment method Treatment solution Treatment Samples References
intervals for UC test

Jack bean Percolation method. Add EICP solution from top of sand EICP solution: 0.67 M 7 days Oven-dried Almajed
column CaCl2, 1.0 M urea, 3 g/ at 50 °C et al. [3]
Premixing method. Mix sand with EICP solution L enzyme 3 days Oven-dried Almajed
at 40 °C et al. [4]
Self- Circulated-percolation process including 4 steps: (1) Urease: 25.4 mM urea/ 24–27 h Oven-dried Hoang
extracted circulated percolation of urease (3 h); (2) drain off the min; CS: 0.3 M CaCl2, at 50 °C et al.
from pore volume urease; (3) circulated percolation of CS 0.3 M urea for 48 h [20, 21]
bacteria (9–12 h); (4) flush with deionized water (2 h), then drain
off all liquid (10 h)
Jack bean Inject EICP solution through the injection tube pre- EICP solution: 1.0 M 24 h Wet Neupane
embedded in sand CaCl2, 1.0 M urea, et al.
15 g/L urease [33]
Self- Pre-mixing method including 4 steps: (1) mixing jack bean – 3 days Wet Park et al.
extracted extract with urea (3 days); (2) adding calcium to yield [37]
from jack calcite solution: (3) mixing sand with calcite solution and
bean compacting into mould; (4) air curing
Jack bean Pre-mixing method including 3 steps: (1) pluviating sand CS: (1) 0.5 M CaCl2, 24 h Dry Yasuhara
into mould after well-mixing with urease powder; (2) 0.5 M urea; (2) 1.0 M et al.
evacuating and applying 50 kPa confining pressure on CaCl2, 1.0 M urea [49]
sand sample; (3) injecting CS solution

achieved in each EICP and MICP treatment, then the
number of calcium carbonate crystals will be more. In
other words, there will be more contact points for cemen-
tation in EICP than those in MICP for the same CCC. As
biocementation of calcium carbonate between the adjacent
soil particles contributes to enhance the strength and
stiffness properties of soil [22], the more cementation
points of calcium carbonate will produce higher strength
for EICP. In summary, the difference in strength
enhancement of sand treated by EICP and MICP could also
be explained by the differences in the properties of the
calcium carbonate crystals and the number and sizes of the
crystals.
3.3 Comparison of one-phase-low-pH EICP
with published EICP treatment methods
The calcium carbonate content (CCC) and unconfined
compressive strength (quc) versus number of treatments
obtained from the one-phase-low-pH EICP method in this
study is compared with those from the published EICP
methods [3, 4, 20, 21, 33, 37, 49], as shown in Fig. 5.
Information about the published EICP methods is sum-
marised in Table 2. It can be seen from Fig. 5a that the
calcium carbonate production in this study is higher com-
pared with three others under the same number of treatments
[3, 4, 20, 21, 49]. Figure 5b shows that the quc obtained from
this study is similar to the results of Almajed et al. [3, 4] and
Hoang et al. [20, 21], but higher than the cases in the
published studies [33, 37, 49] under the same CCC. As the
number of treatments required is less in the one-phase-low-
pH EICP method to achieve the same CCC, it implies that for
the same number of treatments, the quc will be higher. This is
indeed the case as shown in Fig. 5c. However, it needs to be
pointed out that quc is also affected by other factors such as
concentration of bacterial or cementation solutions used and
thus the data in Fig. 5b and c are scattered.
4 Conclusions
In this paper, a one-phase-low-pH EICP method was pro-
posed to improve the efficiency of calcium carbonate
production for EICP. In this method, the EICP-solution
consisting of a mixture of low pH urease solution (pH =
6.5) and cementation solution (calcium chloride and urea)
is injected together into soil. The following conclusions can
be drawn from this study:
1. A higher calcium conversion efficiency and more
uniform distribution of calcium carbonate in the soil
specimens treated was achieved using the one-phase-
low-pH EICP method compared with that using a two-
phase method, and thus producing higher unconfined
compressive strength.
2. When the one-phase-low-pH method is adopted, the
efficiency of calcium carbonate production for EICP
and MICP is almost the same. The injected calcium of

123

each treatment could be converted almost completely
to calcium carbonate after 24 h incubation. The
unconfined compressive strength of the sand column
with EICP treatment is greater than that of MICP
treatment for the same calcium carbonate content. This
can be explained by the differences in the types of
calcium carbonate crystals and the sizes of the crystals.
3. Compared with the published EICP treatment methods,
the one-phase-low-pH method proposed in this study is
more effective as it results in a higher shear strength
and simpler as the number of injections is reduced by
half given the other conditions the same.
Acknowledgements The authors would like to thank Dr Xiaoniu YU
of Nanyang Technological University, Singapore, for the discussion
with him. 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 are
gratefully acknowledged. The first two authors would also like to
thank the support by the National Natural Science Foundation of
China (NSFC) (No. 51708243) and the China Postdoctoral Science
Foundation (Nos. 2016M600595, 2018M632862 and 2018T110769)
for the early stage of their research.
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