Engineering Properties of Biocementation

Coarse- and Fine-Grained Sand Catalyzed

By Bacterial Cells and Bacterial Enzyme
Tung Hoang, M.ASCE 1; James Alleman, M.ASCE 2; Bora Cetin, M.ASCE 3; and Sun-Gyu Choi 4
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Abstract: Biological induced calcite precipitation is a potential method being investigated for improved soil stabilization. In terms of the
associated urea hydrolysis concept, three main strategies have been developed over the last 2 decades: (1) using live urease-producing bac-
teria, (2) using plant-extracted urease, and (3) using bacterial-extracted urease. This paper focused on evaluating the comparative benefits
of two of these methods (i.e., live bacterial cell or extracted bacterial urease methods for induced calcium precipitation) in terms of their
biocementation performance. Cell-based induced carbonate precipitation (ICP) (i.e., MICP) testing was completed on standard Ottawa
coarse-grained sand (#20/30), and bacterial-enzyme-based (i.e., BEICP) testing was conducted individually on both coarse-grained and
fine-grained (#50/70) sands. Distinctly higher unconfined compressive strength (UCS) was achieved with the BEICP method when evaluated
at similar levels of calcium precipitation. Residual permeability levels remained markedly higher after BEICP testing versus MICP. The UCS
of BEICP coarse-grained treated sand was approximately 450–1,500 kPa, whereas that of fine-grained treated sand had a notably lower range
(i.e., 250–900 kPa) when evaluated at similar levels of CaCO3 production. These results indicate that calcium carbonate content is not the sole
factor which impacts the strength of biocemented sand. Additional test-tube investigation of ICP-derived CaCO3 precipitation was used to
evaluate the chemical conversion efficiency for each method, i.e., live cells (i.e., Sporosarcina pasteurii) or bacterial-extracted urease. The
calcite precipitation ratio declined at higher substrate chemical concentrations. However, this ratio increased with higher rates of enzymatic
activity. DOI: 10.1061/(ASCE)MT.1943-5533.0003083. © 2020 American Society of Civil Engineers.

Introduction (enzyme-induced CaCO3 precipitation). These MICP and EICP

Ground stabilization is a fast-growing discipline in geotechnical
engineering. It includes more than 30 techniques classified within
several categories, including replacement; densification through
dynamic energy; consolidation by dewatering; chemical grouting;
admixture stabilization using cement, lime, or fly ash; thermal
stabilization; and so forth (Mitchell 1981; Terashi and Juran 2000;
Chu et al. 2009). Within the last decade, a bio-based environmental
grouting method has been investigated as an additional ground
improvement technique via reduced permeability and enhanced
strength and stiffness of cohesionless soil based on both (1) pore-
filling (bio-clogging); and (2) particle roughening plus interparticle
bridging (biocementation) (Ivanov and Chu 2008). The majority of
these bio-grouting methods rely on a biochemical reaction whose
pH-elevating chemical products then induce calcium carbonate
(CaCO3 ) precipitation. Hamed Khodadadi et al. (2017) identified
these strategies for microbial-induced CaCO3 precipitation (MICP)
as either MICP (using intact ureolytic microbial cells) or EICP
1
Lecturer, Univ. of Danang–Univ. of Science and Technology, Danang
550000, Vietnam. Email: hptung@dut.udn.vn
2
Professor, Dept. of Civil and Environmental Engineering, Univ. of
Notre Dame, Notre Dame, IN 46556. Email: jalleman@nd.edu
3
Assistant Professor, Dept. of Civil and Environmental Engineering,
East Lansing, MI 48824 (corresponding author). Email: cetinbor@msu.edu
4
Senior Researcher, Disaster Prevention Research Division, National
Disaster Management Research Institute, Ulsan 44538, Republic of Korea.
Email: choisg@kaist.ac.kr
Note. This manuscript was submitted on October 28, 2018; approved on
August 22, 2019; published online on January 22, 2020. Discussion period
open until June 22, 2020; separate discussions must be submitted for in-
dividual papers. This paper is part of the Journal of Materials in Civil
Engineering, © ASCE, ISSN 0899-1561.
processes both use a similar mechanism, i.e., urea hydrolysis.
MICP employs live bacterial cells to achieve ureolysis. EICP treat-
ment relies on free urease enzyme which was previously extracted
from jack beans provided by commercial sources (Nafisi et al.
2019; Oliveira and Neves 2019).
MICP-based research has demonstrated multiple soil-
stabilization benefits (i.e., higher strength and stiffness, liquefaction
resistance, and reduced permeability) of sandy soil. These outcomes
have been established at varied experimental scales, ranging from
laboratory column tests (Cheng et al. 2013; Choi et al. 2019a, b;
DeJong et al. 2006) to several higher-level experiments [100 m3
sand (van Paassen et al. 2010a), and 1,000 m3 gravel (van der Star
et al. 2011)], and even field-scale evaluation (Burbank et al. 2011;
Gomez et al. 2015; van Paassen 2011; Saneiyan et al. 2019).
Although the MICP method has been successful within these vari-
ous scales of ground improvement testing, this method still has
several limitations because of nonuniform outcomes. Notably, prob-
lems have been experienced with both nonhomogenous distribu-
tion of bacterial cells and cementation results (Sun et al. 2018;
Whiffin 2004; Whiffin et al. 2007), as well as constraints on the
pore-space transport of microorganism cells, in which they are re-
strained by the presence of fine-grained soil particles (Rebata-Landa
and Santamarina 2006). By comparison, the newer ureolysis meth-
ods using nanoscale and water-soluble urease enzyme biocatalysts
(i.e., enzymes extracted from either plant or live cell sources) for
in situ calcium precipitation may offer beneficial processing bene-
fits. Several researchers have published results of plant-derived
free-enzyme biostabilization studies (e.g., Yasuhara et al. 2012;
Hamdan et al. 2013). However, these researchers commented on
the high cost of purchasing commercially purified plant-derived
urease enzyme for their lab-scale experiments (Almajed et al. 2018;
Hamdan and Kavazanjian 2016; Kavazanjian and Hamdan 2015;

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 Larsen et al. 2008; Nafisi et al. 2019; Nam et al. 2015; Nemati and
Voordouw 2003; Neupane et al. 2013, 2015a; Pasillas et al. 2018;
Phua and Røyne 2018; Simatupang and Okamura 2017). Currently,
only three research groups have reported on the innovative use
of self-extracted urease obtained from agricultural sources. These
studies used jack bean (Park et al. 2014) and watermelon seeds
(Dilrukshi et al. 2016, 2018; Javadi et al. 2018). Unfortunately, this
approach to extracting urease enzyme from plant sources appears
to have a number of drawbacks, including high time- and land-
demand for plant growth, plus additional high processing costs for
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converting plant biomass into soluble extracted enzyme. To date,

very few plant-based EICP studies have compared biostabilization
efficiency relative to that of conventional MICP processing. Sim-
ilarly, only a few of these plant-based EICP studies have addressed
the effect of sand type and size (Hamdan et al. 2013; Hamdan and
Kavazanjian 2016; Kavazanjian et al. 2017; Neupane et al. 2015b;
Nafisi et al. 2019; Pasillas et al. 2018; Simatupang and Okamura
2017).
Hoang et al. (2018) documented a new biogeotechnical ground
improvement method using bacterial enzyme calcium carbonate
precipitation (BEICP). The present paper further demonstrates the
performance of BEICP versus standard MICP processing method in
terms of chemical conversion efficiency and coarse-sand stabiliza-
tion. This paper also provides further insight regarding the impact of
varying sand types (coarse- and fine-grained) in relation to this new
BEICP strategy. The results showed similar gains in chemical con-
version efficiency when using either whole-cell (MICP) or enzyme-
only (BEICP) processing. However, distinct differences were found
when using these alternative methods with varying sand sizes.
Materials and Experimental Procedures
Sand Materials and Sand Column Preparation
Two types of silica (SiO2 )/quartz sand were used, coarse-grained
sand #20/30 and fine-grained sand #50/70. These sand criteria are
described in ASTM C778 (ASTM 2014). These sands contained
more than 98.7% silica. The sand materials initially were washed
with deionized water to remove any soluble chemicals, followed by
oven drying at 105°C for 24 h before being tested. Further evalu-
ation of the sand properties included particle diameter at 10% finer
by mass (D10 ), particle diameter at 50% finer by mass (D50 ), uni-
formity coefficient (Cu ), coefficient of gradation (Cc ), specific
gravity (Gs ), maximum and a minimum void ratio (emax and emin ).
The sand was poorly graded sand (SP) according to the Unified Soil
Classification System (USCS) (ASTM 2010). These properties are
collectively summarized in Table 1. The grain-size distribution
curves of the coarse and fine sands are presented in Fig. 1.
Sand columns were packed separately to include either coarse-
grained or fine-grained sand soil. Prior to loading sand into each test
column, the dry sand was mixed with distilled water to achieve 5%
moisture (Choi et al. 2019b; Hoang et al. 2018). The following se-
quential preparation steps were followed. First, an underlying gravel
stratum with a nominal maximum size of 9.5 mm was layered into
the bottom of the test chamber’s plastic cap. Second, a precut section
Fig. 1. Grain-size distribution curve of tested sands.
of filter pad material (i.e., 3M Scotch Brite Model 7447, Livonia,
Michigan) was placed on top of the gravel layer. Third, a moist-
tamping method was used to pack the specimens, in which the pre-
moistened sand was tamped gently within the PVC columns (50 mm
diameter and 100 mm height). To achieve a similar specific density in
each layer, predetermined amounts of soil were added in 10 succes-
sive layers of equal thickness (10 mm=layer) within each column.
These column compaction steps were carefully conducted to achieve
similar relative density (Dr ) levels within all column samples. The
characteristics of sample columns for coarse and fine sands including
Dr , porosity (n), and untreated permeability (k) are listed in Table 1.
Finally, another filter pad was located on the top of the sand layer to
avoid scouring and distributing flow streams. Fig. 2 provides a sche-
matic of a filled column in operation, including its solution pump.
Urease-Producing Bacteria Suspension and
Chemical Solution
The urease active strain of Sporosarcina pasteurii (ATCC-11859,
American Type Culture Collection, Manassas, Virginia) was pro-
duced as described by Hoang et al. (2018). The isolated strain was
cultivated in a presterilized culturing broth (i.e., an ammonium–
trypticase soy broth (NH4-TSB) growth media which included
trypticase soy broth (20 g=L), ammonium sulfate (10 g=L), and
Tris buffer (0.13 mol=L); the overall solution pH was 9.0. A shak-
ing incubator (Innova Model 4000, New Brunswick Scientific, En-
field, Connecticut) was used to incubate this culture for 48 h with a
continuous shaking speed of 160 rpm at 30°C.
This cultured biomass optical density (OD) at 600 nm was mea-
sured using a visible light spectrophotometer (Hach Model #DR
3900, Loveland, Colorado). When the urease-producing bacteria
(UPB) solution OD was in the range 0.9–1.3, the stock microbial
culture was harvested and stored at 4°C. An electric conductivity
meter was used to measure the urease activity of the UPB solution
(Chu et al. 2012), which ranged from 8 to 13 mM urea=min, similar
to that reported by Hoang et al. (2018). The concentrations of

Table 1. Index properties of sands used in the current study

Properties Column packing
Sand
type D10 (mm) D50 (mm) Cu Cc Gs emax emin USCS Method Dr (%) n (%) k (cm=s)
Coarse 0.61 0.72 1.21 1.02 2.65 0.74 0.51 SP Moist tamping 64.6  0.5 ∼37 1.01 × 10−1
Fine 0.26 0.36 1.46 0.97 2.65 0.87 0.55 SP Moist tamping 64.4  0.5 ∼40 3.2 × 10−2

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Fig. 2. Soil column circulating-percolation treatment. (Reprinted from
Choi et al. 2016b, © ASCE.)
chemical solution used in the current study were 0.3 M, the same
molar concentrations as the soluble CaCl2 and urea.
Biomass Sonication and Enzyme Extraction
Urease enzyme lysed directly from live bacteria of Sporosarcina
pasteurii was used in the present study. The procedures for extrac-
tion and optimization were described by Hoang et al. (2018). Urease
extraction was completed using a repetitive series of cyclic run–cool
(i.e., 10 min on, followed by 10 min off) sonication steps. Six cycles
were applied over a 2-h period, with a 150-mL aliquot of the origi-
nal stock microbial culture placed directly into a sonication bath
(Bransonic Model 220, Sterling Heights, Michigan; 120 V, 125 W,
and 50=60 kHz). The cyclic processing mode was adopted to pro-
vide the lowest temperature buildup (i.e., typically at slightly below
32°C–34°C) and highest residual enzyme activity. After completing
these tests, the remaining lysed solution was subjected to a relative
centrifugal force (RCF) of 5,500 for 20 min to separate out residual
cellular debris solids from the extracted soluble urease. The sonica-
tion method typically produced an approximate urease activity rate
of 25.4 mM urea=min.
Test-Tube Experiments
Two different modes of test-tube experiments were used to examine
and compare calcium consumption efficiency impacted by either
bacterial cells (MICP) or extracted bacterial urease (BEICP). These
tests were performed within transparent 50-mL polypropylene
tubes. Neupane et al. (2013), Yasuhara et al. (2012), and Carmona
et al. (2018) performed similar test-tube experiments to evaluate the
calcium carbonate precipitation ratio at different concentrations of
commercial urease. A 15-mL equimolar solution of urea and CaCl2
was mixed with a 15-mL volume of either live bacteria cells or
extracted bacterial urease. These bioreactive mixtures were evalu-
ated at various urease activity levels. The experimental conditions
are listed in Table 2. To ensure the repeatability of these reactions,
© ASCE 04020030-3
J. Mater. Civ. Eng., 2020, 32(4): 04020030
Table 2. Test-tube experimental conditions
Number of test-tube
sample repetitious
Test substrate
(urea and CaCl2 )
concentrations
Urease
(mole)
Cells/ activitya
enzyme Test ID (mM urea/min) 0.15 0.25 0.5
Cells Cell UA-2 2 3 3 3
Cell UA-5 5 3 3 3
Cell UA-10 10 3 3 3
Enzyme Enzyme UA-2 2 3 3 3
Enzyme UA-5 5 3 3 3
Enzyme UA-10 10 3 3 3
a
Activity rates were derived.
three replicates were tested. The activity rates for these tests were
derived (with ∼2%–3% accuracy) by proportionate dilutions from
an initial free enzyme solution with a known (and higher) enzyme
activity. These test tubes were shaken continuously for 24 h on a
shake table at 25°C  1°C to ensure complete mixing. At the com-
pletion of this shaking period, the product solutions were filtered
through filter paper. These tubes, as well as the filter paper samples,
were oven dried at 100°C  5°C for 24 h. The weight of each cal-
cium carbonate precipitation product (i.e., on the filter paper and
attached to the test tube wall) was quantified on the basis of pretest-
ing and posttesting weights. The total produced CaCO3 contents
were calculated by adding the CaCO3 precipitated onto the test tube
surface and that remaining on the filter paper. The calcium con-
sumed efficiency was computed as the ratio of actual measured
CaCO3 mass to the theoretical CaCO3 mass determined accord-
ing to the input chemical concentration (Neupane et al. 2013;
Al Qabany et al. 2012).
Testing Program and Biotreatment Procedures
Each specimen was treated with either MICP or BEICP processing
to compare the impact of these treatment methods on properties of
coarse- and fine-grained biocemented sand. Sand columns were
treated with either 4, 8, 12, or 16 cycles because Choi et al. (2016a)
and Choi et al. (2019c) used similar levels of ICP cycling. The ex-
perimental program is given in Table 3, with three replicate samples
at each testing condition in order to verify reproducibility. Two
modes of soil stabilization were evaluated using the sand columns:
(1) BEICP, and (2) MICP. A circulated-percolation down-flow pro-
cess was applied to treat these sand columns under partially satu-
rated conditions. The sandy-soil columns were processed using the
following sequential procedure (Fig. 2). First, the catalytic biologi-
cal solution (extracted urease for the BEICP method, and bacterial
cells for the MICP method) was pumped and recycled into the top
of a sand column and gravity-drained from the bottom. A peristaltic
pump (Masterflex Model 77202-50) with silicone tubing (Master-
flex Model 96410-16) was used to recirculate this biological liquid
for 3 h at a rate of approximately 5 mL=min to achieve a 60% sat-
uration level, consistent with prior research by Cheng et al. (2013),
which allowed the bacterial cells or extracted enzyme to sorb onto
or be trapped on the sand particle surfaces. Second, after the 3-h
introduction of the catalytic agents (i.e., either MICP culture or
BEICP enzymes), the pore-volume biological liquid was drained
off the soil column. Third, a mixed chemical solution of urea and
calcium chloride (0.3 M at 1∶1 ratio) was introduced and circu-
lated for 9–12 h. Fourth, the sand column was flushed with cyclic
J. Mater. Civ. Eng.
 Table 3. Experiment details Results and Discussion
Number of specimens
Test ICP Treatment Sand UCS Permeability CaCO3 SEM/ Whole-Cell versus Enzyme-Only Chemical Conversion
group option cycles type test test test EDS Efficiency
1 MICP 4 Coarse 3 3 3 — The chemical conversion ratios of various combinations of en-
2 8 3 3 3 1 zymatic solutions (i.e., either live cells or extracted enzyme) and
3 12 3 3 3 1 substrate reagents were evaluated using a series of test-tube experi-
4 16 3 3 3 1 ments. Fig. 3 shows the efficiencies of chemical conversion in re-
5 BEICP 4 Coarse 3 3 3 — lation to the concentration of CaCl2 for both biological sources.
6 8 3 3 3 1 In general, the chemical conversion decreased as the substrate con-
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7 12 3 3 3 1 centration levels increased. The lowest level of precipitation effi-

8 16 3 3 3 1
9 BEICP 4 Fine 3 3 3 —
10 8 3 3 3 1 MICP and of Neupane et al. (2013) and Putra et al. (2017) for EICP.
11 12 3 3 3 1
deionized water pumping for 2 h to remove soluble byproducts and
then the bottom cap was removed to drain off all liquid for approx-
imately 10 h. After completing this treatment cycle (introducing
urease or bacterial cells, followed by urea/calcium chloride solu-
tion addition), fresh biological solution and chemical were intro-
duced and recirculated through the sand column in each successive
cycle. This stepwise approach to introducing enzyme solution (or
bacterial cells) and urea/calcium chloride solution was repeated at
1 cycle=day for 4, 8, 12, or 16 days of total treatment phases.
Testing of Properties of ICP-Cemented Sand Columns
After achieving the desired cycles of treatment, each column’s
bottom plastic cap and its internal filters were removed. The engi-
neering properties of the biotreated columns then were evaluated
with the following laboratory experiments: permeability, unconfined
compression strength (UCS), calcium carbonate content, and micro-
structure imaging. The latter testing was completed using scanning
electron microscopy (SEM) and energy-dispersive X-ray spectros-
copy (EDS).
The permeability test was conducted on untreated and treated
sand columns. The untreated sandy soil was packed in a permeabil-
ity testing device at a similar relative density as the packed sand
columns (Table 1). The untreated sands were saturated and the per-
meability was tested following the procedure of a constant head test
(ASTM 2006). The permeability of biotreated specimens was
tested on the samples which were still held within the PVC test
columns. The biocemented columns were saturated by applying
15 kPa back-pressure (Cheng et al. 2013). After the initial satura-
tion step, permeability tests were run until steady permeability val-
ues (k) were reached. Tests were stopped when the k values of the
specimen did not vary by more than 20% (data not shown here for
brevity). This state is reached only when specimens are fully satu-
rated (Hoang et al. 2018).
After measuring the permeability, the columnar PVC molds
were cut to separate the biocemented samples, which were oven
dried for 48 h at a moderate temperature (i.e., ∼50°C). The UCS
test was conducted in accordance with ASTM D2324. The elastic
modulus (E50 ) of the biocementation samples was determined by
the slope of the stress–strain curve at 50% of peak stress (Oliveira
and Neves 2019; van Paassen et al. 2010a). After the samples were
broken during UCS testing, approximately 5 g of biocemented sand
was withdrawn from the middle of the sand column for calcium
carbonate content measurement using an acid-rinsed method (Choi
et al. 2017b; Feng and Montoya 2015).
ciency was at 0.5 mol=L of reagent solution.
This result agrees with those of Al Qabany et al. (2012) for
The results by all these researchers suggested that high substrate
(i.e., urea and CaCl2 ) concentrations appeared to restrain the urease
activity of whole bacterial cells or free enzyme. A related hy-
pothesis was offered in the related literature regarding a possibly
negative impact caused by thicker calcium precipitation matrixes
which seemingly retarded ureolytic hydrolysis (Al Qabany et al.
2012; Al Qabany and Soga 2013). Increased urease activity levels
resulted in a higher efficiency of chemical conversion with both
MICP and BEICP (Fig. 3). This finding again is comparable to
that reported in the literature (Neupane et al. 2013; Putra et al.
2017). The present paper offers two different relative perspectives
on the impact of substrate concentration. At lower substrate levels
(i.e., 0.15 mol=L), chemical conversion efficiency increased at
higher urease activity levels. The efficiency decreased as substrate
levels increased. Although the range of the chemical efficiencies at
higher substrates levels was lower, they still retained a sequential
correlation with enzymatic activity. Another significant aspect
of these test-tube studies was the observed correlation between
enzyme form (i.e., whole-cell versus free enzyme) and chemical
conversion efficiency. The chemical conversion efficiency for
live cell testing was higher than that of free enzyme testing for
urease activity levels of 2 and 5 mM urea=min (Fig. 3). This dif-
ference reversed during tests conducted at the higher activity level
(i.e., 10 mM urea=min). After reaching 0.5 mol=L substrate con-
centration, though, both MICP and BEICP tests had lower effi-
ciency with calcium carbonate precipitation. Although the ureolytic
bacterial cells might produce more enzyme in the long term, this
is not expected to impact the result for chemical conversion
efficiency. Notably, other factors might restrict the activity of cells
Fig. 3. Chemical conversion efficiency of biological sources: bacterial
cells versus bacterial enzyme.

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 and/or free-enzymes, such as high pH (Ciurli et al. 1996; Stocks-
Fischer et al. 1999) or surficial coating by calcite precipitation,
which then might retard the nucleation rate of calcite crystal for-
mation (Mitchell and Ferris 2005, 2006). Therefore, actual mea-
surements of chemical conversion were lower than the expected
theoretical chemical conversions.
The highest efficiency of chemical conversion was observed
with 10 mM urea=min at a substrate level of 0.15 mol=L. However,
the 10%–20% increase in the precipitation ratio corresponded to
a 50% activity increase (i.e., from 5 to 10 mM urea=min). The
approximate urease activity of 5 mM urea=min subsequently was
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chosen for future tests given the pragmatic premise that a lower
urease dosing level is more economical when the original bio-
logical solution is to be diluted. In addition, Cheng et al. (2017)
mentioned that a low-urease-activity solution is more effective in
improving mechanical strength. A low concentration of substrate
reagent (0.15 mol=L) was used for the chemical solution in the col-
umn test because it resulted in higher strength and more-uniform
samples for a given amount of calcium carbonate precipitation
(Choi et al. 2019a; Al Qabany and Soga 2013).
Whole-Cell versus Enzyme-Only Impact on
Biostabilized Coarse-Sand Properties
The coarse-grained sand columns were stabilized via intact bacteria
and extracted enzyme ICP methods which received 4, 8, 12, or 16
repetitive treatment cycles to achieve different levels of CaCO3
content. The properties of the biocemented samples were tested to
comparatively evaluate the resulting variations with these alterna-
tive methods in terms of UCS, elastic modulus (E50 ), permeability,
and microstructure of biocemented coarse-grained sand.
UCS and Elastic Modulus
Fig. 4 identifies the peak stress levels of MICP-treated samples;
these results varied considerably, from 200 to 2,300 kPa. However,
the UCS estimates for BEICP-treated sands ranged from 400 to
1,500 kPa and, in one sample, up to approximately 2,400 kPa.
Although the UCS range of treated sand was similar for both bio-
treatment methods, the levels of CaCO3 precipitation were substan-
tially different in the MICP- and BEICP-treated samples. These
precipitation values ranged from 2.5% to 16% CaCO3 for MICP-
treated samples, and from 1.5% to 8% for BEICP-treated specimens.
The result demonstrated that MICP processing consistently pro-
duced a higher amount of CaCO3 for the same number of treatment
cycles compared with BEICP treatment. The increased biocemen-
tation may be caused by whole-cell attachment to sand surfaces, and
Fig. 4. UCS results for MICP- and BEICP-treated samples of coarse
sand.
© ASCE 04020030-5
J. Mater. Civ. Eng., 2020, 32(4): 04020030
filtering and trapping of these intact cells within the sand pore
matrix during down-flow percolation, in which both then induce
CaCO3 nucleation (Ginn et al. 2002). Furthermore, it is plausible
that increased numbers of treatment cycles might increase ionic par-
ticle charging, which then could boost bacterial adhesion (Faibish
et al. 1998; Foppen and Schijven 2006) on sand particles in relation
to an elevated zeta potential (Dick et al. 2006). In contrast, the lower
levels of CaCO3 precipitation in the BEICP-treated samples may
have been caused by soluble urease enzyme flushing and loss during
cyclic drainage, as opposed to surficial binding or trapping reten-
tion. Back-calculated estimates of residual urease activity remaining
in the column, versus that which was removed via flushing, were
approximately 40% of the original rate during BEICP processing for
one to four treatment cycles (data not shown). However, this per-
centage of urease activity retention reached 60%–70% as the num-
ber of treatment cycles increased (data not shown), likely due to
decreased permeability (and additional enzyme trapping) which de-
veloped at higher treatment cycles. Although the CaCO3 content of
BEICP-treated sand did reach ∼8% after 16 cycles, this level still
was far lower than that of MICP-treated sands.
In general, therefore, when considered on the basis of compa-
rable levels of calcium carbonate precipitation, the strength range
of BEICP-treated sand tended to be higher than that of MICP-
treated sand. For example, at 2%–4% and 5%–8% levels of CaCO3 ,
the UCS of BEICP-treated sand was approximately double that
achieved with MICP processing. Although Zhao et al. (2014) also
reported that EICP treatment had a lower range of CaCO3 content,
their tests gave lower UCS results than for MICP processing. How-
ever, they used a different treatment method, i.e., without adding
fresh biological solution, so ongoing degradation of the originally
added enzyme might have decreased their EICP product outcome
below that of MICP.
Although the amount of CaCO3 precipitation undoubtedly is an
important factor impacting biocemented sands, the distribution pat-
terns of the precipitated crystal clusters also might have played a
vital role in product strength (Cheng et al. 2017; Cui et al. 2017;
Hoang et al. 2018; Lin et al. 2016; Terzis and Laloui 2018). These
distribution patterns involve three apparent factors: (1) location of
the calcium crystals relative to sand interparticle contact; (2) thick-
ness of the crystal clusters; and (3) nonbeneficial precipitation of
CaCO3 within sand surfaces. Microstructure analysis provided fur-
ther insight into the influence of these latter three factors, and these
details are presented subsequently.
Fig. 5 compares the present and previously reported UCS results
for CaCO3 precipitation, including the present BEICP results and
the present and prior MICP results. The UCS strength levels re-
corded during this study are in exceptionally good agreement with
previously published MICP findings (Choi et al. 2016b, 2017a;
van Paassen et al. 2010a; Al Qabany and Soga 2013) from studies
which employed a similar substrate concentration (i.e., 0.3 M for
both urea and CaCl2 ). Other UCS data of MICP-treated coarse sand
with 1 M chemical concentration and partial saturation treatment
process by Cheng et al. (2013) are included in Fig. 5 for compari-
son. However, the UCS trend line obtained from BEICP-treated
sand in the present work was recorded at a noticeably higher range
than that of MICP-treated data in terms of comparable calcium dep-
osition levels.
Typical stress–strain curves of biocemented sand obtained from
unconfined compression testing at four and eight cycles of treat-
ment are presented in Fig. 6. Both MICP- and BEICP-treated sand
exhibited a brittle nature comparable to that observed in prior MICP
(Bernardi et al. 2014; Choi et al. 2016a; Mujah et al. 2019) and
EICP (Kavazanjian and Hamdan 2015; Neupane et al. 2015a; Park
et al. 2014) testing. The peak stress of MICP-treated samples after
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Fig. 5. UCS of present BEICP and MICP results versus previous MICP
data.
four-cycle treatment clearly was lower than that of similarly treated
BEICP-treated sand (Fig. 6). A lack of uniformity within MICP-
treated sands resulted in lower UCS strengths. During these sample
tests, failure did not occur within the whole core. Instead, sample
failure occurred within the less solidified and weaker bottom sec-
tion. Similar outcomes were reported by Cheng et al. (2013) and
van Paassen et al. (2010b). Lower strengths of the MICP-treated
samples similarly could have been caused by nonuniform bioce-
mentation conditions after low cycles of treatment (i.e., with lower
calcium cementation at lower depths within treated columns).
The modulus was computed as a secant elastic modulus based
on the strain required to mobilize 50% of the peak stress, E50 . For
example, the maximum stress of BEICP-treated sand with four
treatment cycles was 490 kPa at 0.29% strain (Fig. 6). At this strain
level the E50 of the specimen corresponds to 119.8 MPa. The elastic
modulus of the MICP-treated samples ranged from 20 to 250 MPa,
whereas it varied between 50 and 200 MPa for the BEICP-treated
Fig. 6. Typical stress–strain relationship for MICP- and BEICP-treated
samples of coarse sand.
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Fig. 7. Elastic modulus results for MICP- and BEICP-treated samples
of coarse sand.
sand specimens (Fig. 7). The increasing trend of E50 correlated to
CaCO3 content provided by MICP-treated samples was comparable
to that observed for BEICP treatment. Compared with previous
E50 test results from MICP-treated sand, van Paassen et al. (2010a)
found that E50 results with ICP-treated sand ranged from 100 to
8,500 MPa, significantly higher than the elastic modulus results
in the present study. However, BEICP processing produced an
even higher elastic modulus than the EICP results reported by
Yasuhara et al. (2012) (50–160 MPa) and Neupane et al. (2015a)
(16–18 MPa). van Paassen et al. (2010a) reported the use of a high
substrate concentration (1 M CaCl2 ∶urea) and a high level of treat-
ment (16 days), whereas fewer treatment cycles were injected by
Yasuhara et al. (2012) (4 and 8) and Neupane et al. (2015a) (2).
These data, both past and present, strongly suggest that substrate
strength and number of treatment cycles are the main factors for the
finished strength of biocemented sand.
Permeability
The permeability results for biocemented sand, using both MICP
and BEICP treatment, are graphed in Fig. 8. The findings are again
depicted in relation to calcium precipitation levels. In general,
the ICP-treated sand samples exhibited a reduction in permeabil-
ity in relation to increases in CaCO3 content. The permeability
levels for BEICP-treated sands were slightly lower in the range of
1.5%–4% CaCO3 precipitation. For BEICP processing, the highest
Fig. 8. Permeability results for MICP- and BEICP-treated samples of
coarse sand.
J. Mater. Civ. Eng.
 reduction of permeability occurred at an 8% CaCO3 content. The
impact of MICP processing was more pronounced, with a 3- to
4-log decrease within a 13%–16% CaCO3 precipitation range.
Although MICP exhibited far lower permeability reductions than
did BEICP, both treatment options had a similar pattern of reduction
relative to CaCO3 precipitation. This similarity may be attributable
to multiple factors. First, the sand columns were packed with the
same type of sand (coarse-grained sand, #20/30) and with the same
porosity (Table 2), which in turn would have been expected to
produce similar initial permeabilities. Second, sample permeabil-
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precipitation, induced by either whole cells with MICP or extracted
enzymes with BEICP, plays a crucial role in reducing the per-
meability of biocemented sand.
Microstructural Analyses
The microscale morphology of precipitated calcite crystals within
the coarse-grained biocemented sand matrix was analyzed using
scanning electron microscopy. Photographs obtained with the SEM
evaluation are provided in Fig. 9. Figs. 9(a–c) show that calcium
carbonate precipitated via bacterial cells (i.e., MICP) agglomerated
and formed large crystal clusters with a substantial thickness of

ity would have been reduced with progressive calcium-rich crystal

formation within void spaces between sand grains. Third, escalat- 50–100 μm. One important feature in this regard was that of the
ing CaCO3 precipitation within sand pore space would have led to large CaCO3 clusters precipitated at the contact points between
the lower permeability levels within the specimens. A consequent sand particles, which accumulated within and progressively filled
observation was that progressively increased calcium carbonate interstitial gaps between the adjacent sand particles and through

Fig. 9. SEM images of bio-treated samples for coarse sand: (a–c) MICP at 12-cycle levels; and (d–f) BEICP at 12-cycle levels.

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J. Mater. Civ. Eng., 2020, 32(4): 04020030

 which agglomeration then clogged pore spaces within the sand
matrix. The crystal cluster patterns observed in this study agree
with observations reported by previous researchers (Cheng et al.
2013, 2017; Cui et al. 2017; Mujah et al. 2019). In contrast, the
small crystal clusters produced via bacterial enzyme (i.e., BEICP)
processing accumulated as noticeably smaller calcium-bearing crys-
tals [Figs. 9(d–f)]. The average size of these smaller BEICP-based
cluster ranged from 5 to 20 μm. These smaller BEICP-derived crys-
tal clusters were observed primarily at the sand-grain contact points
and only intermittently on the sand particle surfaces. Because of the
smaller size of these crystals, less pore volume was filled within the
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BEICP-treated sands.
These observed differences in the size of ICP-derived calcium
formation between the MICP and BEICP processes were explained
by Hoang et al. (2018). Most calcium crystal sizes ranged from 5 to
10 μm for MICP-treated sand [Fig. 9(c)], whereas BEICP crystals
were noticeably smaller, 1–4 μm [Fig. 9(f)]. The premise behind
this change in crystal precipitation size is that of the nucleation
sources, derived from either whole-cell or enzyme-only mecha-
nisms. Mitchell and Ferris (2006) employed urea hydrolysis using
whole-cell S. pastuerii bacteria for MICP processing, and observed
calcium crystal diameters of 4.2 and 7.4 μm in 1 and 7 days, re-
spectively. In contrast, nanosized crystal formation (∼20 nm) was
reported by Sondi and Salopek-Sondi (2005), who used enzyme-
only ureolytic treatment for ICP precipitation. These findings sug-
gest that the mode of urea hydrolysis is a key factor affecting the
size of calcite crystals in the ICP process. Perspectives regarding
the distribution patterns of calcium crystal clusters have been re-
ported for MICP processing under various conditions. Al Qabany
and Soga (2013) and Choi et al. (2019c) reported the impacts of
substrate concentrations, retention times, and chemical addition
flow rates. Cheng et al. (2013, 2017) investigated the effect of vary-
ing degrees of saturation, levels of urease activity, processing tem-
peratures, and other environmental factors. To further evaluate the
distribution patterns of crystal clusters of MICP versus BEICP
processing, therefore, EDS overlay imaging was conducted for both
sample specimens following 16 cycles of treatment (Fig. 10).
Although the sand-particle contact points were bridged by
BEICP-deposited crystals, the pore space of the sand matrix re-
mained fairly open [Fig. 10(a)]. However, MICP processing pro-
duced a noticeably higher level of calcium deposition [Fig. 10(b)].
Fig. 10 shows calcite clusters deposited at three locations: (1) sand
particle contact points, (2) sand grain surfaces, and (3) sand pore-
space volume. These findings regarding differences in calcium
precipitation via MICP and BEICP processing appear to directly
impact the finished mechanical properties in terms of strength
and stiffness of biocemented sand. One such apparent correlation
is that the strength of biocemented sands was more influenced by
the location of calcium crystal deposition than by the total mass
of CaCO3 buildup. This premise agrees with previous studies by
Cheng et al. (2013), Cui et al. (2017), Al Qabany and Soga (2013).
For example, to reach 1,500 kPa of UCS, BEICP-treated sam-
ples needed approximately 6%–8% CaCO3 precipitation, whereas
MICP-treated sands required about 10%–11% CaCO3 content to
achieve similar mechanical strength (Fig. 4). Therefore, in terms of
strength achieved per mass of deposited calcium, the efficacy of
BEICP processing appear to be higher than that of MICP treatment.
Furthermore, a more specific rationale for the high efficiency of
increased UCS results with BEICP processing is that calcium de-
posited at the contact points of sand particles governs the strength
of sand. Similar perspectives were presented by Cheng et al. (2017),
Martinez and DeJong (2009).
Another related aspect of this behavior is that the amount quan-
tity of calcium deposition had a major impact on the reduction of
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permeability produced by MICP and BEICP. This correlation in
shown in the trends for permeability reduction in Fig. 8. The visual
evidence in Fig. 10 confirmed that far higher levels of deposited
calcium were attached to sand grain surfaces and pore-space vol-
umes during MICP processing [Fig. 10(b)] versus BEICP process-
ing [Fig. 10(e)]. Similar observations were made by previous
researchers (i.e., Cheng et al. 2013; Al Qabany and Soga 2013).
One further noteworthy observation is the significant difference
between MICP [Fig. 10(c)] and BEICP [Fig. 10(e)] processing
in terms of their relative chloride deposition levels. Although both
modes of treatment involved tap-water flushing within 2 h after
every treatment cycle to remove residual chemicals, Fig. 10(e)
clearly shows a substantial residual presence of chloride. This
chloride residual was attributed to the far lower permeability of
MICP-treated samples after multiple treatment cycles. The reduced
permeability means that chloride likely was not flushed from the
sample during the postprocessing tap-water rinse step, and then was
baked into a solid, chloride-rich deposit inside the MICP cores
during the final oven drying. This covert chloride crystallization
behavior could well have contributed to a falsely higher measure-
ment of MICP mechanical strength levels compared with what
would have been expected had long-term soaking and flushing
of the chloride been achieved. This unexpected MICP finding sug-
gests yet another benefit of BEICP processing, in where no such
chloride deposition occurred because tap-water flushing was far
more effective due to the higher residual permeability.
Effect of Sand Grain Size on BEICP-Treated Samples
The following details and discussion address the influence of sand
particle size on BEICP-treated columns. The type and size of sand
grains subjected to ICP processing are key factors in terms of bio-
cementation success. Previous researchers addressed the effects of
sand grain size ranging from fine to coarse using MICP processing
(Bernardi et al. 2014; Cheng et al. 2013, 2017; Kaur et al. 2016; Lin
et al. 2016; Martinez et al. 2013; Mortensen et al. 2011; Mwandira
et al. 2017; Zhao et al. 2014; Rebata-Landa and Santamarina
2006; Terzis and Laloui 2018, 2019; Weil et al. 2018) and EICP
processing (Hamdan et al. 2013; Kavazanjian and Hamdan 2015;
Nafisi et al. 2019; Neupane et al. 2015b). The present paper adds
further insight to this prior understanding, in terms of BEICP’s per-
formance with coarse and fine sand. As with the previous tests of
coarse-grained sand, these BEICP fine-sand tests were conducted
with 4, 8, and 12 cycles. Figs. 11 and 12 show the resultant UCS
and elastic modulus. Fig. 13 further compares UCS results for
BEICP with other published EICP results. In addition, Fig. 14
shows permeability results and Figs. 15 and 16 provide a set of
SEM images.
UCS and Elastic Modulus
The results obtained with BEICP-based treatment revealed a
more pronounced strength (Fig. 11) and elastic modulus (Fig. 12)
stabilization when applied to cohesionless materials. The UCS
of BEICP-treated coarse-grained sand was between 450 and
1,500 kPa for calcium carbonate precipitation from 2% to 6%.
BEICP processing of fine sand provided ranges of UCS from
200 to 900 kPa at similar CaCO3 contents. Similar patterns of
increased biocemented stiffness were observed in relation to in-
creased calcium carbonate deposition for both sand types. The
Young’s modulus of coarse biocemented sand varied between 75
and 125 MPa, whereas that of fine biocemented sand varied from
25 to 75 MPa when correlated with ranges of CaCO3 varying from
2% to 6% for both materials. The trend toward lower UCS and
stiffness for BEICP-treated fine sand compared with coarse sand
at a similar level of CaCO3 content was consistent with results
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(a) (d)

(b) (e)

(c) (f)

Fig. 10. SEM and EDS images of bio-treated samples for coarse sand: (a–c) MICP at 16-cycle levels; and (d–f) BEICP at 16-cycle levels.

Fig. 11. UCS results of BEICP-treated samples for coarse- and fine- Fig. 12. Elastic modulus results of BEICP-treated samples for coarse-
grained sands. and fine-grained sands.

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Fig. 13. UCS of present BEICP results versus previous EICP data.
UCS was compared based on the total precipitated CaCO3 content.
Fig. 14. Permeability results of BEICP-treated samples for coarse- and
fine-grained sands.
previously reported by Gomez et al. (2013), Lin et al. (2016), Zhao
et al. (2014), and Terzis and Laloui (2018) for MICP processing
and by Hamdan et al. (2013) and Kavazanjian and Hamdan (2015)
for EICP processing. In contrast, Cheng et al. (2013) mentioned
that at a similar CaCO3 content, fine MICP-treated sand achieved
higher values of cohesion and friction angle than did MICP-treated
coarse sand.
The overall bulk mass of calcium carbonate precipitation could
not be considered the sole factor governing the level of stabilization
for biocemented sand [Lin et al. (2016], Terzis and Laloui (2018)).
Overall, the UCS of BEICP-treated coarse sand tended to be higher
than that of BEICP-treated fine sand at comparable levels of cal-
cium carbonate content. For example, at 2%–3% of CaCO3 precipi-
tation, the UCS values of coarse-grained bio-treated sand ranged
from 450 to 700 kPa, whereas the UCS of fine-grained bio-treated
sand ranged from 380 to 540 kPa. In addition, at 5%–6% levels
of CaCO3 , the strength of biocemented coarse sand was approxi-
mately twice the UCS of BEICP-treated fine sand. The fact that
finer sand had higher CaCO3 content and lower strength matches
observations made by previous studies on MICP and EICP process-
ing (Kavazanjian and Hamdan 2015; Lin et al. 2016; Terzis and
Laloui 2018).
Cheng et al. (2013) characterized the nature of this same behav-
ior on the basis of a so-called hinge mode of calcium deposition at
the point of grain contact. Their corresponding assumption was that
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moisture retention is higher in this hinge area under partially satu-
rated–column conditions. Although they did not offer any further
hypotheses about the extent of hinge development and correspond-
ing calcium deposition, their paper’s findings suggest that higher
calcium deposition, higher hinge volume, and higher moisture re-
tention were all more prominent when dealing with fine-grain sand.
They also indicated that for a partially saturated treatment, the cal-
cium carbonate crystal clusters precipitated mainly at the contact
points of grains where solution menisci layers formed. Therefore,
the CaCO3 contents were higher in the fine BEICP-treated sand
which was treated by the partial saturation method (percolation–
circulation technique).
Terzis and Laloui (2018) investigated crucial microscopic char-
acteristics of biocemented sand in relation to apparent biostabi-
lization strength, covering such factors as the particle sizes of the
crystalline bond lattice, bond-grain contacts, and particle orienta-
tions. Their study indicated that larger-sized biocemented sands
had higher strength and stiffness than smaller-sized MICP-treated
sand at a similar CaCO3 content. According to Terzis and Laloui
(2018), MICP-treated large sand had larger mean diameters of
crystal clusters precipitated at sand grain contact points than that
achieved with biocemented smaller sand. They concluded that this
biocemented coarse sand grain behavior might lead to a higher
resistance to particle shearing and increased particle interlocking.
In addition, the spatial orientation and population of calcium crys-
tal clusters in large biocemented sands were found to be more
homogenously distributed than calcium bonds precipitated with
smaller-grained sands. As a more homogenous distribution of cal-
cium crystals developed within the spatial orientation space, a
higher overall stress resistance developed due to intergranular con-
tact and cementation. Although the UCS and Young’s modulus of
coarse-grained BEICP-treated sand were higher than those of fine-
grained biocemented sand at a similar level of CaCO3 , further stud-
ies of stress–strain behavior at various confining stress, friction
angles, and cohesion should be investigated for both sand materials.
Fig. 13 compares this study’s BEICP UCS results with re-
sults of prior investigators using EICP biostabilization methods
(Kavazanjian and Hamdan 2015; Neupane et al. 2015b; Oliveira
et al. 2016; Park et al. 2014; Yasuhara et al. 2012). In this case,
the present study’s BEICP-derived UCS results were considerably
higher than the previously published EICP findings at comparable
calcium deposition levels.
Permeability
The permeability results for coarse-grained and fine-grained
BEICP-based sand treatment are given in Fig. 14, including values
for both untreated materials and biostabilized samples. As expected,
a permeability reduction realized during BEICP sand processing
correlated with an increase in CaCO3 content for both fine and
coarse sand. However, the trend of reduced permeability observed
with fine-grained biocemented sands steadily declined with higher
calcium deposition levels, whereas the trend for coarse-grained sand
had much less reduction in permeability as calcium carbonate pre-
cipitation increased. This trend of lower permeability in fine-grained
bio-treated sand compared with coarse-grained sand was consistent
with the data of Cheng et al. (2013) from MICP treatment. Fine-
grained BEICP-treated sand typically had lower permeability values
than the coarse-grained BEICP-treated sands at similar levels of
CaCO3 content. In addition, both materials were packed at a similar
relative density in the untreated state. This indicates that the per-
meability reduction of BEICP-treated coarse and fine sand might
have been controlled by factors other than average or bulk CaCO3
content (e.g., the distribution patterns of calcium carbonate pre-
cipitation). The SEM analysis in the next section provides a visual
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Fig. 15. SEM images of BEICP-treated samples: (a–c) coarse-grained sand at 8-cycle levels; and (d–f) fine-grained sand at 8-cycle levels.

perspective of calcite crystal distribution patterns observed with
both coarse- and fine-grained BEICP-treated sand.
Microstructural Analyses
Figs. 15 and 16 provide a set of SEM images for BEICP-treated
coarse-grained and fine-grained sands after 8 and 12 treatment
cycles, respectively. These images depict calcium cluster deposi-
tion occurring at the contact points and surface of sand grains, but
pore-space deposition with these BEICP samples is noticeably less
than that with MCIP samples [Figs. 9(a) and 10(a)]. A similar pat-
tern of reduced pore-space volume deposition was reported by
Cheng et al. (2013) for MICP biostabilization applied in a partially
saturated state. During this sort of unsaturated processing con-
ditions, and with pore-space volume primarily filled with air, the
down-flowing substrate solution is retained as a moist film spread
across the grain surfaces and in a retained menisci layer formed due
to surface tension at the point of sand grain contact. The size of a
single calcium crystal initially formed by BEICP processing tended
to be quite small, ranging from ∼1 to 4 μm, for both bio-treated
coarse and fine sands [Figs. 15(c and f)]. This is because the pre-
cipitation particle was built up from nanosized crystals generated
from BEICP’s nanoscale free enzyme catalyst.
However, the amount and distribution pattern of calcium crystal
precipitation differed widely between the coarse and fine materials.
The CaCO3 levels with #50/70 sand samples [Figs. 15(d–f)] likely
were higher than those in #20/30 sand [Figs. 15(a–c)]. As was dis-
cussed previously, the average mass of CaCO3 was not the sole
factor governing the strength of biocemented sand. The calcium
carbonate distribution pattern was another main factor influencing
the processed sample strength. The crystal clusters concentrated

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Fig. 16. SEM images of BEICP-treated samples: (a and b) coarse-grained sand at 12-cycle levels; and (c and d) fine-grained sand at 12-cycle levels.
mainly at the contact points of sand grains in coarse-grained BEICP-
treated sands, whereas these clusters predominantly coated the grain
surfaces of fine-grained sands. Cheng et al. (2013), Cui et al. (2017),
Hoang et al. (2018), Lin et al. (2016), Terzis and Laloui (2018)
all mentioned that surface-coating calcium crystals tend to provide
noneffective grain bonding (i.e., compared with effective interpar-
ticle bridging bonds, whose effectiveness likely governs stabiliza-
tion strength). Therefore, the UCS and Young’s modulus of the fine
BEICP-improved sands were lower than those of coarse-grained
sands (Figs. 11 and 12).
The distribution pattern of calcium clusters likely impacted the
reduction of permeability in BEICP-treated sands. Fig. 15 shows the
clusters deposited almost exclusively at the contact points of sand
grains and sand surfaces after 8 cycles, whereas there was continued
deposition both at the contact points and in the internal void space
after 12 cycles (Fig. 16). However, after 12 cycles of treatment, the
amount of calcium crystal filling the void space during fine-sand
BEICP treatment [Figs. 16(c and d)] likely was higher than that tak-
ing place within coarse-grained sands [Figs. 16(a and b)]. In this
case, therefore, more pore-volume filling, and a resultant further re-
duction in permeability, is expected.
Conclusions and Recommendations
This paper provided a comparative investigation of whole-cell
(i.e., MICP) and extracted bacterial enzyme (i.e., BEICP) methods
focusing on two key factors: (1) chemical conversion efficiency,
and (2) engineering properties. Both MICP and BEICP assess-
ments were conducted using coarse sands, whereas only fine sands
© ASCE 04020030-12
J. Mater. Civ. Eng., 2020, 32(4): 04020030
were evaluated using BEICP. The following key conclusions were
identified from this research effort:
• Chemical conversation efficiency for both MICP and BEICP
methods decreased as the concentration of applied substrate
chemicals increased. However, in both cases the chemical con-
version efficiency increased as the urease activity level increased.
MICP processing (using whole-cell enzymes) produced higher
levels of chemical conversion efficiency when evaluated at ur-
ease activity levels between 2 and 5 mM urea=min. However, the
chemical conversion efficiency for BEICP (using bacterial-
extracted free enzyme), was higher than that of MICP processing
at a 10 mM urea=min rate.
• BEICP processing demonstrated higher UCS levels than MICP
with coarse sands and published EICP results at comparable cal-
cium deposition levels. BEICP processing also generated smaller
calcium crystal sizes at comparable levels of treatment cycles.
For example, BEICP completed at an eight-cycle treatment
mode produced crystals of 1–4 μm. MICP produced a noticeably
higher level of calcium deposition within sand pore-space vol-
ume than at the sand surface and meniscus areas, whereas BEICP
tended to realize a higher proportion of calcium deposition at
sand contact points and adjacent meniscus zones.
• MICP produced a higher reduction in permeability because of a
tendency toward higher pore-space calcium deposition. Conver-
sely, BEICP processing tended to have a lower impact on redu-
cing permeability. This particular behavior may be viewed as a
distinct benefit, because multiple treatment cycle applications
would not be as prone to column clogging.
• The observed correlation between sand grain size, levels
of CaCO3 precipitation, and distribution patterns of calcium
J. Mater. Civ. Eng.
 deposition clustering were prominent factors in relation to
strength and permeability outcomes. BEICP processing of fine-
grained sands produced lower UCS levels compared with coar-
segrained sands at similar levels of CaCO3 precipitation. This
pattern for BEICP was consistent with prior EICP publications
and prior MICP publications.
Two recommendations are given for future research regarding
the mechanisms and performance of BEICP processing. First, test-
ing should be conducted to specifically quantify protein mass levels
when using extracted free-enzyme removed from viable ureolytic
cells. This approach would provide an enhanced means of com-
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pleting a specific characterization of enzymatic reactions relative
to chemical conversion efficiency. Second, further investigation is
warranted to qualify the use of BEICP processing within complex,
natural soil systems.
References
Almajed, A., H. Khodadadi, and E. Kavazanjian Jr. 2018. “Sisal fiber
reinforcement of EICP-treated soil.” In Proc., IFCEE 2018, 29–36.
Reston, VA: ASCE.
Al Qabany, A., and K. Soga. 2013. “Effect of chemical treatment used in
MICP on engineering properties of cemented soils.” Géotechnique
63 (4): 331–339. https://doi.org/10.1680/geot.SIP13.P.022.
Al Qabany, A., K. Soga, and C. Santamarina. 2012. “Factors affecting
efficiency of microbially induced calcite precipitation.” J. Geotech.
Geoenviron. Eng. 138 (8): 992–1001. https://doi.org/10.1061/(ASCE)
GT.1943-5606.0000666.
ASTM. 2006. Standard test method for permeability of granular soils (con-
stant head). ASTM D2434–68. West Conshohocken, PA: ASTM.
ASTM. 2010. Standard practice for classification of soils for engineering
purposes (unified soil classification system). ASTM D2487. West
Conshohocken, PA: ASTM.
ASTM. 2014. Standard specification for sand. ASTM C778. West
Conshohocken, PA: ASTM.
Bernardi, D., J. T. Dejong, B. M. Montoya, and B. C. Martinez. 2014.
“Bio-bricks: Biologically cemented sandstone bricks.” Constr. Build.
Mater. 55 (Mar): 462–469. https://doi.org/10.1016/j.conbuildmat.2014
.01.019.
Burbank, M. B., T. J. Weaver, T. L. Green, B. C. Williams, and R. L.
Crawford. 2011. “Precipitation of calcite by indigenous microorgan-
isms to strengthen liquefiable soils.” Geomicrobiol. J. 28 (4): 301–312.
https://doi.org/10.1080/01490451.2010.499929.
Carmona, J. P. S. F., P. J. V. Oliveira, L. J. L. Lemos, and A. M. G. Pedro.
2018. “Improvement of a sandy soil by enzymatic calcium carbonate
precipitation.” Proc. Inst. Civ. Eng. Geotech. Eng. 171 (1): 3–15.
https://doi.org/10.1680/jgeen.16.00138.
Cheng, L., R. Cord-Ruwisch, and M. A. Shahin. 2013. “Cementation of
sand soil by microbially induced calcite precipitation at various degrees
of saturation.” Can. Geotech. J. 50 (1): 81–90. https://doi.org/10.1139
/cgj-2012-0023.
Cheng, L., M. A. Shahin, and D. Mujah. 2017. “Influence of key environ-
mental conditions on microbially induced cementation for soil stabili-
zation.” J. Geotech. Geoenviron. Eng. 143 (1): 04016083. https://doi
.org/10.1061/(ASCE)GT.1943-5606.0001586.
Choi, S., T. Hoang, E. J. Alleman, and J. Chu. 2019a. “Splitting tensile
strength of fiber-reinforced and biocemented sand.” J. Mater. Civ. Eng.
31 (9): 1–6. https://doi.org/10.1061/(ASCE)MT.1943-5533.0002841.
Choi, S., T. Hoang, and S. Park. 2019b. “Undrained behavior of microbially
induced calcite precipitated sand with polyvinyl alcohol fiber.” Appl.
Sci. 9 (6): 1214. https://doi.org/10.3390/app9061214.
Choi, S., S. Wu, and J. Chu. 2016a. “Biocementation for sand using an
eggshell as calcium source.” J. Geotech. Geoenviron. Eng. 142 (10):
06016010. https://doi.org/10.1061/(ASCE)GT.1943-5606.0001534.
Choi, S. G., J. Chu, R. C. Brown, K. Wang, and Z. Wen. 2017a. “Sustain-
able biocement production via microbially induced calcium carbonate
precipitation: Use of limestone and acetic acid derived from pyrolysis
© ASCE 04020030-13
J. Mater. Civ. Eng., 2020, 32(4): 04020030
of lignocellulosic biomass.” ACS Sustainable Chem. Eng. 5 (6): 5183–
5190. https://doi.org/10.1021/acssuschemeng.7b00521.
Choi, S. G., J. Chu, and T. H. Kwon. 2019c. “Effect of chemical concen-
trations on strength and crystal size of biocemented sand.” Geomech.
Eng. 17 (5): 465–473. https://doi.org/10.12989/gae.2019.17.5.465.
Choi, S.-G., S.-S. Park, S. Wu, and J. Chu. 2017b. “Methods for calcium
carbonate content measurement of biocemented soils.” J. Mater. Civ.
Eng. 29 (11): 06017015. https://doi.org/10.1061/(ASCE)MT.1943
-5533.0002064.
Choi, S.-G., K. Wang, and J. Chu. 2016b. “Properties of biocemented, fiber
reinforced sand.” Constr. Build. Mater. 120 (Sep): 623–629. https://doi
.org/10.1016/j.conbuildmat.2016.05.124.
Chu, J., V. Stabnikov, and V. Ivanov. 2012. “Microbially induced calcium
carbonate precipitation on surface or in the bulk of soil.” Geomicrobiol.
J. 29 (6): 544–549. https://doi.org/10.1080/01490451.2011.592929.
Chu, J., S. Varaksin, U. Klotz, and P. Menge. 2009. “Construction proc-
esses.” In Proc., 17th Int. Conf. on Soil Mechanics and Geotechnical
Engineering: The Academia and Practice of Geotechnical Engineering.
Amsterdam, Netherlands: IOS Press.
Ciurli, S., C. Marzadori, S. Benini, S. Deiana, and C. Gessa. 1996. “Urease
from the soil bacterium Bacillus pasteurii: Immobilization on
Ca-polygalacturonate.” Soil Biol. Biochem. 28 (6): 811–817. https://doi
.org/10.1016/0038-0717(96)00020-X.
Cui, M. J., J. J. Zheng, R. J. Zhang, H. J. Lai, and J. Zhang. 2017. “In-
fluence of cementation level on the strength behaviour of bio-cemented
sand.” Acta Geotech. 12 (5): 971–986. https://doi.org/10.1007/s11440
-017-0574-9.
DeJong, J. T., M. B. Fritzges, and K. Nüsslein. 2006. “Microbially induced
cementation to control sand response to undrained shear.” J. Geotech.
Geoenviron. Eng. 132 (11): 1381–1392. https://doi.org/10.1061/(ASCE)
1090-0241(2006)132:11(1381).
Dick, J., W. De Windt, B. De Graef, H. Saveyn, P. Van Der Meeren,
N. De Belie, and W. Verstraete. 2006. “Bio-deposition of a calcium
carbonate layer on degraded limestone by Bacillus species.” Biodegra-
dation 17 (4): 357–367. https://doi.org/10.1007/s10532-005-9006-x.
Dilrukshi, R. A. N., K. Nakashima, and S. Kawasaki. 2018. “Soil improve-
ment using plant-derived urease-induced calcium carbonate precipita-
tion.” Soils Found. 58 (4): 894–910. https://doi.org/10.1016/j.sandf
.2018.04.003.
Dilrukshi, R. A. N., J. Watanabe, and S. Kawasaki. 2016. “Strengthening of
sand cemented with calcium phosphate compounds using plant-derived
urease.” Int. J. GEOMATE 11 (25): 2461–2467. https://doi.org/10
.21660/2016.25.5149.
Faibish, R. S., M. Elimelech, and Y. Cohen. 1998. “Effect of interparticle
electrostatic double layer interactions on permeate flux decline in cross-
flow membrane filtration of colloidal suspensions: An experimental in-
vestigation.” J. Colloid Interface Sci. 204 (1): 77–86. https://doi.org/10
.1006/jcis.1998.5563.
Feng, K., and B. M. Montoya. 2015. “Influence of confinement and cemen-
tation level on the behavior of microbial-induced calcite precipitated
sands under monotonic drained loading.” J. Geotech. Geoenviron. Eng.
142 (1): 04015057. https://doi.org/10.1061/(ASCE)GT.1943-5606
.0001379.
Foppen, J. W. A., and J. F. Schijven. 2006. “Evaluation of data from the
literature on the transport and survival of Escherichia coli and thermo-
tolerant coliforms in aquifers under saturated conditions.” Water Res.
40 (3): 401–426. https://doi.org/10.1016/j.watres.2005.11.018.
Ginn, T. R., B. D. Wood, K. E. Nelson, T. D. Scheibe, E. M. Murphy, and
T. P. Clement. 2002. “Processes in microbial transport in the natural
subsurface.” Adv. Water Resour. 25 (8–12): 1017–1042. https://doi
.org/10.1016/S0309-1708(02)00046-5.
Gomez, M. G., B. C. Martinez, and J. T. Dejong. 2013. “Bio-mediated soil
improvement field study to stabilize mine sands.” In Proc., GeoMontreal.
Montreal: Ontario Water Works Association.
Gomez, M. G., B. C. Martinez, J. T. DeJong, C. E. Hunt, L. A. deVlaming,
D. W. Major, and S. M. Dworatzek. 2015. “Field-scale bio-cementation
tests to improve sands.” Proc. Inst. Civ. Eng. Ground Improv. 168 (3):
206–216. https://doi.org/10.1680/grim.13.00052.
J. Mater. Civ. Eng.
 Hamdan, N., and E. Kavazanjian. 2016. “Enzyme-induced carbonate
mineral precipitation for fugitive dust control.” Géotechnique 66 (7):
546–555. https://doi.org/10.1680/jgeot.15.P.168.
Hamdan, N., E. Kavazanjian Jr., and S. O’Donnell. 2013. “Carbonate
cementation via plant derived urease.” In Proc., 18th Int. Conf. on Soil
Mechanics and Geotechnical Engineering, 2489–2492. Paris: French
Society for Soil Mechanics and Geotechnical Engineering.
Hamed Khodadadi, T., E. Kavazanjian, L. Van Paassen, and J. Dejong.
2017. “Bio-grout materials: A review.” In Proc., Grouting 2017.
Reston, VA: ASCE.
Hoang, T., J. Alleman, B. Cetin, K. Ikuma, and S.-G. Choi. 2018. “Sand
Downloaded from ascelibrary.org by Nanyang Technological University- Library on 02/04/20. Copyright ASCE. For personal use only; all rights reserved.
and silty-sand soil stabilization using bacterial enzyme induced calcite
precipitation (BEICP).” Can. Geotech. J. 56 (6): 808–822. https://doi
.org/10.1139/cgj-2018-0191.
Ivanov, V., and J. Chu. 2008. “Applications of microorganisms to geotech-
nical engineering for bioclogging and biocementation of soil in situ.”
Rev. Environ. Sci. Biotechnol. 7 (2): 139–153. https://doi.org/10.1007
/s11157-007-9126-3.
Javadi, N., H. Khodadadi, N. Hamdan, and E. Kavazanjian Jr. 2018. “EICP
treatment of soil by using urease enzyme extracted from watermelon
seeds.” In Proc., IFCEE 2018, 115–124. Reston, VA: ASCE.
Kaur, N., M. S. Reddy, and A. Mukherjee. 2016. “Significant indicators
for biomineralisation in sand of varying grain sizes.” Constr. Build.
Mater. 104 (Feb): 198–207. https://doi.org/10.1016/j.conbuildmat.2015
.12.023.
Kavazanjian, E., Jr., A. Almajed, and N. Hamdan. 2017. “Bio-inspired soil
improvement using EICP soil columns and soil nails.” In Proc., Grout-
ing 2017 GSP, 13–22. Reston, VA: ASCE.
Kavazanjian, E., and N. Hamdan. 2015. “Enzyme induced carbonate pre-
cipitation (EICP) columns for ground improvement.” In Proc., IFCEE
2015, edited by M. Iskander, M. T. Suleiman, J. B. Anderson, and D. F.
Laefer, 2252–2261. Reston, VA: ASCE.
Larsen, J., M. Poulsen, T. Lundgaard, and M. Agerbaek. 2008. “Plugging
of fractures in chalk reservoirs by enzymatic-induced calcium carbonate
precipitation.” SPE Production Oper. 23 (4): 478–483. https://doi.org
/10.2118/108589-PA.
Lin, H., M. T. Suleiman, D. G. Brown, and E. Kavazanjian. 2016.
“Mechanical behavior of sands treated by microbially induced carbon-
ate precipitation.” J. Geotech. Geoenviron. Eng. 142 (2): 04015066.
https://doi.org/10.1061/(ASCE)GT.1943-5606.0001383.
Martinez, B. C., and J. T. DeJong. 2009. “Bio-mediated soil improvement:
Load transfer mechanisms at the micro- and macro- scales.” In Proc.,
US-China Workshop on Ground Improvement Technologies 2009,
242–251. Reston, VA: ASCE.
Martinez, B. C., J. T. DeJong, T. R. Ginn, B. M. Montoya, T. H. Barkouki,
C. Hunt, B. Tanyu, and D. Major. 2013. “Experimental optimization
of microbial-induced carbonate precipitation for soil improvement.”
J. Geotech. Geoenviron. Eng. 139 (Apr): 587–598. https://doi.org/10
.1061/(ASCE)GT.1943-5606.0000787.
Mitchell, A. C., and F. G. Ferris. 2005. “The coprecipitation of Sr into cal-
cite precipitates induced by bacterial ureolysis in artificial groundwater:
Temperature and kinetic dependence.” Geochim. Cosmochim. Acta
69 (17): 4199–4210. https://doi.org/10.1016/j.gca.2005.03.014.
Mitchell, A. C., and F. G. Ferris. 2006. “The influence of Bacillus pasteurii
on the nucleation and growth of calcium carbonate.” Geomicrobiol. J.
23 (3–4): 213–226. https://doi.org/10.1080/01490450600724233.
Mitchell, J. K. 1981. “Soil improvement state of the art report.” In Vol. 4 of
Proc., 11th Int. Conf. on SMFE. Boca Raton, FL: CRC Press.
Mortensen, B. M., M. J. Haber, J. T. Dejong, L. F. Caslake, and D. C.
Nelson. 2011. “Effects of environmental factors on microbial induced
calcium carbonate precipitation.” J. Appl. Microbiol. 111 (2): 338–349.
https://doi.org/10.1111/j.1365-2672.2011.05065.x.
Mujah, D., L. Cheng, and M. A. Shahin. 2019. “Microstructural and
geomechanical study on biocemented sand for optimization of MICP
process.” J. Mater. Civ. Eng. 31 (4): 04019025. https://doi.org/10.1061
/(ASCE)MT.1943-5533.0002660.
Mwandira, W., K. Nakashima, and S. Kawasaki. 2017. “Bioremediation
of lead-contaminated mine waste by Pararhodobacter sp. based on
the microbially induced calcium carbonate precipitation technique and
© ASCE 04020030-14
J. Mater. Civ. Eng., 2020, 32(4): 04020030
its effects on strength of coarse and fine grained sand.” Ecol. Eng.
109 (Sep): 57–64. https://doi.org/10.1016/j.ecoleng.2017.09.011.
Nafisi, A., S. Safavizadeh, and B. M. Montoya. 2019. “Influence of microbe
and enzyme-induced treatments on cemented sand shear response.”
J. Goetech. Geoenviron. Eng. 145 (9): 06019008. https://doi.org/10
.1061/(ASCE)GT.1943-5606.0002111.
Nam, I., C. Chon, K. Jung, and S. Choi. 2015. “Calcite precipitation
by ureolytic plant (Canavalia ensiformis) extracts as effective bioma-
terials.” KSCE J. Civ. Eng. 19 (6): 1620–1625. https://doi.org/10.1007
/s12205-014-0558-3.
Nemati, M., and G. Voordouw. 2003. “Modification of porous media
permeability, using calcium carbonate produced enzymatically in situ.”
Enzyme Microb. Technol. 33 (5): 635–642. https://doi.org/10.1016
/S0141-0229(03)00191-1.
Neupane, D., H. Yasuhara, N. Kinoshita, and Y. Ando. 2015a. “Distribution
of mineralized carbonate and its quantification method in enzyme medi-
ated calcite precipitation technique.” Soils Found. 55 (2): 447–457.
https://doi.org/10.1016/j.sandf.2015.02.018.
Neupane, D., H. Yasuhara, N. Kinoshita, and H. Putra. 2015b. “Distribution
of grout material within 1-m sand column in insitu calcite precipitation
technique.” Soils Found. 55 (6): 1512–1518. https://doi.org/10.1016/j
.sandf.2015.10.015.
Neupane, D., H. Yasuhara, N. Kinoshita, and T. Unno. 2013. “Applicability
of enzymatic calcium carbonate precipitation as a soil-strengthening
technique.” J. Geotech. Geoenviron. Eng. 139 (12): 2201–2211. https://
doi.org/10.1061/(ASCE)GT.1943-5606.0000959.
Oliveira, P. J. V., L. D. Freitas, and J. P. S. F. Carmona. 2016. “Effect of soil
type on the enzymatic calcium carbonate precipitation process used for
soil improvement.” J. Mater. Civ. Eng. 29 (4): 04016263. https://doi.org
/10.1061/(ASCE)MT.1943-5533.0001804.
Oliveira, P. J. V., and J. P. G. Neves. 2019. “Effect of organic matter content
on enzymatic biocementation process applied to coarse-grained soils.”
J. Mater. Civ. Eng. 31 (7): 04019121. https://doi.org/10.1061/(ASCE)
MT.1943-5533.0002774.
Park, S.-S., S.-G. Choi, and I.-H. Nam. 2014. “Effect of microbially in-
duced calcite precipitation on strength of cemented sand.” J. Mater.
Civ. Eng. 26 (8): 06014017. https://doi.org/10.1061/(ASCE)MT.1943
-5533.0001029.
Pasillas, J. N., H. Khodadadi, K. Martin, P. Bandini, C. M. Newtson, and
E. Kavazanjian Jr. 2018. “Viscosity-enhanced EICP treatment of soil.”
In Proc., IFCEE 2018, 145–154. Reston, VA: ASCE.
Phua, Y. J., and A. Røyne. 2018. “Bio-cementation through controlled
dissolution and recrystallization of calcium carbonate.” Constr. Build.
Mater. 167 (Apr): 657–668. https://doi.org/10.1016/j.conbuildmat.2018
.02.059.
Putra, H., H. Yasuhara, and N. Kinoshita. 2017. “Applicability of nat-
ural zeolite for NH-Forms removal in enzyme-mediated calcite pre-
cipitation technique.” Geosciences 7 (3): 61. https://doi.org/10.3390
/geosciences7030061.
Rebata-Landa, V., and J. C. Santamarina. 2006. “Mechanical limits to
microbial activity in deep sediments.” Geochem. Geophys. Geosyst.
7 (11): 1–12. https://doi.org/10.1029/2006GC001355.
Saneiyan, S., D. Ntarlagiannis, J. Ohan, J. Lee, F. Colwell, and S. Burns.
2019. “Induced polarization as a monitoring tool for in-situ micro-
bial induced carbonate precipitation (MICP) processes.” Ecol. Eng.
127 (Feb): 36–47. https://doi.org/10.1016/j.ecoleng.2018.11.010.
Simatupang, M., and M. Okamura. 2017. “Liquefaction resistance of sand
remediated with carbonate precipitation at different degrees of satura-
tion during curing.” Soils Found. 57 (4), 619–631. https://doi.org/10
.1016/j.sandf.2017.04.003.
Sondi, I., and B. Salopek-Sondi. 2005. “Influence of the primary structure
of enzymes on the formation of CaCO3 polymorphs: A comparison of
plant (Canavaliaensiformis) and bacterial (Bacilluspasteurii) ureases.”
Langmuir 21 (19): 8876–8882. https://doi.org/10.1021/la051129v.
Stocks-Fischer, S., J. K. Galinat, and S. S. Bang. 1999. “Microbiological
precipitation of CaCO3 .” Soil Biol. Biochem. 31 (11): 1563–1571.
https://doi.org/10.1016/S0038-0717(99)00082-6.
Sun, X., L. Miao, D. Ph, T. Tong, and C. Wang. 2018. “Improvement of
microbial-induced calcium carbonate precipitation technology for sand
J. Mater. Civ. Eng.
 solidification.” J. Mater. Civ. Eng. 30 (11): 04018301. https://doi.org
/10.1061/(ASCE)MT.1943-5533.0002507.
Terashi, M., and I. Juran. 2000. “Ground improvement—State of the art.”
In Proc., ISRM Int. Symp. Lisbon, Portugal: International Society for
Rock Mechanics and Rock Engineering.
Terzis, D., and L. Laloui. 2018. “3-D micro-architecture and mechanical
response of soil cemented via microbial-induced calcite precipitation.”
Sci. Rep. 8 (1): 1416. https://doi.org/10.1038/s41598-018-19895-w.
Terzis, D., and L. Laloui. 2019. “Cell-free soil bio-cementation with
strength, dilatancy and fabric characterization.” Acta Geotech. 14 (3):
639–656. https://doi.org/10.1007/s11440-019-00764-3.
van der Star, W. R. L., W. K. van Wijngaarden-van Rossum, L. A. van
Downloaded from ascelibrary.org by Nanyang Technological University- Library on 02/04/20. Copyright ASCE. For personal use only; all rights reserved.
Paassen, and L. R. van Baalen. 2011. “Stabilization of gravel deposits
using microorganisms.” In Proc., 15th European Conf. on Soil Mechan-
ics and Geotechnical Engineering, edited by A. Anagnostopoulos,
85–90. Amsterdam, Netherlands: IOS Press.
van Paassen, L. A. 2011. “Bio-mediated ground improvement: From
laboratory experiment to pilot applications.” In Proc., Geo-Frontiers,
4099–4108. Reston, VA: ASCE.
van Paassen, L. A., R. Ghose, T. J. M. van der Linden, W. R. L. van der Star,
and M. C. M. van Loosdrecht. 2010a. “Quantifying biomediated ground
improvement by ureolysis: Large-scale biogrout experiment.” J. Geo-
tech. Geoenviron. Eng. 136 (12): 1721–1728. https://doi.org/10.1061
/(ASCE)GT.1943-5606.0000382.
© ASCE 04020030-15
J. Mater. Civ. Eng., 2020, 32(4): 04020030
van Paassen, L. A., M. C. M. Van Loosdrecht, M. Pieron, A. Mulder,
D. J. M. Ngan-Tillard, and T. J. M. Van Der Linden. 2010b. “Strength
and deformation of biologically cemented sandstone.” In Proc., Rock
Engineering in Difficult Ground Conditions—Soft Rocks and Karst,
edited by I. Vrkljan, 405–410. Boca Raton, FL: CRC Press.
Weil, M. H., J. T. Dejong, B. C. Martinez, and B. M. Mortensen. 2018.
“Seismic and resistivity measurements for real-time monitoring of mi-
crobially induced calcite precipitation in sand.” Geotech. Test. J. 35 (2):
330–341. https://doi.org/10.1520/GTJ103365.
Whiffin, V. S. 2004. “Microbial CaCO3 precipitation for the production of
biocement.” Ph.D. dissertation, School of Biological Sciences and Bio-
technology, Murdoch Univ.
Whiffin, V. S., L. A. van Paassen, and M. P. Harkes. 2007. “Microbial car-
bonate precipitation as a soil improvement technique.” Geomicrobiol. J.
24 (5): 417–423. https://doi.org/10.1080/01490450701436505.
Yasuhara, H., D. Neupane, K. Hayashi, and M. Okamura. 2012. “Experi-
ments and predictions of physical properties of sand cemented by
enzymatically-induced carbonate precipitation.” Soils Found. 52 (3):
539–549. https://doi.org/10.1016/j.sandf.2012.05.011.
Zhao, Q., L. Li, C. Li, M. Li, F. Amini, and H. Zhang. 2014. “Factors
affecting improvement of engineering properties of MICP-treated
soil catalyzed by bacteria and urease.” J. Mater. Civ. Eng. 26 (12):
04014094. https://doi.org/10.1061/(ASCE)MT.1943-5533.0001013.
J. Mater. Civ. Eng.