Science of the Total Environment 672 (2019) 722–731

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Short Communication

Microbial induced carbonate precipitation for immobilizing Pb

contaminants: Toxic effects on bacterial activity and
immobilization efficiency
Ning-Jun Jiang a,b,1, Rui Liu a,1, Yan-Jun Du a,⁎, Yu-Zhang Bi a
a
Jiangsu Key Laboratory of Urban Underground Engineering & Environmental Safety, Institute of Geotechnical Engineering, Southeast University, Nanjing 210096, China
b
Department of Civil and Environmental Engineering, University of Hawaii at Manoa, Honolulu, HI 96822, USA

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Pb contaminants are immobilized by

microbial induced carbonate precipita-
tion.
• Pb has toxic effects on bacterial activity
and immobilization efficiency.
• Abiotic and biotic precipitation and
bio-sorption are immobilization
mechanisms.

a r t i c l e i n f o a b s t r a c t

Article history: Microbial induced carbonate precipitation (MICP) is a natural bio-mediated process, which has been explored for
Received 22 October 2018 soil stabilization and heavy metals immobilization in soil and groundwater. Previous studies have shown that
Received in revised form 14 March 2019 MICP is capable of immobilizing various heavy metals including lead (Pb). However, most studies focus merely on
Accepted 19 March 2019
the immobilization of heavy metals with relatively low concentration. This study: (1) presents results of an investi-
Available online 20 March 2019
gation into the toxic effects of Pb on bacterial activity and immobilization efficiency within a wide range of Pb con-
Editor: Filip M.G. Tack centrations; and (2) identifies controlling biotic and abiotic factors of Pb immobilization by MICP. In the first series of
tests, bacterial strains (Sporosarcina pasteurii) are inoculated into nutrient solutions containing 0–50 mM Pb(NO3)2
Keywords: and incubated at 30 °C. Biochemical parameters are measured over time, which include pH, electrical conductivity,
Microbial induced carbonate precipitation urease activity, and viable cell number. In the second series of tests, grown bacterial strains are mixed with urea, cal-
Sporosarcina pasteurii cium salts and Pb(NO3)2 in solution. Viable cell number, produced ammonium concentration, aqueous Pb concen-
Urease activity tration of the mixed solution, and total precipitation mass are measured. The results show that the presence of Pb
Lead contamination has marginal effect on bacterial growth and associated urease activity at Pb concentration b 30 mM. The calcium
source and initial bacteria concentration are found to remarkably influence Pb immobilization efficiency in terms
of Pb removal percentage. Supplementary geochemical simulation results indicate that the Pb immobilization mech-
anisms includes abiotic precipitation, biotic precipitation and bio-sorption.
© 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Institute of Geotechnical Engineering, Southeast University, Nanjing 210096, China.
E-mail addresses: jiangn@hawaii.edu (N.-J. Jiang), liurui1926@seu.edu.cn (R. Liu), duyanjun@seu.edu.cn (Y.-J. Du), biyuzhang@imde.ac.cn (Y.-Z. Bi).
1
Co-first authors. These authors contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2019.03.294
0048-9697/© 2019 Elsevier B.V. All rights reserved.
 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
1. Introduction
Heavy metal contamination in soil and groundwater is a major risk
to the ecosystem and human health, since (1) heavy metals accumulate
in air, water, soil and biosphere; and (2) industrial activities have
strengthened this effect (Nagajyoti et al., 2010; Wei and Yang, 2010;
Xia et al. 2019a,b). In the past few decades, various in-situ and ex-situ
methods have been developed to treat heavy metal contaminated soil
and groundwater. These methods include but not limited to electroki-
netic extraction, soil flushing, solidification/stabilization (S/S),
phytoremediation, and bioremediation. Among these remediation
methods, bioremediation utilizes microorganisms to detoxify contami-
nants via bio-sorption, extracellular chemical precipitation, and bioac-
cumulation (Srivastava and Kowshik, 2018). Bioremediation has been
used widely in treating wastewater and petroleum polluted soils by
stimulating native microbes to degrade contaminants, mostly toxic
organics.
Microbially induced carbonate precipitation (MICP) is an emerging
bio-mediated technique and its potential applications in civil and envi-
ronmental engineering have been extensively investigated. It is re-
ported that MICP can be used to strengthen sandy soil, improve soil
resistance to erosion, mitigate liquefaction, and seal concrete cracks
(Dejong et al., 2013). While most of these studies are conducted under
laboratory-scale conditions, a few field trials are also reported. For in-
stance, Phillips et al. (2016) promoted MICP in the field to seal fractured
sandstone with the help of oil field technology. van Paassen (2011) ap-
plied MICP in a field trial using injection and extraction wells to improve
the mechanical stability of horizontal boreholes.
MICP is initiated through the activity of enzymes such as urease
(Castanier et al., 1993; Castanier et al., 1999; Gollapudi et al., 1995), car-
bonic anhydrase (Dhami et al., 2017) or asparaginase (Li et al., 2015).
One of the most popular pathways of achieving MICP is through hydro-
lysis of urea by urease enzyme, which is originated from some soil bac-
teria or plants. Eqs. (1)–(4) show the biochemical reactions involving
ureolytic-driven MICP (van Paassen, 2009):
ðNH2 Þ2 CO þ H2 O→2NH3 þ CO2 ð1Þ
þ −
2NH3 þ 2H2 O↔2NH4 þ 2OH ð2Þ
CO2 þ 2OH− ↔HCO3 – þ OH– ↔CO3 2– þ H2 O ð3Þ
Ca2þ þ CO3 2– ↔CaCO3 ðsÞ ð4Þ
Among various MICP pathways, ureolytic-driven MICP outstands
since ureolytic bacteria are widespread in natural soils (Gomez et al.,
2014) and this process is highly energy efficient in forming carbonate
precipitates (DeJong et al., 2010). So in this paper, “MICP” refers to the
ureolytic-driven MICP process. While MICP is extensively studied for
improving mechanical and hydraulic performances of granular soils
and other construction materials, it is also attempted as a bioremedia-
tion method for immobilizing heavy metal contaminants and radioac-
tive wastes (Fujita et al., 2004). Qian et al. (2017) used urease-
producing fungi to produce carbonate precipitates and immobilize Cr
and Pb contained in farmland soils at 100 mg/kg and 200 mg/kg, respec-
tively. 95% exchangeable Cr and Pb in the soil were reported to be
immobilized. Li et al. (2016) introduced another urease-producing mi-
crobe T. tumescens capable of immobilizing multiple heavy metals (Ni,
Cu Pb, Co, Zn and Cd) in aqueous solution with a removal percentage
of approximately 90%. Fujita et al. (2010) examined the potential of na-
tive ureolytic microbes for fixing 90Sr in groundwater. Achal et al.
(2012b) mixed extracted ureolytic bacteria from mining site with Pb-
contaminated soil (100 mg/kg) to trigger MICP and yielded a Pb re-
moval percentage of 83%. Co-precipitation of heavy metals in carbonate
minerals after MICP treatment was reported to be the dominant immo-
bilization mechanism in most of these studies.
723
Although it is promising to apply MICP in heavy metal immobilization,
especially Pb, most previous studies focus on heavy metal concentration
no higher than 5 mM in aqueous solution or 400 mg/kg in soil (Kang
et al., 2015; Li et al., 2013; Li et al., 2016; Mugwar and Harbottle, 2016;
Mwandira et al., 2017; Qian et al., 2017; Zhu et al., 2017). While these
studies have demonstrated the effectiveness of MICP for immobilizing ex-
changeable Pb, the detrimental effects of high Pb concentration on bacte-
rial activity and MICP effectiveness have not yet been well addressed.
According to the investigation by Duan et al. (2016), nearly 20% of sites
in China are contaminated by lead, whose concentration is higher than
the regulatory limit value (80 mg/kg). A lead concentration of
30,430 mg/kg in soil was even detected close to a lead and zinc melting
plant in Fujian Province, China. Therefore, it is of great importance to ex-
amine the MICP immobilization efficiency at high Pb concentration.
To conduct MICP in-situ, both bio-augmentation and bio-stimulation
techniques can be used. The distinction between bio-augmentation and
bio-stimulation is that bio-augmentation introduces exogenous bacteria
strains, and bio-stimulation enriches indigenous bacteria to promote
certain biogeochemical processes (Dhami et al., 2017; Gomez et al.,
2017). Bio-stimulation saves the cost of incubating bacteria but still
gives comparable performances in soils (Gomez et al., 2017), while
bio-augmentation serves better where natural nutrients are poor
(Dhami et al., 2017). In this study, we adopts the bio-augmentation
pathway for the experiment.
This study aims to: (1) investigate the survivability of Sporosarcina
pasteurii and Pb immobilization efficiency by MICP within a wide
range of Pb concentrations; and (2) identify controlling factors that af-
fect Pb immobilization efficiency by MICP. In this study, bacterial viabil-
ity and Pb immobilization efficiency via MICP were investigated with
initial Pb concentration up to 50 mM in a series of aqueous solution ex-
periments. Bacterial viability was evaluated based on electrical conduc-
tivity (EC), pH, viable cell numbers and urease activity at various Pb
concentrations. Pb immobilization efficiency via MICP was assessed
based on Pb removal percentage at various Pb concentrations, initial
bacteria concentrations and calcium sources.
2. Materials and testing methods
2.1. Bacteria and chemicals
Sporosarcina pasteurii (S. pasteurii, ATCC 6452) was used in this
study for MICP. S. pasteurii is non-pathogenic bacterium with high
level of urease activity. Bacterial solution was made by inoculating bac-
terial colonies into the NH4-YE solution medium (20 g/L yeast extract,
10 g/L (NH4)2SO4, and 0.13 M Tris-base) and then subjected to
shaking-incubation at 30 °C for approximately 24 h until the optical
density at 600 nm (OD600) reached 1.5. The OD600 value was measured
via a UV–Visible spectrometer (Shanghai Analytical Instruments Co Ltd.,
Shanghai, China). The prepared bacterial solution was then stored at 4
°C. Prior to its use in the experiment, it was firstly re-harvested in a
fresh growth medium. When bacteria cells reached the exponential
growth stage during resuscitation, they were inoculated into the solu-
tion for formal test. Stock solutions (1 M) were prepared using analyti-
cally pure reagents including Pb(NO3)2, CaCl2, Ca(Ac)2 (i.e. calcium
acetate) and urea. To make solutions, the predetermined amount of re-
agents was mixed with proper amount of distilled water using a mag-
netic stirrer. The solution was then diluted with distilled water to
1.0 M. NH4-YE medium, CaCl2 and calcium acetate (Ca(Ac)2) solutions
were sterilized by autoclaving at 121 °C for 20 min, while Pb(NO3)2
and urea solutions were filter-sterilized with 0.22-μm filter membrane
(Zhao et al., 2017). Stock solutions were stored at 4 °C before use.
2.2. Bacterial activity tests
Bacterial activity test was conducted to monitor the growth of newly
inoculated S. pasteurii in Pb-containing NH4-YE liquid medium, which
724 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731

aimed to reveal the effects of Pb concentration on bacterial survivability
and urease activity. The overview of the bacterial activity test is sche-
matically shown in Fig. 1. Bacterial solution was firstly inoculated (1%
(v/v)) into the NH4-YE solution medium amended with Pb(NO3)2. The
concentration of Pb(NO3)2 ranged from 0 to 50 mM. The final bacterial
solution containing bacteria, nutrient and Pb(NO3)2 was then incubated
at 30 °C with shaking (200 rpm) for 72 h. pH, EC and urease activity of
the final bacterial solution were measured at 0, 1, 2, 4, 8, 24, 48 and
72 h. Viable cell number in terms of colony forming unit (CFU) was
measured at 0, 12, 24, 48 and 72 h. In particular, EC measured the elec-
trolytic ion concentrations in aqueous solution. Bacterial activity
(i.e., metabolism) involved the decomposition of large organic mole-
cules in culture medium into electrolytic ions (e.g., the consumption
of protein by bacteria), leading to increased EC in the solution
(Krishnamurti and Kate, 1951). Therefore, EC was used as an indicator
of bacterial metabolic activity in this study.
More specifically, pH and EC were measured using a handheld pH
meter (Shanghai Leici PHB-4 handheld pH meter, China) and a bench-
top conductivity meter (Thermo Scientific Orion Star A215 pH/Conduc-
tivity benchtop multiparameter, USA), respectively. Urease activity was
measured based on the method presented by Van Paassen (2009) and
was expressed as the amount of hydrolyzed urea per minute. 1 mL
final bacterial solution was mixed with 9 mL 1.11 M urea, with EC mea-
sured at 1 min and 8 min after mixing at room temperature. Eq. (5)
gives the equation to calculate urease activity:
EC8 −EC1  
UA ¼  10  11 mM urea min−1
7 differently.
where UA is urease activity and EC1 and EC8 are electrical conductivity
at 1 and 8 min, respectively. The viable cell number was obtained
using the plate counting method (Sanders, 2012). Viable cell number
was preferred over OD600 measurement in this study, since OD600 was
proportional to total cell number rather than viable one. Firstly, final
bacterial solution underwent serial dilution and was thereby spread
on fresh NH4-YE agar plates. Number of bacterial colonies on the agar
plate was counted after 24 h incubation at 30 °C. Viable cell number in
final bacterial solution was expressed in CFU and was equal to counted
colony number on the plate multiplied by a dilution factor (102, 104, 106,
and 108 in this study) (Kang and So, 2016). All abovementioned tests
had three biological replicates with coefficient of variance (COV) b10%.
2.3. Pb immobilization tests
The objective of the Pb immobilization test was to determine the Pb
immobilization efficiency via MICP at different initial Pb and bacterial
concentrations, and calcium sources. The overview of the Pb immobili-
zation test is shown in Fig. 1. Three experimental settings were designed
to examine the effects of calcium source and initial bacteria concentra-
tion on the Pb immobilization efficiency via MICP at different initial Pb
concentrations. In Case 1, 3 mL bacterial solution (OD600 = 1.5) was
mixed with 27 mL aqueous solution of 0–50 mM Pb(NO3)2 and
200 mM urea/CaCl2 mixture. In Case 2, the procedures were the same
as Case 1 except that CaCl2 was substituted by Ca(Ac)2. In Case 3, Pb
(NO3)2, urea and Ca(Ac)2 were dissolved in the bacterial solution to
make a total volume of 30 mL with 0–50 mM Pb(NO3)2 and urea/Ca
(Ac)2 at 200 mM. It can be seen that Case 1 and Case 2 were differed
in terms of calcium source while Case 2 and Case 3 were differed in
terms of initial bacterial concentration. The 200 mM urea/Calcium con-
centration was selected that did not adversely affect the viability of
S. pasteurii. It should be noted, however, that the different anions of cal-
cium sources might impact the bacterial viability and MICP efficiency
ð5Þ
The mixed solution was incubated at 30 °C with 2 mL solution sam-
ples taken at 0, 24 and 48 h for the measurement of viable cell number,
produced ammonium concentration (which came from the urea hydro-
lysis) and remaining Pb concentration in solution. The ammonium con-
centration in solution was determined using the modified Nessler
method (Whiffin et al., 2007). The solution sample was firstly acidified
to convert all ammonia into ammonium. Then, it was diluted 100–500

Fig. 1. Schematic diagram of the bacterial activity test (marked in red) and Pb immobilization test (marked in blue) (DW: distilled water and BS: bacterial solution). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
times to minimize the interference of Pb and Ca in the solution. 2 mL di-
luted solution was subsequently mixed with 100 μL Nessler solution for
1 min before subjected to the spectrophotometer measurement at the
wavelength of 425 nm. Absorbance reading was then converted to the
ammonium concentration by referring to a calibration curve obtained
using standard NH4Cl. The Pb concentration was determined using a
Thermo Scientific iCE 3000 Series atomic absorption spectrometer
after the sample solution was acidified to yield a pH b2.0. At the end
of the test, produced precipitation was separated from aqueous solution
using the vacuum filtration method (Keykha et al., 2018) and dried at 30
°C for 3 days before subjected to mass measurement. Total precipitation
mass was obtained by measuring mass of unused filter membrane be-
fore filtration and mass of filter membrane with precipitation settled
on it after drying. The filters were weighed individually before and
after filtration. All abovementioned tests had three biological replicates
with coefficient of variance (COV) b10%.
2.4. Geochemical modelling on Pb precipitation
Precipitation speciation in the Pb immobilization tests was simu-
lated using Visual MINTEQ software. Although Visual MINTEQ was un-
able to simulate bacterial activities due to limitation of its database, it
could model the evolution of precipitation speciation with increased
amount of hydrolyzed urea. More specifically in the simulation, urea hy-
–
drolysis was modeled as an incremental titration of NH+ 4 and CO3 with a
stoichiometric ratio of 2:1 (Gat et al., 2017), which were input as initial
conditions for various simulation cases. The initial conditions in Case 1,
Case 2 and Case 3 are summarized in Table 1. Then the evolution of var-
ious precipitation phases could be obtained at different amount of hy-
drolyzed urea (from 0 to 200 mM). In this way, the biotic effect of
MICP on Pb precipitation was obtained.
3. Results and analysis
3.1. Bacterial activity tests
Bacterial metabolic activities utilized nutrients as the energy source
through breaking down macromolecules. This process led to an increase
in ion concentration and thereby an increase in EC value (Krishnamurti
and Kate, 1951). Therefore in this test, EC value was used as an indicator
of bacterial metabolic activity. Although the initial EC value depended
on Pb(NO3)2 concentration, the further increase in EC value, more spe-
cifically the EC change rate, reflected the bacterial metabolic activity.
Fig. 2(a) shows the evolution of EC value with time in the bacterial activ-
ity test. At Pb concentration between 0 and 30 mM, EC increased
steadily from 9 ~ 10 mS/cm to 12 ~ 16 mS/cm in 72 h, with fastest
change rate occurring at 30 mM. This indicates that bacterial metabolic
activity reached peak at 30 mM of Pb. However, when Pb concentration
was higher than 30 mM, EC increased from around 9.5 mS/cm to around
12.5 mS/cm within a very short period, but totally stopped changing
after 8 h. This indicates that, at high Pb concentration (N30 mM), bacte-
rial metabolic activity was intensively stimulated upon contact with Pb
contaminants, but diminished quickly in a few hours. Though not exper-
imentally determined, the faster EC increase in the initial hours at
higher Pb concentration was likely due to higher salinity brought by
more Pb contaminants and stronger bacterial metabolism for
Table 1
Initial conditions for the geochemical modelling.
Chemical components Case 1 (mM) Case 2 and Case 3 (mM)
2+
Calcium (Ca ) 200 200
Lead (Pb2+) 0–50 0–50
Nitrate (NO-3) 0–100 0–100
Chloride (Cl−) 400 0 2013).
Acetate (CH3COO−) 0 400
Urea ((NH2)2CO) 0–200 0–200
725
synthesizing exopolysaccharide (EPS) in response to heavy metal toxic-
ity (Gupta and Diwan, 2017). Fig. 2(b) shows the change of solution pH
with time in the bacterial activity test. At Pb concentration no higher
than 30 mM, pH decreased from (8.5~9.5) to (7.5–~8) in 72 h. The
drop in pH at initial hours was probably due to acid-base equilibria
with the addition of Pb(NO3)2, while continuous pH decrease later
was assumed to result from the production of carbonic acid by respira-
tion and organic acids by the consumption of carbohydrates (Jimenez-
Lopez et al., 2008) during bacterial metabolism. Higher Pb concentra-
tion corresponded to a faster pH drop. Then, when Pb concentration
was over 30 mM, pH displayed a sharp drop from (9~9.25) to 8.25 in
24 h and then remained almost unchanged. Fig. 2(c) shows the change
in viable cell number in terms of CFU in solution with time. The viable
cell number at 0 h (immediately after bacteria contacts with Pb) was be-
tween 106 and 107 CFU/mL. At Pb concentration no higher than 30 mM,
viable cell number went up to 109 CFU/mL at 24 h, indicating that bac-
teria were in the exponential growth stage. After 24 h, viable cell num-
ber remained stable, indicating that bacteria were in the stationary
growth stage. However, when Pb concentration reached 40 mM, viable
cell number dropped from 107 to 104 CFU/mL at the end of the test, in-
dicating that bacteria were in the death condition. At 50 mM Pb, viable
cells were not detected even after 12 h.
The ability of ureolytic bacteria to induce MICP and immobilize
heavy metal contamination depended primarily on their urease activity
(Anbu et al., 2016). Fig. 2(d) shows the evolution of urease activity with
time. Urease activity fluctuated between 0 and 4 mM hydrolyzed urea/
min in initial 4 h regardless of Pb concentration, possibly due to adaptive
process of bacteria after being introduced to a new medium. Then at Pb
concentration lower than 30 mM, urease activity steadily increased to
8–12 mM hydrolyzed urea/min at the end of the test. In contrast, urease
activity was mostly below 2 mM hydrolyzed urea/min at Pb
concentration ≥ 30 mM.
3.2. Pb immobilization tests
Fig. 3(a) displays the change of viable cell number in solution during
the Pb immobilization test. In Case 1, viable cell number at 0 h dropped
from 5 × 106 to 5 × 103 CFU/mL when Pb concentration increased to
30 mM. It then recovered slightly to 104 CFU/mL when Pb concentration
reached 50 mM. However, at 24 and 48 h, viable cells could not be de-
tected when Pb was present in solution. In Case 2, viable cell number
steadily dropped from around 107 CFU/mL to b104 CFU/mL when Pb
concentration reached 50 mM. Similarly as in Case 1, no viable cells
existed after 24 and 48 h as long as Pb contaminant was present. Finally
in Case 3, viable cell number maintained stable although reduction was
observed at 40 and 50 mM Pb after 24 and 48 h. The significant differ-
ence between Case 2 and Case 3 in terms of viable cell number at 24
and 48 h was likely due to the amount of yeast extract presented in
the solution since yeast extract was a rich carbon source, providing
amino-acids essential for bacterial growth (Bornside and Kallio, 1956).
Thus, bacteria cells in Case 3 had sustained rich carbon and amino-
acid source to maintain high cell concentration.
It should be noted that there were substantial differences in terms of
viable cell number in the bacteria activity test and Pb immobilization
test. Without direct experimental evidence, we could not definitely ex-
plain this phenomenon. One hypothesis was that both abiotically and
biotically formed Pb precipitation in the Pb immobilization test was
likely to encapsulate bacterial cells, cutting off their nutrient supply. En-
capsulated cells lost viability faster. Therefore, the measured viable cell
number in the Pb immobilization test was mostly smaller than that in
the bacterial activity test. Moreover, the forms of carbonate precipita-
tion in the two cases were also likely to be different, which might con-
tribute to different viable cell numbers in the two test (Gorospe et al.,
Fig. 3(b) presents the ammonium concentration in solution at differ-
ent initial Pb concentrations. As (NH4)2SO4 was originally included in
726 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731

t t

t t

Fig. 2. Variables in the bacteria activity test: (a) EC, (b) pH, (c) viable cell number and (d) urease activity in solution (Number of replicates = 3; COV b 10%).

Fig. 3. Variables in the Pb immobilization test: (a) viable cell number, (b) ammonium concentration and (c) Pb removal percentage in solution. (Number of replicates = 3; COV b 10%).
 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
the bacterial solution, there was a baseline ammonium concentration
(around 15 mM) in the solution. Any additional ammonium concentra-
tion above the baseline value was due to urea hydrolysis. In Case 1, the
ammonium concentration from ureolysis at 0 h was 20 mM (the base-
line value has been subtracted) at 10 mM initial Pb. With the increase
in initial Pb concentration, the ammonium concentration from ureolysis
dropped steadily and reached 5 mM at 50 mM initial Pb. With time, a
general decreasing trend of ammonium concentration was observed,
which was likely due to the volatilization of ammonia under alkaline
conditions (Gat et al., 2017). Case 2 showed very similar trend as Case
1, except that the ammonium concentration from ureolysis at 0 h was
18 mM at 10 mM initial Pb and dropped steadily to 8 mM at 50 mM ini-
tial Pb. The ammonium concentration results in Case 1 and Case 2 indi-
cated that ureolysis and MICP reactions did happen initially, but soon
stopped due to insufficient cell concentration and presence of Pb. Differ-
ently in Case 3, the ammonium concentration from ureolysis at 0 h
ranged from 18 mM to 36 mM, which was not significantly influenced
by initial Pb concentration. Then, the ammonium concentration in-
creased with time regardless of initial Pb concentration. At 48 h, the
measured ammonium concentration from ureolysis was 110 mM and
65 mM at 10 mM and 50 mM initial Pb, respectively. The results in
Case 3 indicated that substantial amount of urea was hydrolyzed and
MICP reactions did happen throughout the entire testing period.
Fig. 3(c) presents Pb removal percentage in solution at different Pb
concentrations. Pb removal percentage represents Pb immobilization
efficiency and was expressed as Eq. (6):
C I −C R
Pb removal percentage ¼  100%
CI
where CI stands for initial Pb concentration and CR stands for remaining
Pb concentration in aqueous solution. In Case 1, Pb removal percentage
dropped from 85~95% to around 80% when initial Pb concentration in-
creased to 20 mM. With Pb concentration increased to 50 mM, Pb re-
moval percentage reached around 95%. Pb removal percentage at 0 h
and 24 h were close, while it was slightly lower at 48 h. It was not
clear why Pb removal percentage dropped slightly with time. One hy-
pothesis might be that some Pb were initially subjected to bio-
absorption (accumulation of metal cations on negatively charged cell
surface (Kotrba et al., 2011)) and were counted as immobilized specia-
tion. However, with elapsed time, viable cell number declined especially
at high initial Pb concentrations, leading to the Pb desorption. Further
studies are needed to confirm this hypothesis. In Case 2, Pb removal per-
centage declined from around 45% to around 20% at 0 h when the initial
Pb concentration increased to 20 mM, and rebounded to around 40%
with initial Pb concentration increases to 50 mM. In Case 3, Pb removal
percentage was close to 100% when initial Pb concentration increased to
30 mM, then decreased slightly to 95–97% when initial Pb concentration
reached 50 mM. With increasing time, Pb removal percentage slightly
went up, which was different from the trend in Case 1 and Case 2.
Finally, the produced precipitation mass in the three cases are
shown in Fig. 4. It is clearly that at an identical Pb concentration, Case
3 had the greatest precipitation mass while Case 2 had the least. This
conclusion was consistent with the results of Pb removal percentage
discussed before. In our study, the precipitation phases were not identi-
fied experimentally due to insufficient mass of collected samples. How-
ever, the following geochemical model simulation results could help
understand the Pb speciation at various initial Pb concentrations in
the three cases.
3.3. Geochemical modelling on precipitation
The geochemical modelling results revealed the change of Pb precip-
itation phases with increased amount of hydrolyzed urea. The simula-
tion result of Case 1, in which CaCl2 was used, is shown in Fig. 5. The
results of Case 2 and Case 3, in which Ca(Ac)2 was used, are shown in
727
0.40
Case 1
0.35 Case 2
Case 3
0.30
0.25
?
0.20
0.15
0.10
0.05
0.00
0 10 20 30 40 50
Pb concentration (mM)
Fig. 4. Total precipitation mass at different initial Pb concentration in the Pb
immobilization test (Number of replicates = 3; COV b 10%).
Fig. 6. In the simulation of Case 1, it can be seen that when hydrolyzed
urea concentration was 0, the Pb precipitation was in the form of
PbCl2, with Pb removal percentage increased from 42% to 85% when ini-
tial Pb concentration increased from 10 to 50 mM. The initial PbCl2 pre-
cipitation was purely abiotic. With slight increase in hydrolyzed urea
concentration (0–17 mM), which was a biotic effect, the Pb removal
percentage abruptly increased to N85%. The Pb precipitation also trans-
ð6Þ
formed into PbCO3 and CaCO3, with intermediate phases in transition
((PbCl)2CO3 and Pb3(CO3)2(OH)2).
In the simulation of Case 2 and Case 3, as shown in Fig. 6, it can be
seen that when there was no hydrolyzed urea, the Pb precipitation
was in the form of Pb(OH)2, with Pb removal percentage dropped
from 17.8% to 8.1% when initial Pb concentration increased from
10 mM to 50 mM. The initial Pb(OH)2 precipitation was purely abiotic.
With moderate increase in hydrolyzed urea concentration
(12–60 mM), which was a biotic effect, the Pb removal percentage
abruptly increased to N97%. The Pb precipitation also transformed into
PbCO3 and CaCO3, with intermediate phase Pb3(CO3)2(OH)2 in
transition.
In order to verify the geochemical model, measured ammonium
concentrations from ureolysis were input into the model to calculate
Pb2+ removal percentage in solution. The comparison with experimen-
tal results is shown in Fig. 3(c). It can be seen that in all three cases, the
simulated results were close to the ones from the experiment, indicat-
ing the validity of the developed geochemical model. The moderate var-
iations between simulated and experimental results were possibly due
to the volatilization of ammonia and pH variation during the test.
One thing that needs to be mentioned is that bio-sorption was also a
contributor to Pb removal and it was likely that larger viable cell num-
ber corresponded to more Pb being absorbed biologically (Kotrba
et al., 2011). Thus, it was rational to assume that bio-sorption played a
more important role in Case 3 than Case 1 and Case 2 due to its
overwhelmingly higher viable bacteria concentration. However, it was
unknown to what extent the bio-sorption might impact Pb removal per-
centage, as it could not be individually determined in the experiment.
4. Discussion
4.1. Effect of viable cell number on urease activity
Urease activity is the most critical parameter to evaluate whether
ureolytic bacteria have sufficient capacity to trigger MICP. Urease activ-
ity is found to be dominantly controlled by viable cell number in the so-
lution during the bacterial activity test (See Fig. 7). More specifically,
substantial urease activity is only present when viable cell number is
728 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
Fig. 5. Geochemical modelling results for Case 1 at various initial Pb concentrations: (a) 10 mM, (b) 20 mM, (c) 30 mM, (d) 40 mM, (e) 50 mM.
N108 CFU/mL. This reveals how elevated Pb contamination level nega-
tively affects urease activity of S. pasteurii by reducing the amount of vi-
able cells. This correlation is consistent with the results reported by
Achal et al. (2012a), with our study covers wider range of Pb concentra-
tion, which is a novelty of the current study. According to Srivastava and
Kowshik (2018), heavy metal contaminants at high concentration can
make bacteria incapable of producing enough exopolysaccharides
(EPS) to absorb heavy metals and thereby bacteria cells are deactivated
and then become dead. This leads to a reduction in viable cell number
and consequently reduced urease activity.
Mugwar and Harbottle (2016) conducted similar tests on toxicity ef-
fect of Pb on S. pasteurii, where 1 mM of Pb (in total concentration) was
found to suppress OD600. This threshold value was remarkably lower
than 30 mM in our study. On the other hand, Kang et al. (2015) showed
that several ureolytic bacteria strains could withstand Pb concentration
up to 12.5–60 mM. It is likely that the difference in the threshold Pb con-
centration is due to the variance of Pb bioavailability in different culture
media. In particular, yeast extract may reduce Pb bioavailability and in-
crease threshold Pb concentration.
It should be noted that previous studies also show that the activity of
extracellular urease is remarkably affected by pH of surrounding envi-
ronment (Stocks-Fischer et al., 1999). However, in our study, the intra-
cellular and extracellular urease cannot be distinguished and
intracellular urease activity is dependent on the viable cell number.
Thus, the urease activity is not directly linked to pH in our study.
4.2. Effect of viable cell number and calcium source on Pb immobilization
via MICP
Fig. 8 shows the relationship between Pb removal percentage and vi-
able cell number in the Pb immobilization test. It is found that Pb re-
moval percentage has a positive relationship with viable cell number
in all three cases, though different calcium sources lead to slightly differ-
ent increase pattern. This indicates that it is important to maintain suf-
ficient viable cells in order to reach higher Pb removal percentage,
regardless of Pb concentration.
The viable cell number and calcium source change the Pb removal
percentage in three ways: abiotic precipitation, biotic precipitation
 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
Fig. 6. Geochemical modelling results for Case 2 and 3 at various initial Pb concentrations: (a) 10 mM, (b) 20 mM, (c) 30 mM, (d) 40 mM, (e) 50 mM.
Fig. 7. The effect of viable cell number on urease activity in the bacteria activity test.
729
and bio-sorption, according to the experimental and geochemical simu-
lation results obtained in this study. Firstly, the calcium resource deter-
mines the initial abiotic precipitation. When CaCl2 is present, the initial
abiotic precipitation is PbCl2, which can achieve 42% to 85% Pb removal
percentage at the Pb range used in this study. When Ca(Ac)2 is present,
the precipitation is Pb(OH)2 with the Pb removal percentage ranging
from 8% to 18%. Secondly, the viable cell number determines the
ureolysis-induced and bio-sorption-induced precipitations, both are bi-
otic but cannot be separated in the current study. Therefore, in a Pb-
containing aqueous environment where substantial viable cells can sur-
vive and actively function for a long time, the biotic precipitation will be
significant, as shown in Fig. 8(a) and (b). The comparison between Case
2 and Case 3 clearly shows the difference in biotic precipitation when vi-
able cell number varies.
To the knowledge of the authors, there are few existing studies that
explicitly illustrate the relationship between Pb immobilization per-
centage and viable ureolytic bacteria cell number. Li et al. (2013) re-
ported the evolution of Pb removal rate with time when treated by
different urease producing bacteria. Pb removal rate exceeded 80%
730 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731

100 of numerous bacterial cells, the precipitates are likely to be nucleated

(a)
Pb removal percentage in solution (%)

based on these cells (Ferris et al., 1994, 1995). The initially formed abi-
Upper bound
otic precipitates are likely to be partially converted into biotic precipi-
tates which form the outer layer and encapsulate the initial abiotic
90 precipitates, preventing their further conversion. Accordingly, we have
proposed a hypothesized multi-layer precipitation structure, as sche-
matically illustrated in Fig. 9. In this structure, abiotic precipitates
10 mM (PbCl2 in Case 1 and Pb(OH)2 in Case 2 and Case 3) nucleate based on
80 20 mM bacterial cells and are wrapped by biotic precipitates in the sequence
Lower bound 30 mM
of (PbCl)2CO3, PbCO3, and CaCO3 in Case 1 and Pb3(CO3)2(OH)2, PbCO3
40 mM
50 mM and CaCO3 in Case 2 and Case 3. The CaCO3 on the outer perimeter can
protect Pb-related precipitations from being flushed or acid leached.
103 104 105 This hypothesized multi-layer precipitation structure is similar to the
Viable cell numbers in solution (CFU/mL) conceptual model of ureolysis driven calcite precipitation approach for
remediation of 90Sr contaminated medium proposed by Fujita et al.
(2010). Although geochemical modelling results support this hypothe-
100 (b)
Pb removal percentage in solution (%)

sis, further studies, in particular physicochemical, mineralogical and mi-

Upper bound crostructural analyses, are needed to confirm it.
80 Case 3
Case 2
60 5. Conclusions
Lower bound
40 In this study, the bacterial activity and Pb immobilization tests were
10 mM
20 mM conducted to examine the effectiveness and mechanisms of MICP for Pb
20 30 mM
40 mM
50 mM
0
101 102 103 104 105 106 107 108 109 (a)
Viable cell number in solution (CFU/mL)

C
Fig. 8. The effect of viable cell number on Pb removal percentage in the Pb immobilization

aC
test: (a) Case 1; (b) Case 2 and Case 3.

Pb

O3
(P

C
bC

O3
l) 2
after 4 h. However, the relationship between Pb removal rate and OD600

C
Pb
was not explicitly given. Achal et al. (2012) reported the different pre- Negative

O3
(C
cipitate phases of Pb when MICP was used for Pb immobilization. In par- charged
l) 2
ticular, carbonate fraction was identified as dominant precipitate.
However, biotic and abiotic precipitations were not explicitly distin-
C

guished. Fujita et al. (2010) performed a geochemical modelling to spe-

el
l

cifically reveal the evolution of different precipitate fractions of heavy

metal when subjected to ureolysis immobilization. However, the results
were only limited to 90Sr in his study.

4.3. Mechanisms of Pb immobilization via MICP

(b)
From the above discussion, it can be seen that the efficiency of Pb im-
mobilization via MICP is controlled by both abiotic and biotic mecha-
nisms. Abiotically, the low Ksp value of PbCl2 leads to its precipitation
when Cl− ions are presented in the solution in Case 1. In Case 2 and
Case 3, the initial aqueous chemical composition leads to an acid-base
C
Pb

aC

equilibrium in favor of the precipitation of Pb(OH)2. Biotically, the grad-

Pb
3

O3
(C

ual urea hydrolysis leads to the conversion of existing and formation of

C
O3
O3

new precipitates. If ureolysis can substantially happen, then the final

Negative
Pb

) 2(

precipitates are primarily PbCO3 and CaCO3, with Pb removal percent-

charged
O
(O

age nearing 100%. Meanwhile, bio-sorption, whose significance

H
H

)2

depends on viable cell concentration, can further enhance Pb

)2

immobilization.
According to the geochemical model results as demonstrated in
C
el

Figs. 5 and 6, with the increase in the degree of ureolysis, major precip-
l

itates form in the following sequence: PbCl2 → (PbCl)2CO3 → PbCO3

→ CaCO3 (Case 1) and Pb(OH)2 → Pb3(CO3)2(OH)2 → PbCO3 → CaCO3
(Case 2 and Case 3). Theoretically in an aqueous solution with high de-
gree of ureolysis, PbCl2 and (PbCl)2CO3 should be fully converted into
PbCO3 and CaCO3 in Case 1 while Pb(OH)2 and Pb3(CO3)2(OH)2 into
PbCO3 and CaCO3 in Case 2 and Case 3. However, due to the presence Fig. 9. Hypothesized multi-layer precipitation structure: (a) Case 1; (b) Case 2 and Case 3.
 N.-J. Jiang et al. / Science of the Total Environment 672 (2019) 722–731
immobilization. Based on the results, the following conclusions can be
drawn:
(1) S. pasteurii exhibited compatible resistance to Pb toxicity when
Pb concentration was no higher than 30 mM, with viable cell
number remained at 108–109 CFU/mL and urease activity at
8–12 hydrolyzed mM urea/min.
(2) Pb removal percentage in solution was higher at its higher initial
bacterial concentration. At identical initial bacterial concentra-
tion, removal percentage was higher when CaCl2 was used as cal-
cium source as compared to Ca(Ac)2. The highest Pb removal
percentage (N95%) was observed in Case 3 which had the largest
viable cell number and greatest total precipitation mass.
(3) Ureolytic activity in the presence of Pb was largely determined
by the concentration of viable cells, which were directly related
to Pb toxicity in solution. High urease activity was only possible
when viable cell number exceeded 108 CFU/mL. Pb immobiliza-
tion efficiency was determined by abiotic precipitation, biotic
precipitation and bio-sorption. Abiotic precipitation was depen-
dent on calcium source and initial Pb concentration. Biotic pre-
cipitation depended on the degree of ureolysis. Bio-sorption
was largely controlled by the viable cell number.
Acknowledgements
This work is supported by the National Key Research and Develop-
ment Program of China (Grant Nos. 2018YFC1803100 and
2018YFC1802300), Environmental Protection Scientific Research Pro-
ject of Jiangsu Province (Grant No. 2016031), National Natural Science
Foundation of China (Grant Nos. 41472258 and 41877248), Natural Sci-
ence Foundation of Jiangsu Province (Grant No. BK20170394), China
Postdoctoral Science Foundation (Grant No. 2017M621595), and Indo-
U.S. Science and Technology Forum (Grant No. IUSSTF/AUG/JC/047/
2018).
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